Research ArticleAstragaloside IV Inhibits NF-120581B Activation and InflammatoryGene Expression in LPS-Treated Mice

Wei-Jian Zhang and Balz Frei

Linus Pauling Institute and Department of Biochemistry and Biophysics Oregon State University307 Linus Pauling Science Center Corvallis OR 97331 USA

Correspondence should be addressed to Wei-Jian Zhang weijianzhangoregonstateedu

Received 16 December 2014 Revised 27 February 2015 Accepted 28 February 2015

Academic Editor Yi Fu Yang

Copyright copy 2015 W-J Zhang and B Frei This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In this study we investigated the role of astragaloside IV (AS-IV) one of the major active constituents purified from the Chinesemedicinal herb Astragalus membranaceus in LPS-induced acute inflammatory responses in mice in vivo and examined possibleunderlying mechanisms Mice were assigned to four groups vehicle-treated control animals AS-IV-treated animals (10mgkg bwAS-IV daily ip injection for 6 days) LPS-treated animals and AS-IV plus LPS-treated animals We found that AS-IV treatmentsignificantly inhibited LPS-induced increases in serum levels ofMCP-1 and TNF by 82 and 49 respectively AS-IV also inhibitedLPS-induced upregulation of inflammatory gene expression in different organs Lung mRNA levels of cellular adhesion moleculesMCP-1 TNF120572 IL-6 and TLR4were significantly attenuated and lung neutrophil infiltration and activationwere strongly inhibitedas reflected by decreased myeloperoxidase content when the mice were pretreated with AS-IV Similar results were observed inheart aorta kidney and liver FurthermoreAS-IV significantly suppressed LPS-inducedNF-120581BandAP-1DNA-binding activities inlung and heart In conclusion our data provide new in vivo evidence thatAS-IV effectively inhibits LPS-induced acute inflammatoryresponses by modulating NF-120581B and AP-1 signaling pathways Our results suggest that AS-IV may be useful for the prevention ortreatment of inflammatory diseases

1 Introduction

Endothelial cells are a primary target of inflammatoryresponses and their injury can lead to vasculopathy andorgan dysfunction [1] The bacterial endotoxin LPS directlyelicits several acute inflammatory responses in endothelialcells including production of cellular adhesion moleculessuch as E-selectin vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1 (ICAM-1)and other proinflammatory mediators such as TNF120572 IL-6and monocyte chemoattractant protein-1 (MCP-1) Togetherthese proinflammatory mediators elicit leukocyte adhesionto the vasculature and transmigration into the underlyingtissue causing endothelial injury and dysfunction associatedwith sepsis [1] Although these proinflammatory mediatorsare required for an adequate host-defense responsetheir dysregulation can lead to refractory hypotension

cardiovascular hyporeactivity intravascular coagulationmultiple organ failure and death [2 3]

The regulation of inflammatory gene transcription hasbeen shown to be controlled by specific signaling pathwaysand transcription factors such as NF-120581B and AP-1 [4 5] Inparticular the NF-120581B pathway affects host defense againstinfectious agents by upregulating inflammatory genes thatcause acute neutrophilic inflammation and the systemicinflammatory response syndrome [5] Under normal con-dition NF-120581B is located in the cytoplasm in an inactiveform in association with its inhibitor I120581B In response tostimulation for example by LPS I120581B is phosphorylated byI120581B kinase Following phosphorylation I120581B is ubiquitinatedand degraded allowing NF-120581B to translocate to the nucleusbind to DNA promoter regions and induce inflammatorygene transcription Various agents that blockNF-120581B signalinghave been shown to decrease expression of proinflammatory

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 274314 11 pageshttpdxdoiorg1011552015274314

2 Mediators of Inflammation

mediators [6ndash9] Thus the inhibition of NF-120581B activation isexpected to be protective in pathological inflammatory states

Astragalus membranaceus is one of the most widely usedChinese medicinal herbs for the treatment of many diseasesincluding cardiovascular disease nephritis hepatitis anddiabetes [10] It also has been available in Europe and theUS for many years as a food supplement Evidence frompharmacological research and clinical practice suggests thatAstragalus possesses a wide spectrum of activities includingimmunomodulation [11 12] cardiovascular protection [13ndash15] anti-inflammatory effects [16ndash18] hepatoprotection [1920] antidiabetes [21] anticancer [22] and neuroprotection[23]

The biologically active constituents of Astragalus rootsrepresent three classes of chemical compounds saponinspolysaccharides and flavonoids [24] By chemical degra-dation and 13C nuclear magnetic resonance examina-tion the structure of astragaloside IV (AS-IV) was deter-mined as 3-O-120573-D-xylopyranosyl-6-O-120573-D-glucopyranosyl-cycloastragenol (C

41H68O14 MW = 7849) [25] As one of

the major active constituents of Astragalus AS-IV is used asthe characteristic marker for quality evaluation of Astragalusin the Chinese Pharmacopeia and has been shown to exertpotent cardioprotective and anti-inflammatory effects [6 1026ndash30] We have previously shown that AS-IV inhibits LPS-and TNF120572-induced adhesion molecule expression and NF-120581B activation in cultured human endothelial cells [6] How-ever in vivo evidence supporting such activity is currentlylacking Therefore in this study we investigated whether AS-IV can inhibit LPS-induced acute inflammatory responses inexperimental mice

2 Materials and Methods

21 Animals and Experimental Procedures Female C57BL6Jmice 12 weeks old and weighing 20ndash22 g were purchasedfrom Jackson Laboratory (Bar Harbor ME) and housedin specific pathogen-free conditions and a temperature-and humidity-controlled environment (12-h lightdark cycle)with unlimited access to tap water and Purina 5001 chow diet(Harlan Teklad Madison WI) The investigation conformedto the Guide for the Care and Use of Laboratory Animals byNIH and all animal procedures were reviewed and approvedby the Oregon State University Institutional Animal Care andUse Committee

AS-IV was purchased from Quality Phytochemicals LLC(Edison NJ) and a stock solution was prepared with propy-lene glycol (SigmaAldrich St LouisMO) and further dilutedwith Hankrsquos buffered saline solution (HBSS) LPS (serotype055B5 from Escherichia coli Sigma Aldrich) stock solutionwas prepared in HBSS Mice were randomly assigned to4 groups as follows (i) control animals received daily ipinjection of the vehicle propylene glycol with HBSS for 6days followed by a single ip HBSS injection (ii) AS-IV-treated animals received 10mgkg bw AS-IV daily ip for 6days followed by a single ip HBSS injection (iii) LPS-treatedanimals received daily ip injection of propylene glycol withHBSS for 6 days followed by a single ip injection of 05 120583ggbw LPS and (iv) AS-IV plus LPS-treated animals received

AS-IV daily ip for 6 days followed by single ip injectionof LPS Animals were sacrificed 3 hours after HBSS or LPSinjection Based on our previous observations the 3-h timepoint and LPS dose of 05 120583gg bw were chosen [31] In somestudies mice were randomly assigned to receive ip injectionof LPS and sacrificed after 1 3 8 or 24 h Each group consistedof 4 to 5 animals After sacrifice blood and tissues werecollected for further analysis

22 Serum Inflammatory Mediators Serum concentrationsof MCP-1 TNF120572 sVCAM-1 and sICAM-1 were measuredby quantitative colorimetric sandwich ELISA (RampD SystemsMinneapolisMN)The sensitivity of the assays is 2 pgmL forMCP-1 5 pgmL for TNF120572 and 30 pgmL for sVCAM-1 andsICAM-1

23 Tissue mRNA Levels of Inflammatory Mediators TotalRNAwas isolated fromdifferent organs using TRIzol Reagent(Life Technologies Foster City CA) cDNA synthesis wasperformed using the high capacity cDNA archive kit (LifeTechnologies) mRNA levels of VCAM-1 ICAM-1 E-selectinP-selectin MCP-1 TNF120572 IL-6 myeloperoxidase (MPO)Toll-like receptor-4 (TLR4) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) were quantitated by real-timeqPCR All primers and probes were purchased as kits (Assayson Demand Life Technologies) The assays are supplied asa 20x mixture of PCR primers and TaqMan minor groovebinder 6-FAM dye-labeled probes with a nonfluorescentquencher at the 31015840 end TaqMan quantitative PCR (40 cyclesat 95∘C for 15 sec and 60∘C for 1min) was performed usingTaqMan Universal PCR Master Mix (Life Technologies) in96-well plates with an ABI Prism 7500 Sequence DetectionSystem (Life Technologies) To obtain relative quantificationtwo standard curves were constructed in each plate with onetarget gene and the internal control GAPDH gene Standardcurves were generated by plotting the threshold cycle numbervalues against the log of the amount of input cDNA and usedto quantify the expression of the target genes and GAPDHgene in the same sample After normalization to internalGAPDH in each sample results were expressed as percentageof GAPDH or fold of control

24 Lung Protein Concentration of Myeloperoxidase A partof the right lung lobe was homogenized and the cytosolicfraction of lung tissue homogenate was prepared usingnuclear extract kits (Active Motif Carlsbad CA) LungcytosolicMPOwas quantified using theMPO enzyme-linkedimmunosorbent assay kit (Hycult Biotechnology PlymouthMeeting PA) according to the manufacturerrsquos instructionsThe lung MPO concentration of each sample was normal-ized with cytosolic protein concentration and expressed asngMPOmg tissue protein

25 Nuclear Transcription Factors Nuclear extracts were pre-pared from lung and heart using nuclear extract kits (ActiveMotif) For analysis of nuclear transcription factor activationELISA-based assays (Active Motif) were used to determinethe DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos)

Mediators of Inflammation 3

Seru

m M

CP-1

(ng

mL)

0

20

40

60

80

100

LPSAS-IV

lowast

+

+ +

+

minus

minus

minus

minus

(a)

0

100

200

300

LPSAS-IV

Seru

m T

NF120572

(pg

mL)

lowast

+

+

minus

minus +

minus +

minus

(b)

Figure 1 AS-IV inhibits LPS-induced increases in serumMCP-1 and TNF120572 in mice Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPSThree hours after the HBSS or LPS injection the animals were sacrificed and bloodwas collected Serum MCP-1 (a) and TNF120572 (b) were measured by ELISA Data shown are mean values plusmn SEM of five animals per grouplowast119875 lt 005 compared to animals treated with LPS only

The specificity of binding was confirmed by competition witheither wild-type or mutant oligonucleotides

26 Statistical Analysis The data were calculated as means plusmnSEM and analyzed by ANOVA with Fisher PLSD post hoctest Statistical significance was set at 119875 lt 005

3 Results and Discussion

The pathophysiology of acute inflammation triggered by thebacterial endotoxin LPS is characterized by the productionof multiple proinflammatory cytokines and chemokinesexpression of adhesion molecules and infiltration of neu-trophils and monocytes into inflamed tissues Because of thecomplexity of the pathology of septic shock major effortshave focused on identifying novel anti-inflammatory drugsthat prevent the proinflammatory process at the early stage ofgene expression of key inflammatory mediators [32]

We have previously shown that AS-IV inhibits LPS- andTNF120572-induced adhesion molecule expression and conse-quent adherence of monocytes by modulating NF-120581B signal-ing pathway in cultured human endothelial cells [6] We nowprovide new evidence showing that AS-IV exhibits stronganti-inflammatory activities in vivo by attenuating LPS-induced acute inflammatory responses through inhibition ofNF-120581B- andAP-1-mediated inflammatory signaling pathwaysin mice

31 AS-IV Inhibits LPS-Induced Increases in SerumMCP-1 andTNF120572 in Mice Treatment of mice with AS-IV for 6 dayshad no effect on body weight changes compared to HBSS-treated control animals The mean body weight was 208 plusmn06 g before and 208 plusmn 05 g after 6-day AS-IV treatment

In control animals the mean body weight was 202 plusmn 03 gbefore and 199 plusmn 04 g after 6-day HBSS treatment We firstinvestigated the effect of AS-IV on LPS-induced systemicinflammatory responses by determining serum levels ofMCP-1 TNF120572 and the soluble cellular adhesion moleculessVCAM-1 and sICAM-1 Treatment ofmice with AS-IV alonefor 6 days had no effect on serum levels of these inflammatorymediators compared to HBSS-treated control animals Asexpected 3 hours after LPS injection significant increasesin the serum levels of MCP-1 and TNF120572 were observed(Figure 1) Interestingly pretreatment of animals with AS-IV significantly inhibited LPS-induced increases in serumMCP-1 (Figure 1(a)) andTNF120572 (Figure 1(b)) by 82 and 49respectively Specifically MCP-1 levels were 152 plusmn 20 ngmLin mice treated with AS-IV plus LPS compared to 818 plusmn95 ngmL in animals treatedwith LPS only TNF120572 levels were142 plusmn 9 pgmL and 279 plusmn 35 pgmL respectively (119875 lt 005119899 = 5) However AS-IV did not inhibit the LPS-inducedincreases in serum sVCAM-1 and sICAM-1 concentrations(data not shown)

32 AS-IV Inhibits LPS-Induced Upregulation of Inflamma-tory Gene Expression in Mouse Lung and Other TissuesTo investigate whether AS-IV inhibits LPS-induced acuteinflammatory responses in mouse organs we assessed geneexpression of inflammatory mediators in lung heart aortakidney and liver of LPS-exposed mice using real-time qPCRanalysis Treatment of mice with AS-IV alone did not affectgene expression of cellular adhesion molecules and proin-flammatory mediators (Figures 2ndash6) As expected treatmentof mice with LPS for 3 hours strongly upregulated inflam-matory gene expression in all tissues examined (Figures 2ndash6) Pretreatment of mice with AS-IV significantly inhibited

4 Mediators of Inflammation

Lung

mRN

A le

vels

( o

f GA

PDH

)

VCAM-1ICAM-1

0

50

100

150

lowast

lowast

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

(a)

E-selectinP-selectin

0

50

100

150

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(b)

MCP-1

IL-6

0

50

100

150

lowast

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

Lung

mRN

A le

vels

( o

f GA

PDH

)

(c)

0

30

60

90

120TLR4

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(d)

Figure 2 AS-IV inhibits LPS-induced inflammatory gene expression in mouse lung Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPS Three hours after the HBSS or LPS injection the animals were sacrificed andtissues were collected Total RNA was isolated from lung Inflammatory gene expression was quantified using real-time quantitative PCRAfter normalization to the internal control gene GAPDH the results for each target gene were expressed as percentage of GAPDH Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

the LPS-induced increase in lung mRNA levels of adhesionmolecules VACM-1 by 73 ICAM-1 by 60 E-selectinby 79 and P-selectin by 71 and other proinflammatorymediators MCP-1 by 85 TNF120572 by 42 IL-6 by 83 andTLR4 by 30 (119875 lt 005 119899 = 5) (Figure 2) Similar inhibitoryeffects of AS-IV were also observed in heart (Figure 4) aorta(Figure 5) kidney (Figure 6) and liver (data not shown)

33 AS-IV Inhibits LPS-Induced Neutrophil Infiltration andActivation in Lung As polymorphonuclear neutrophils

(PMN) and other phagocytic cells such as monocyte-macrophages play critical roles in acute inflammation andtissue injury we assessed lung myeloperoxidase (MPO)a well-documented PMN-specific biomarker [31 33 34]after LPS challenge MPO is rapidly released when PMNare activated which triggers transcriptional upregulationof mRNA and new protein synthesis of MPO Thereforeupregulation of MPO mRNA levels also reflects PMNactivation during acute lung inflammatory responses [31 33]As shown in Figure 3(a) lung MPO mRNA was at very low

Mediators of Inflammation 5

Time after LPS injection (h)0 3 6 9 12 15 18 21 24

Lung

MPO

mRN

A (f

old

of co

ntro

l)

0

20

40

60

(a)

0

20

40

60

Lung

MPO

mRN

A (f

old

of co

ntro

l)

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

lowast

0

100

200

300

400

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

MPO

pro

tein

(ng

mg

tissu

e pro

tein

)

(c)

Figure 3 AS-IV inhibits LPS-induced gene and protein expression of myeloperoxidase (MPO) in mouse lung For panel (a) mice wererandomly assigned to receive ip injection of LPS and sacrificed after 1 3 8 or 24 h For panels (b) and (c) mice were treated as describedin the legend of Figure 2 Total RNA and cytosolic protein were isolated from lung LungMPO gene expression was quantified as describedin the legend of Figure 2 Lung MPO protein was determined using an ELISA kit as described in detail in Section 2 The mRNA data areshown as fold of control after normalization to the internal control gene GAPDH Data shown are means plusmn SEM of 4 to 5 animals per grouplowast119875 lt 005 compared to animals treated with LPS only

levels in control animals however LPS treatment inducedtime-dependent upregulation of lung MPO gene expressionwhich peaked at 3 h and remained elevated for up to 8 h andthen declined after 24 h These data indicate infiltration andactivation of interstitial PMN in the lungs of LPS-treatedmice in a time-dependent manner which is consistent withour previous results showing that TNF120572 treatment inducedincreases in MPOmRNA and protein levels enzyme activityand morphological accumulation of PMN in mouse lungtissues [33] However pretreatment of mice with AS-IVsignificantly inhibited the LPS-induced increase in MPOmRNA level by 27 (119875 lt 005 119899 = 5) (Figure 3(b)) Thiswas further confirmed by the lung MPO protein level whichincreased from 6 plusmn 1 ngmg tissue protein in control animalsto 325 plusmn 67 ngmg tissue protein in LPS-treated animals andsignificantly reduced by AS-IV treatment by 80 to 71 plusmn9 ngmg tissue protein (119875 lt 005 119899 = 5) (Figure 3(c)) Asthe lung is the main target for activated PMN during acuteinflammation [31 33 34] these data support the notion thatAS-IV inhibits PMN infiltrationrecruitment to the lung

and subsequent tissue damage during LPS-induced acuteinflammatory responses

34 AS-IV Inhibits LPS-Induced NF-120581B and AP-1 DNA-Binding Activity in Mouse Lung and Heart It is well rec-ognized that NF-120581B plays a prominent role in LPS-inducedtranscriptional regulation of most inflammatory genes thatcontribute to the development of septic shock multipleorgan failure and death [5 32] Of clinical relevance NF-120581Bactivation was increased in patients with acute inflammationand sepsis and correlated with clinical severity and mortality[35] To investigate possible signaling pathways mediatingthe inhibitory effect of AS-IV on LPS-induced inflammatorygene transcription we assessed the nuclear content of theNF-120581B subunit p65 and the AP-1 subunit c-fos as indicatorsof nuclear translocation and activation of these transcriptionfactors The DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) was detectable at low levels in lung and hearttissues of control and AS-IV-only-treated animals AS-IValone did not cause NF-120581B or AP-1 activation in either lung

6 Mediators of Inflammation

Hea

rt m

RNA

leve

ls (

of G

APD

H)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

0

50

100

150

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(b)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(c)

0

30

60

90

120

TLR4

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(d)

Figure 4 AS-IV inhibits LPS-induced inflammatory gene expression inmouse heartMice were treated as described in the legend of Figure 2Total RNA was isolated from heart and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

or heart (Figure 7) LPS markedly increased NF-120581B activityin lung and heart by 158- and 115-fold respectively AS-IV treatment significantly suppressed LPS-induced activationof NF-120581B by 42 and 54 respectively (119875 lt 005 119899 =5) (Figure 7(a)) LPS also strongly increased AP-1 activationby 96- and 253-fold respectively while AS-IV treatmentsignificantly diminished LPS-induced AP-1 activity by 41and 49 respectively in both lung and heart (119875 lt 005119899 = 5) (Figure 7(b))

Our data provide new evidence that the inhibitory effectof AS-IV on LPS-induced inflammatory gene expression ismainly through modulating NF-120581B and AP-1 DNA-binding

activity These data indicate that the ability of AS-IV to sup-press the NF-120581B and AP-1 pathways is the major underlyingmechanism contributing to its anti-inflammatory potential invivo It is well documented that the ldquoclassicrdquo TLR4 pathwayplays a major role in LPS signaling during sepsis [36] TLR4recognizes LPS from Gram-negative bacteria and mediatesthe innate immune response by activating I120581B kinase (IKK)and mitogen-activated protein kinase kinases (MKK) whichin turn activate NF-120581B and AP-1 respectively [5 36] Ourresults further show that AS-IV inhibits LPS-induced TLR4gene expression in lung and heart by 30 and 65 respec-tively (Figures 2 and 4) which is consistent with its inhibitory

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Mediators of Inflammation

mediators [6ndash9] Thus the inhibition of NF-120581B activation isexpected to be protective in pathological inflammatory states

Astragalus membranaceus is one of the most widely usedChinese medicinal herbs for the treatment of many diseasesincluding cardiovascular disease nephritis hepatitis anddiabetes [10] It also has been available in Europe and theUS for many years as a food supplement Evidence frompharmacological research and clinical practice suggests thatAstragalus possesses a wide spectrum of activities includingimmunomodulation [11 12] cardiovascular protection [13ndash15] anti-inflammatory effects [16ndash18] hepatoprotection [1920] antidiabetes [21] anticancer [22] and neuroprotection[23]

The biologically active constituents of Astragalus rootsrepresent three classes of chemical compounds saponinspolysaccharides and flavonoids [24] By chemical degra-dation and 13C nuclear magnetic resonance examina-tion the structure of astragaloside IV (AS-IV) was deter-mined as 3-O-120573-D-xylopyranosyl-6-O-120573-D-glucopyranosyl-cycloastragenol (C

41H68O14 MW = 7849) [25] As one of

the major active constituents of Astragalus AS-IV is used asthe characteristic marker for quality evaluation of Astragalusin the Chinese Pharmacopeia and has been shown to exertpotent cardioprotective and anti-inflammatory effects [6 1026ndash30] We have previously shown that AS-IV inhibits LPS-and TNF120572-induced adhesion molecule expression and NF-120581B activation in cultured human endothelial cells [6] How-ever in vivo evidence supporting such activity is currentlylacking Therefore in this study we investigated whether AS-IV can inhibit LPS-induced acute inflammatory responses inexperimental mice

2 Materials and Methods

21 Animals and Experimental Procedures Female C57BL6Jmice 12 weeks old and weighing 20ndash22 g were purchasedfrom Jackson Laboratory (Bar Harbor ME) and housedin specific pathogen-free conditions and a temperature-and humidity-controlled environment (12-h lightdark cycle)with unlimited access to tap water and Purina 5001 chow diet(Harlan Teklad Madison WI) The investigation conformedto the Guide for the Care and Use of Laboratory Animals byNIH and all animal procedures were reviewed and approvedby the Oregon State University Institutional Animal Care andUse Committee

AS-IV was purchased from Quality Phytochemicals LLC(Edison NJ) and a stock solution was prepared with propy-lene glycol (SigmaAldrich St LouisMO) and further dilutedwith Hankrsquos buffered saline solution (HBSS) LPS (serotype055B5 from Escherichia coli Sigma Aldrich) stock solutionwas prepared in HBSS Mice were randomly assigned to4 groups as follows (i) control animals received daily ipinjection of the vehicle propylene glycol with HBSS for 6days followed by a single ip HBSS injection (ii) AS-IV-treated animals received 10mgkg bw AS-IV daily ip for 6days followed by a single ip HBSS injection (iii) LPS-treatedanimals received daily ip injection of propylene glycol withHBSS for 6 days followed by a single ip injection of 05 120583ggbw LPS and (iv) AS-IV plus LPS-treated animals received

AS-IV daily ip for 6 days followed by single ip injectionof LPS Animals were sacrificed 3 hours after HBSS or LPSinjection Based on our previous observations the 3-h timepoint and LPS dose of 05 120583gg bw were chosen [31] In somestudies mice were randomly assigned to receive ip injectionof LPS and sacrificed after 1 3 8 or 24 h Each group consistedof 4 to 5 animals After sacrifice blood and tissues werecollected for further analysis

22 Serum Inflammatory Mediators Serum concentrationsof MCP-1 TNF120572 sVCAM-1 and sICAM-1 were measuredby quantitative colorimetric sandwich ELISA (RampD SystemsMinneapolisMN)The sensitivity of the assays is 2 pgmL forMCP-1 5 pgmL for TNF120572 and 30 pgmL for sVCAM-1 andsICAM-1

23 Tissue mRNA Levels of Inflammatory Mediators TotalRNAwas isolated fromdifferent organs using TRIzol Reagent(Life Technologies Foster City CA) cDNA synthesis wasperformed using the high capacity cDNA archive kit (LifeTechnologies) mRNA levels of VCAM-1 ICAM-1 E-selectinP-selectin MCP-1 TNF120572 IL-6 myeloperoxidase (MPO)Toll-like receptor-4 (TLR4) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) were quantitated by real-timeqPCR All primers and probes were purchased as kits (Assayson Demand Life Technologies) The assays are supplied asa 20x mixture of PCR primers and TaqMan minor groovebinder 6-FAM dye-labeled probes with a nonfluorescentquencher at the 31015840 end TaqMan quantitative PCR (40 cyclesat 95∘C for 15 sec and 60∘C for 1min) was performed usingTaqMan Universal PCR Master Mix (Life Technologies) in96-well plates with an ABI Prism 7500 Sequence DetectionSystem (Life Technologies) To obtain relative quantificationtwo standard curves were constructed in each plate with onetarget gene and the internal control GAPDH gene Standardcurves were generated by plotting the threshold cycle numbervalues against the log of the amount of input cDNA and usedto quantify the expression of the target genes and GAPDHgene in the same sample After normalization to internalGAPDH in each sample results were expressed as percentageof GAPDH or fold of control

24 Lung Protein Concentration of Myeloperoxidase A partof the right lung lobe was homogenized and the cytosolicfraction of lung tissue homogenate was prepared usingnuclear extract kits (Active Motif Carlsbad CA) LungcytosolicMPOwas quantified using theMPO enzyme-linkedimmunosorbent assay kit (Hycult Biotechnology PlymouthMeeting PA) according to the manufacturerrsquos instructionsThe lung MPO concentration of each sample was normal-ized with cytosolic protein concentration and expressed asngMPOmg tissue protein

25 Nuclear Transcription Factors Nuclear extracts were pre-pared from lung and heart using nuclear extract kits (ActiveMotif) For analysis of nuclear transcription factor activationELISA-based assays (Active Motif) were used to determinethe DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos)

Mediators of Inflammation 3

Seru

m M

CP-1

(ng

mL)

0

20

40

60

80

100

LPSAS-IV

lowast

+

+ +

+

minus

minus

minus

minus

(a)

0

100

200

300

LPSAS-IV

Seru

m T

NF120572

(pg

mL)

lowast

+

+

minus

minus +

minus +

minus

(b)

Figure 1 AS-IV inhibits LPS-induced increases in serumMCP-1 and TNF120572 in mice Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPSThree hours after the HBSS or LPS injection the animals were sacrificed and bloodwas collected Serum MCP-1 (a) and TNF120572 (b) were measured by ELISA Data shown are mean values plusmn SEM of five animals per grouplowast119875 lt 005 compared to animals treated with LPS only

The specificity of binding was confirmed by competition witheither wild-type or mutant oligonucleotides

26 Statistical Analysis The data were calculated as means plusmnSEM and analyzed by ANOVA with Fisher PLSD post hoctest Statistical significance was set at 119875 lt 005

3 Results and Discussion

The pathophysiology of acute inflammation triggered by thebacterial endotoxin LPS is characterized by the productionof multiple proinflammatory cytokines and chemokinesexpression of adhesion molecules and infiltration of neu-trophils and monocytes into inflamed tissues Because of thecomplexity of the pathology of septic shock major effortshave focused on identifying novel anti-inflammatory drugsthat prevent the proinflammatory process at the early stage ofgene expression of key inflammatory mediators [32]

We have previously shown that AS-IV inhibits LPS- andTNF120572-induced adhesion molecule expression and conse-quent adherence of monocytes by modulating NF-120581B signal-ing pathway in cultured human endothelial cells [6] We nowprovide new evidence showing that AS-IV exhibits stronganti-inflammatory activities in vivo by attenuating LPS-induced acute inflammatory responses through inhibition ofNF-120581B- andAP-1-mediated inflammatory signaling pathwaysin mice

31 AS-IV Inhibits LPS-Induced Increases in SerumMCP-1 andTNF120572 in Mice Treatment of mice with AS-IV for 6 dayshad no effect on body weight changes compared to HBSS-treated control animals The mean body weight was 208 plusmn06 g before and 208 plusmn 05 g after 6-day AS-IV treatment

In control animals the mean body weight was 202 plusmn 03 gbefore and 199 plusmn 04 g after 6-day HBSS treatment We firstinvestigated the effect of AS-IV on LPS-induced systemicinflammatory responses by determining serum levels ofMCP-1 TNF120572 and the soluble cellular adhesion moleculessVCAM-1 and sICAM-1 Treatment ofmice with AS-IV alonefor 6 days had no effect on serum levels of these inflammatorymediators compared to HBSS-treated control animals Asexpected 3 hours after LPS injection significant increasesin the serum levels of MCP-1 and TNF120572 were observed(Figure 1) Interestingly pretreatment of animals with AS-IV significantly inhibited LPS-induced increases in serumMCP-1 (Figure 1(a)) andTNF120572 (Figure 1(b)) by 82 and 49respectively Specifically MCP-1 levels were 152 plusmn 20 ngmLin mice treated with AS-IV plus LPS compared to 818 plusmn95 ngmL in animals treatedwith LPS only TNF120572 levels were142 plusmn 9 pgmL and 279 plusmn 35 pgmL respectively (119875 lt 005119899 = 5) However AS-IV did not inhibit the LPS-inducedincreases in serum sVCAM-1 and sICAM-1 concentrations(data not shown)

32 AS-IV Inhibits LPS-Induced Upregulation of Inflamma-tory Gene Expression in Mouse Lung and Other TissuesTo investigate whether AS-IV inhibits LPS-induced acuteinflammatory responses in mouse organs we assessed geneexpression of inflammatory mediators in lung heart aortakidney and liver of LPS-exposed mice using real-time qPCRanalysis Treatment of mice with AS-IV alone did not affectgene expression of cellular adhesion molecules and proin-flammatory mediators (Figures 2ndash6) As expected treatmentof mice with LPS for 3 hours strongly upregulated inflam-matory gene expression in all tissues examined (Figures 2ndash6) Pretreatment of mice with AS-IV significantly inhibited

4 Mediators of Inflammation

Lung

mRN

A le

vels

( o

f GA

PDH

)

VCAM-1ICAM-1

0

50

100

150

lowast

lowast

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

(a)

E-selectinP-selectin

0

50

100

150

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(b)

MCP-1

IL-6

0

50

100

150

lowast

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

Lung

mRN

A le

vels

( o

f GA

PDH

)

(c)

0

30

60

90

120TLR4

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(d)

Figure 2 AS-IV inhibits LPS-induced inflammatory gene expression in mouse lung Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPS Three hours after the HBSS or LPS injection the animals were sacrificed andtissues were collected Total RNA was isolated from lung Inflammatory gene expression was quantified using real-time quantitative PCRAfter normalization to the internal control gene GAPDH the results for each target gene were expressed as percentage of GAPDH Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

the LPS-induced increase in lung mRNA levels of adhesionmolecules VACM-1 by 73 ICAM-1 by 60 E-selectinby 79 and P-selectin by 71 and other proinflammatorymediators MCP-1 by 85 TNF120572 by 42 IL-6 by 83 andTLR4 by 30 (119875 lt 005 119899 = 5) (Figure 2) Similar inhibitoryeffects of AS-IV were also observed in heart (Figure 4) aorta(Figure 5) kidney (Figure 6) and liver (data not shown)

33 AS-IV Inhibits LPS-Induced Neutrophil Infiltration andActivation in Lung As polymorphonuclear neutrophils

(PMN) and other phagocytic cells such as monocyte-macrophages play critical roles in acute inflammation andtissue injury we assessed lung myeloperoxidase (MPO)a well-documented PMN-specific biomarker [31 33 34]after LPS challenge MPO is rapidly released when PMNare activated which triggers transcriptional upregulationof mRNA and new protein synthesis of MPO Thereforeupregulation of MPO mRNA levels also reflects PMNactivation during acute lung inflammatory responses [31 33]As shown in Figure 3(a) lung MPO mRNA was at very low

Mediators of Inflammation 5

Time after LPS injection (h)0 3 6 9 12 15 18 21 24

Lung

MPO

mRN

A (f

old

of co

ntro

l)

0

20

40

60

(a)

0

20

40

60

Lung

MPO

mRN

A (f

old

of co

ntro

l)

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

lowast

0

100

200

300

400

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

MPO

pro

tein

(ng

mg

tissu

e pro

tein

)

(c)

Figure 3 AS-IV inhibits LPS-induced gene and protein expression of myeloperoxidase (MPO) in mouse lung For panel (a) mice wererandomly assigned to receive ip injection of LPS and sacrificed after 1 3 8 or 24 h For panels (b) and (c) mice were treated as describedin the legend of Figure 2 Total RNA and cytosolic protein were isolated from lung LungMPO gene expression was quantified as describedin the legend of Figure 2 Lung MPO protein was determined using an ELISA kit as described in detail in Section 2 The mRNA data areshown as fold of control after normalization to the internal control gene GAPDH Data shown are means plusmn SEM of 4 to 5 animals per grouplowast119875 lt 005 compared to animals treated with LPS only

levels in control animals however LPS treatment inducedtime-dependent upregulation of lung MPO gene expressionwhich peaked at 3 h and remained elevated for up to 8 h andthen declined after 24 h These data indicate infiltration andactivation of interstitial PMN in the lungs of LPS-treatedmice in a time-dependent manner which is consistent withour previous results showing that TNF120572 treatment inducedincreases in MPOmRNA and protein levels enzyme activityand morphological accumulation of PMN in mouse lungtissues [33] However pretreatment of mice with AS-IVsignificantly inhibited the LPS-induced increase in MPOmRNA level by 27 (119875 lt 005 119899 = 5) (Figure 3(b)) Thiswas further confirmed by the lung MPO protein level whichincreased from 6 plusmn 1 ngmg tissue protein in control animalsto 325 plusmn 67 ngmg tissue protein in LPS-treated animals andsignificantly reduced by AS-IV treatment by 80 to 71 plusmn9 ngmg tissue protein (119875 lt 005 119899 = 5) (Figure 3(c)) Asthe lung is the main target for activated PMN during acuteinflammation [31 33 34] these data support the notion thatAS-IV inhibits PMN infiltrationrecruitment to the lung

and subsequent tissue damage during LPS-induced acuteinflammatory responses

34 AS-IV Inhibits LPS-Induced NF-120581B and AP-1 DNA-Binding Activity in Mouse Lung and Heart It is well rec-ognized that NF-120581B plays a prominent role in LPS-inducedtranscriptional regulation of most inflammatory genes thatcontribute to the development of septic shock multipleorgan failure and death [5 32] Of clinical relevance NF-120581Bactivation was increased in patients with acute inflammationand sepsis and correlated with clinical severity and mortality[35] To investigate possible signaling pathways mediatingthe inhibitory effect of AS-IV on LPS-induced inflammatorygene transcription we assessed the nuclear content of theNF-120581B subunit p65 and the AP-1 subunit c-fos as indicatorsof nuclear translocation and activation of these transcriptionfactors The DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) was detectable at low levels in lung and hearttissues of control and AS-IV-only-treated animals AS-IValone did not cause NF-120581B or AP-1 activation in either lung

6 Mediators of Inflammation

Hea

rt m

RNA

leve

ls (

of G

APD

H)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

0

50

100

150

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(b)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(c)

0

30

60

90

120

TLR4

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(d)

Figure 4 AS-IV inhibits LPS-induced inflammatory gene expression inmouse heartMice were treated as described in the legend of Figure 2Total RNA was isolated from heart and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

or heart (Figure 7) LPS markedly increased NF-120581B activityin lung and heart by 158- and 115-fold respectively AS-IV treatment significantly suppressed LPS-induced activationof NF-120581B by 42 and 54 respectively (119875 lt 005 119899 =5) (Figure 7(a)) LPS also strongly increased AP-1 activationby 96- and 253-fold respectively while AS-IV treatmentsignificantly diminished LPS-induced AP-1 activity by 41and 49 respectively in both lung and heart (119875 lt 005119899 = 5) (Figure 7(b))

Our data provide new evidence that the inhibitory effectof AS-IV on LPS-induced inflammatory gene expression ismainly through modulating NF-120581B and AP-1 DNA-binding

activity These data indicate that the ability of AS-IV to sup-press the NF-120581B and AP-1 pathways is the major underlyingmechanism contributing to its anti-inflammatory potential invivo It is well documented that the ldquoclassicrdquo TLR4 pathwayplays a major role in LPS signaling during sepsis [36] TLR4recognizes LPS from Gram-negative bacteria and mediatesthe innate immune response by activating I120581B kinase (IKK)and mitogen-activated protein kinase kinases (MKK) whichin turn activate NF-120581B and AP-1 respectively [5 36] Ourresults further show that AS-IV inhibits LPS-induced TLR4gene expression in lung and heart by 30 and 65 respec-tively (Figures 2 and 4) which is consistent with its inhibitory

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 3

Seru

m M

CP-1

(ng

mL)

0

20

40

60

80

100

LPSAS-IV

lowast

+

+ +

+

minus

minus

minus

minus

(a)

0

100

200

300

LPSAS-IV

Seru

m T

NF120572

(pg

mL)

lowast

+

+

minus

minus +

minus +

minus

(b)

Figure 1 AS-IV inhibits LPS-induced increases in serumMCP-1 and TNF120572 in mice Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPSThree hours after the HBSS or LPS injection the animals were sacrificed and bloodwas collected Serum MCP-1 (a) and TNF120572 (b) were measured by ELISA Data shown are mean values plusmn SEM of five animals per grouplowast119875 lt 005 compared to animals treated with LPS only

The specificity of binding was confirmed by competition witheither wild-type or mutant oligonucleotides

26 Statistical Analysis The data were calculated as means plusmnSEM and analyzed by ANOVA with Fisher PLSD post hoctest Statistical significance was set at 119875 lt 005

3 Results and Discussion

The pathophysiology of acute inflammation triggered by thebacterial endotoxin LPS is characterized by the productionof multiple proinflammatory cytokines and chemokinesexpression of adhesion molecules and infiltration of neu-trophils and monocytes into inflamed tissues Because of thecomplexity of the pathology of septic shock major effortshave focused on identifying novel anti-inflammatory drugsthat prevent the proinflammatory process at the early stage ofgene expression of key inflammatory mediators [32]

We have previously shown that AS-IV inhibits LPS- andTNF120572-induced adhesion molecule expression and conse-quent adherence of monocytes by modulating NF-120581B signal-ing pathway in cultured human endothelial cells [6] We nowprovide new evidence showing that AS-IV exhibits stronganti-inflammatory activities in vivo by attenuating LPS-induced acute inflammatory responses through inhibition ofNF-120581B- andAP-1-mediated inflammatory signaling pathwaysin mice

31 AS-IV Inhibits LPS-Induced Increases in SerumMCP-1 andTNF120572 in Mice Treatment of mice with AS-IV for 6 dayshad no effect on body weight changes compared to HBSS-treated control animals The mean body weight was 208 plusmn06 g before and 208 plusmn 05 g after 6-day AS-IV treatment

In control animals the mean body weight was 202 plusmn 03 gbefore and 199 plusmn 04 g after 6-day HBSS treatment We firstinvestigated the effect of AS-IV on LPS-induced systemicinflammatory responses by determining serum levels ofMCP-1 TNF120572 and the soluble cellular adhesion moleculessVCAM-1 and sICAM-1 Treatment ofmice with AS-IV alonefor 6 days had no effect on serum levels of these inflammatorymediators compared to HBSS-treated control animals Asexpected 3 hours after LPS injection significant increasesin the serum levels of MCP-1 and TNF120572 were observed(Figure 1) Interestingly pretreatment of animals with AS-IV significantly inhibited LPS-induced increases in serumMCP-1 (Figure 1(a)) andTNF120572 (Figure 1(b)) by 82 and 49respectively Specifically MCP-1 levels were 152 plusmn 20 ngmLin mice treated with AS-IV plus LPS compared to 818 plusmn95 ngmL in animals treatedwith LPS only TNF120572 levels were142 plusmn 9 pgmL and 279 plusmn 35 pgmL respectively (119875 lt 005119899 = 5) However AS-IV did not inhibit the LPS-inducedincreases in serum sVCAM-1 and sICAM-1 concentrations(data not shown)

32 AS-IV Inhibits LPS-Induced Upregulation of Inflamma-tory Gene Expression in Mouse Lung and Other TissuesTo investigate whether AS-IV inhibits LPS-induced acuteinflammatory responses in mouse organs we assessed geneexpression of inflammatory mediators in lung heart aortakidney and liver of LPS-exposed mice using real-time qPCRanalysis Treatment of mice with AS-IV alone did not affectgene expression of cellular adhesion molecules and proin-flammatory mediators (Figures 2ndash6) As expected treatmentof mice with LPS for 3 hours strongly upregulated inflam-matory gene expression in all tissues examined (Figures 2ndash6) Pretreatment of mice with AS-IV significantly inhibited

4 Mediators of Inflammation

Lung

mRN

A le

vels

( o

f GA

PDH

)

VCAM-1ICAM-1

0

50

100

150

lowast

lowast

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

(a)

E-selectinP-selectin

0

50

100

150

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(b)

MCP-1

IL-6

0

50

100

150

lowast

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

Lung

mRN

A le

vels

( o

f GA

PDH

)

(c)

0

30

60

90

120TLR4

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(d)

Figure 2 AS-IV inhibits LPS-induced inflammatory gene expression in mouse lung Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPS Three hours after the HBSS or LPS injection the animals were sacrificed andtissues were collected Total RNA was isolated from lung Inflammatory gene expression was quantified using real-time quantitative PCRAfter normalization to the internal control gene GAPDH the results for each target gene were expressed as percentage of GAPDH Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

the LPS-induced increase in lung mRNA levels of adhesionmolecules VACM-1 by 73 ICAM-1 by 60 E-selectinby 79 and P-selectin by 71 and other proinflammatorymediators MCP-1 by 85 TNF120572 by 42 IL-6 by 83 andTLR4 by 30 (119875 lt 005 119899 = 5) (Figure 2) Similar inhibitoryeffects of AS-IV were also observed in heart (Figure 4) aorta(Figure 5) kidney (Figure 6) and liver (data not shown)

33 AS-IV Inhibits LPS-Induced Neutrophil Infiltration andActivation in Lung As polymorphonuclear neutrophils

(PMN) and other phagocytic cells such as monocyte-macrophages play critical roles in acute inflammation andtissue injury we assessed lung myeloperoxidase (MPO)a well-documented PMN-specific biomarker [31 33 34]after LPS challenge MPO is rapidly released when PMNare activated which triggers transcriptional upregulationof mRNA and new protein synthesis of MPO Thereforeupregulation of MPO mRNA levels also reflects PMNactivation during acute lung inflammatory responses [31 33]As shown in Figure 3(a) lung MPO mRNA was at very low

Mediators of Inflammation 5

Time after LPS injection (h)0 3 6 9 12 15 18 21 24

Lung

MPO

mRN

A (f

old

of co

ntro

l)

0

20

40

60

(a)

0

20

40

60

Lung

MPO

mRN

A (f

old

of co

ntro

l)

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

lowast

0

100

200

300

400

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

MPO

pro

tein

(ng

mg

tissu

e pro

tein

)

(c)

Figure 3 AS-IV inhibits LPS-induced gene and protein expression of myeloperoxidase (MPO) in mouse lung For panel (a) mice wererandomly assigned to receive ip injection of LPS and sacrificed after 1 3 8 or 24 h For panels (b) and (c) mice were treated as describedin the legend of Figure 2 Total RNA and cytosolic protein were isolated from lung LungMPO gene expression was quantified as describedin the legend of Figure 2 Lung MPO protein was determined using an ELISA kit as described in detail in Section 2 The mRNA data areshown as fold of control after normalization to the internal control gene GAPDH Data shown are means plusmn SEM of 4 to 5 animals per grouplowast119875 lt 005 compared to animals treated with LPS only

levels in control animals however LPS treatment inducedtime-dependent upregulation of lung MPO gene expressionwhich peaked at 3 h and remained elevated for up to 8 h andthen declined after 24 h These data indicate infiltration andactivation of interstitial PMN in the lungs of LPS-treatedmice in a time-dependent manner which is consistent withour previous results showing that TNF120572 treatment inducedincreases in MPOmRNA and protein levels enzyme activityand morphological accumulation of PMN in mouse lungtissues [33] However pretreatment of mice with AS-IVsignificantly inhibited the LPS-induced increase in MPOmRNA level by 27 (119875 lt 005 119899 = 5) (Figure 3(b)) Thiswas further confirmed by the lung MPO protein level whichincreased from 6 plusmn 1 ngmg tissue protein in control animalsto 325 plusmn 67 ngmg tissue protein in LPS-treated animals andsignificantly reduced by AS-IV treatment by 80 to 71 plusmn9 ngmg tissue protein (119875 lt 005 119899 = 5) (Figure 3(c)) Asthe lung is the main target for activated PMN during acuteinflammation [31 33 34] these data support the notion thatAS-IV inhibits PMN infiltrationrecruitment to the lung

and subsequent tissue damage during LPS-induced acuteinflammatory responses

34 AS-IV Inhibits LPS-Induced NF-120581B and AP-1 DNA-Binding Activity in Mouse Lung and Heart It is well rec-ognized that NF-120581B plays a prominent role in LPS-inducedtranscriptional regulation of most inflammatory genes thatcontribute to the development of septic shock multipleorgan failure and death [5 32] Of clinical relevance NF-120581Bactivation was increased in patients with acute inflammationand sepsis and correlated with clinical severity and mortality[35] To investigate possible signaling pathways mediatingthe inhibitory effect of AS-IV on LPS-induced inflammatorygene transcription we assessed the nuclear content of theNF-120581B subunit p65 and the AP-1 subunit c-fos as indicatorsof nuclear translocation and activation of these transcriptionfactors The DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) was detectable at low levels in lung and hearttissues of control and AS-IV-only-treated animals AS-IValone did not cause NF-120581B or AP-1 activation in either lung

6 Mediators of Inflammation

Hea

rt m

RNA

leve

ls (

of G

APD

H)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

0

50

100

150

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(b)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(c)

0

30

60

90

120

TLR4

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(d)

Figure 4 AS-IV inhibits LPS-induced inflammatory gene expression inmouse heartMice were treated as described in the legend of Figure 2Total RNA was isolated from heart and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

or heart (Figure 7) LPS markedly increased NF-120581B activityin lung and heart by 158- and 115-fold respectively AS-IV treatment significantly suppressed LPS-induced activationof NF-120581B by 42 and 54 respectively (119875 lt 005 119899 =5) (Figure 7(a)) LPS also strongly increased AP-1 activationby 96- and 253-fold respectively while AS-IV treatmentsignificantly diminished LPS-induced AP-1 activity by 41and 49 respectively in both lung and heart (119875 lt 005119899 = 5) (Figure 7(b))

Our data provide new evidence that the inhibitory effectof AS-IV on LPS-induced inflammatory gene expression ismainly through modulating NF-120581B and AP-1 DNA-binding

activity These data indicate that the ability of AS-IV to sup-press the NF-120581B and AP-1 pathways is the major underlyingmechanism contributing to its anti-inflammatory potential invivo It is well documented that the ldquoclassicrdquo TLR4 pathwayplays a major role in LPS signaling during sepsis [36] TLR4recognizes LPS from Gram-negative bacteria and mediatesthe innate immune response by activating I120581B kinase (IKK)and mitogen-activated protein kinase kinases (MKK) whichin turn activate NF-120581B and AP-1 respectively [5 36] Ourresults further show that AS-IV inhibits LPS-induced TLR4gene expression in lung and heart by 30 and 65 respec-tively (Figures 2 and 4) which is consistent with its inhibitory

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Mediators of Inflammation

Lung

mRN

A le

vels

( o

f GA

PDH

)

VCAM-1ICAM-1

0

50

100

150

lowast

lowast

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

(a)

E-selectinP-selectin

0

50

100

150

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(b)

MCP-1

IL-6

0

50

100

150

lowast

lowast

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

Lung

mRN

A le

vels

( o

f GA

PDH

)

(c)

0

30

60

90

120TLR4

lowast

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

mRN

A le

vels

( o

f GA

PDH

)

(d)

Figure 2 AS-IV inhibits LPS-induced inflammatory gene expression in mouse lung Mice were randomly assigned to 4 groups as follows (i)control animals received daily ip injection of the vehicle HBSS for 6 days followed by a single ip HBSS injection (ii) AS-IV-treated animalsreceived 10mgkg bw AS-IV daily ip for 6 days followed by a single ip HBSS injection (iii) LPS-treated animals received daily ip injectionof vehicle HBSS for 6 days followed by a single ip injection of 05120583gg bw LPS and (iv) AS-IV plus LPS-treated animals received AS-IVdaily ip for 6 days followed by single ip injection of LPS Three hours after the HBSS or LPS injection the animals were sacrificed andtissues were collected Total RNA was isolated from lung Inflammatory gene expression was quantified using real-time quantitative PCRAfter normalization to the internal control gene GAPDH the results for each target gene were expressed as percentage of GAPDH Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

the LPS-induced increase in lung mRNA levels of adhesionmolecules VACM-1 by 73 ICAM-1 by 60 E-selectinby 79 and P-selectin by 71 and other proinflammatorymediators MCP-1 by 85 TNF120572 by 42 IL-6 by 83 andTLR4 by 30 (119875 lt 005 119899 = 5) (Figure 2) Similar inhibitoryeffects of AS-IV were also observed in heart (Figure 4) aorta(Figure 5) kidney (Figure 6) and liver (data not shown)

33 AS-IV Inhibits LPS-Induced Neutrophil Infiltration andActivation in Lung As polymorphonuclear neutrophils

(PMN) and other phagocytic cells such as monocyte-macrophages play critical roles in acute inflammation andtissue injury we assessed lung myeloperoxidase (MPO)a well-documented PMN-specific biomarker [31 33 34]after LPS challenge MPO is rapidly released when PMNare activated which triggers transcriptional upregulationof mRNA and new protein synthesis of MPO Thereforeupregulation of MPO mRNA levels also reflects PMNactivation during acute lung inflammatory responses [31 33]As shown in Figure 3(a) lung MPO mRNA was at very low

Mediators of Inflammation 5

Time after LPS injection (h)0 3 6 9 12 15 18 21 24

Lung

MPO

mRN

A (f

old

of co

ntro

l)

0

20

40

60

(a)

0

20

40

60

Lung

MPO

mRN

A (f

old

of co

ntro

l)

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

lowast

0

100

200

300

400

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

MPO

pro

tein

(ng

mg

tissu

e pro

tein

)

(c)

Figure 3 AS-IV inhibits LPS-induced gene and protein expression of myeloperoxidase (MPO) in mouse lung For panel (a) mice wererandomly assigned to receive ip injection of LPS and sacrificed after 1 3 8 or 24 h For panels (b) and (c) mice were treated as describedin the legend of Figure 2 Total RNA and cytosolic protein were isolated from lung LungMPO gene expression was quantified as describedin the legend of Figure 2 Lung MPO protein was determined using an ELISA kit as described in detail in Section 2 The mRNA data areshown as fold of control after normalization to the internal control gene GAPDH Data shown are means plusmn SEM of 4 to 5 animals per grouplowast119875 lt 005 compared to animals treated with LPS only

levels in control animals however LPS treatment inducedtime-dependent upregulation of lung MPO gene expressionwhich peaked at 3 h and remained elevated for up to 8 h andthen declined after 24 h These data indicate infiltration andactivation of interstitial PMN in the lungs of LPS-treatedmice in a time-dependent manner which is consistent withour previous results showing that TNF120572 treatment inducedincreases in MPOmRNA and protein levels enzyme activityand morphological accumulation of PMN in mouse lungtissues [33] However pretreatment of mice with AS-IVsignificantly inhibited the LPS-induced increase in MPOmRNA level by 27 (119875 lt 005 119899 = 5) (Figure 3(b)) Thiswas further confirmed by the lung MPO protein level whichincreased from 6 plusmn 1 ngmg tissue protein in control animalsto 325 plusmn 67 ngmg tissue protein in LPS-treated animals andsignificantly reduced by AS-IV treatment by 80 to 71 plusmn9 ngmg tissue protein (119875 lt 005 119899 = 5) (Figure 3(c)) Asthe lung is the main target for activated PMN during acuteinflammation [31 33 34] these data support the notion thatAS-IV inhibits PMN infiltrationrecruitment to the lung

and subsequent tissue damage during LPS-induced acuteinflammatory responses

34 AS-IV Inhibits LPS-Induced NF-120581B and AP-1 DNA-Binding Activity in Mouse Lung and Heart It is well rec-ognized that NF-120581B plays a prominent role in LPS-inducedtranscriptional regulation of most inflammatory genes thatcontribute to the development of septic shock multipleorgan failure and death [5 32] Of clinical relevance NF-120581Bactivation was increased in patients with acute inflammationand sepsis and correlated with clinical severity and mortality[35] To investigate possible signaling pathways mediatingthe inhibitory effect of AS-IV on LPS-induced inflammatorygene transcription we assessed the nuclear content of theNF-120581B subunit p65 and the AP-1 subunit c-fos as indicatorsof nuclear translocation and activation of these transcriptionfactors The DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) was detectable at low levels in lung and hearttissues of control and AS-IV-only-treated animals AS-IValone did not cause NF-120581B or AP-1 activation in either lung

6 Mediators of Inflammation

Hea

rt m

RNA

leve

ls (

of G

APD

H)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

0

50

100

150

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(b)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(c)

0

30

60

90

120

TLR4

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(d)

Figure 4 AS-IV inhibits LPS-induced inflammatory gene expression inmouse heartMice were treated as described in the legend of Figure 2Total RNA was isolated from heart and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

or heart (Figure 7) LPS markedly increased NF-120581B activityin lung and heart by 158- and 115-fold respectively AS-IV treatment significantly suppressed LPS-induced activationof NF-120581B by 42 and 54 respectively (119875 lt 005 119899 =5) (Figure 7(a)) LPS also strongly increased AP-1 activationby 96- and 253-fold respectively while AS-IV treatmentsignificantly diminished LPS-induced AP-1 activity by 41and 49 respectively in both lung and heart (119875 lt 005119899 = 5) (Figure 7(b))

Our data provide new evidence that the inhibitory effectof AS-IV on LPS-induced inflammatory gene expression ismainly through modulating NF-120581B and AP-1 DNA-binding

activity These data indicate that the ability of AS-IV to sup-press the NF-120581B and AP-1 pathways is the major underlyingmechanism contributing to its anti-inflammatory potential invivo It is well documented that the ldquoclassicrdquo TLR4 pathwayplays a major role in LPS signaling during sepsis [36] TLR4recognizes LPS from Gram-negative bacteria and mediatesthe innate immune response by activating I120581B kinase (IKK)and mitogen-activated protein kinase kinases (MKK) whichin turn activate NF-120581B and AP-1 respectively [5 36] Ourresults further show that AS-IV inhibits LPS-induced TLR4gene expression in lung and heart by 30 and 65 respec-tively (Figures 2 and 4) which is consistent with its inhibitory

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 5

Time after LPS injection (h)0 3 6 9 12 15 18 21 24

Lung

MPO

mRN

A (f

old

of co

ntro

l)

0

20

40

60

(a)

0

20

40

60

Lung

MPO

mRN

A (f

old

of co

ntro

l)

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

lowast

0

100

200

300

400

LPSAS-IV +

+ +

+

minus

minus

minus

minus

Lung

MPO

pro

tein

(ng

mg

tissu

e pro

tein

)

(c)

Figure 3 AS-IV inhibits LPS-induced gene and protein expression of myeloperoxidase (MPO) in mouse lung For panel (a) mice wererandomly assigned to receive ip injection of LPS and sacrificed after 1 3 8 or 24 h For panels (b) and (c) mice were treated as describedin the legend of Figure 2 Total RNA and cytosolic protein were isolated from lung LungMPO gene expression was quantified as describedin the legend of Figure 2 Lung MPO protein was determined using an ELISA kit as described in detail in Section 2 The mRNA data areshown as fold of control after normalization to the internal control gene GAPDH Data shown are means plusmn SEM of 4 to 5 animals per grouplowast119875 lt 005 compared to animals treated with LPS only

levels in control animals however LPS treatment inducedtime-dependent upregulation of lung MPO gene expressionwhich peaked at 3 h and remained elevated for up to 8 h andthen declined after 24 h These data indicate infiltration andactivation of interstitial PMN in the lungs of LPS-treatedmice in a time-dependent manner which is consistent withour previous results showing that TNF120572 treatment inducedincreases in MPOmRNA and protein levels enzyme activityand morphological accumulation of PMN in mouse lungtissues [33] However pretreatment of mice with AS-IVsignificantly inhibited the LPS-induced increase in MPOmRNA level by 27 (119875 lt 005 119899 = 5) (Figure 3(b)) Thiswas further confirmed by the lung MPO protein level whichincreased from 6 plusmn 1 ngmg tissue protein in control animalsto 325 plusmn 67 ngmg tissue protein in LPS-treated animals andsignificantly reduced by AS-IV treatment by 80 to 71 plusmn9 ngmg tissue protein (119875 lt 005 119899 = 5) (Figure 3(c)) Asthe lung is the main target for activated PMN during acuteinflammation [31 33 34] these data support the notion thatAS-IV inhibits PMN infiltrationrecruitment to the lung

and subsequent tissue damage during LPS-induced acuteinflammatory responses

34 AS-IV Inhibits LPS-Induced NF-120581B and AP-1 DNA-Binding Activity in Mouse Lung and Heart It is well rec-ognized that NF-120581B plays a prominent role in LPS-inducedtranscriptional regulation of most inflammatory genes thatcontribute to the development of septic shock multipleorgan failure and death [5 32] Of clinical relevance NF-120581Bactivation was increased in patients with acute inflammationand sepsis and correlated with clinical severity and mortality[35] To investigate possible signaling pathways mediatingthe inhibitory effect of AS-IV on LPS-induced inflammatorygene transcription we assessed the nuclear content of theNF-120581B subunit p65 and the AP-1 subunit c-fos as indicatorsof nuclear translocation and activation of these transcriptionfactors The DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) was detectable at low levels in lung and hearttissues of control and AS-IV-only-treated animals AS-IValone did not cause NF-120581B or AP-1 activation in either lung

6 Mediators of Inflammation

Hea

rt m

RNA

leve

ls (

of G

APD

H)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

0

50

100

150

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(b)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(c)

0

30

60

90

120

TLR4

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(d)

Figure 4 AS-IV inhibits LPS-induced inflammatory gene expression inmouse heartMice were treated as described in the legend of Figure 2Total RNA was isolated from heart and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

or heart (Figure 7) LPS markedly increased NF-120581B activityin lung and heart by 158- and 115-fold respectively AS-IV treatment significantly suppressed LPS-induced activationof NF-120581B by 42 and 54 respectively (119875 lt 005 119899 =5) (Figure 7(a)) LPS also strongly increased AP-1 activationby 96- and 253-fold respectively while AS-IV treatmentsignificantly diminished LPS-induced AP-1 activity by 41and 49 respectively in both lung and heart (119875 lt 005119899 = 5) (Figure 7(b))

Our data provide new evidence that the inhibitory effectof AS-IV on LPS-induced inflammatory gene expression ismainly through modulating NF-120581B and AP-1 DNA-binding

activity These data indicate that the ability of AS-IV to sup-press the NF-120581B and AP-1 pathways is the major underlyingmechanism contributing to its anti-inflammatory potential invivo It is well documented that the ldquoclassicrdquo TLR4 pathwayplays a major role in LPS signaling during sepsis [36] TLR4recognizes LPS from Gram-negative bacteria and mediatesthe innate immune response by activating I120581B kinase (IKK)and mitogen-activated protein kinase kinases (MKK) whichin turn activate NF-120581B and AP-1 respectively [5 36] Ourresults further show that AS-IV inhibits LPS-induced TLR4gene expression in lung and heart by 30 and 65 respec-tively (Figures 2 and 4) which is consistent with its inhibitory

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Mediators of Inflammation

Hea

rt m

RNA

leve

ls (

of G

APD

H)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

0

50

100

150

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(b)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(c)

0

30

60

90

120

TLR4

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

Hea

rt m

RNA

leve

ls (

of G

APD

H)

(d)

Figure 4 AS-IV inhibits LPS-induced inflammatory gene expression inmouse heartMice were treated as described in the legend of Figure 2Total RNA was isolated from heart and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

or heart (Figure 7) LPS markedly increased NF-120581B activityin lung and heart by 158- and 115-fold respectively AS-IV treatment significantly suppressed LPS-induced activationof NF-120581B by 42 and 54 respectively (119875 lt 005 119899 =5) (Figure 7(a)) LPS also strongly increased AP-1 activationby 96- and 253-fold respectively while AS-IV treatmentsignificantly diminished LPS-induced AP-1 activity by 41and 49 respectively in both lung and heart (119875 lt 005119899 = 5) (Figure 7(b))

Our data provide new evidence that the inhibitory effectof AS-IV on LPS-induced inflammatory gene expression ismainly through modulating NF-120581B and AP-1 DNA-binding

activity These data indicate that the ability of AS-IV to sup-press the NF-120581B and AP-1 pathways is the major underlyingmechanism contributing to its anti-inflammatory potential invivo It is well documented that the ldquoclassicrdquo TLR4 pathwayplays a major role in LPS signaling during sepsis [36] TLR4recognizes LPS from Gram-negative bacteria and mediatesthe innate immune response by activating I120581B kinase (IKK)and mitogen-activated protein kinase kinases (MKK) whichin turn activate NF-120581B and AP-1 respectively [5 36] Ourresults further show that AS-IV inhibits LPS-induced TLR4gene expression in lung and heart by 30 and 65 respec-tively (Figures 2 and 4) which is consistent with its inhibitory

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 7

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

VCAM-1ICAM-1

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowastlowast

(a)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

200

E-selectinP-selectin

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(b)

Aort

ic m

RNA

( o

f GA

PDH

)

0

50

100

150

MCP-1

IL-6

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

lowast

TNF120572

(c)

Figure 5 AS-IV inhibits LPS-induced inflammatory gene expression inmouse aortaMice were treated as described in the legend of Figure 2Total RNA was isolated from aorta and inflammatory gene expression was quantified as described in the legend of Figure 2 Data shown aremeans plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

effects on NF-120581B and AP-1 activation in the same tissuesThese data suggest that the inhibition of TLR4 expressionmight be one of the mechanisms by which AS-IV affects theupstream targets of these pathways Some in vitro studieshave shown that AS-IV activates the PI3KAkt pathway [29]which is known to negatively regulate LPS-induced acuteinflammatory responses by modulating the NF-120581B and AP-1 pathways [9 37 38] Therefore PI3KAkt activation may beone of the underlying mechanisms for the anti-inflammatoryactivity of AS-IV Further as a novel antioxidant [39 40] AS-IV may modulate LPS-induced formation of reactive oxygen

species and subsequent activation of the redox-sensitive NF-120581B and AP-1 pathways at different levels [40] Howeverall these mechanisms observed in vitro need to be furtherinvestigated in vivo

A limitation of our study is that we had to apply AS-IVby ip injection because it is poorly bioavailable Previousstudies on dogs and rats have found that only 74 and 37respectively of orally supplemented AS-IV were absorbed[41 42] This is mainly due to AS-IVrsquos low fat solubilityand low transmittance in the small intestine [43] In factin Chinese medicine AS-IV is clinically used as intravenous

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Mediators of Inflammation

Kidn

ey m

RNA

( G

APD

H)

0

30

60

90

120

VCAM-1ICAM-1

LPS

AS-IV +

+ +

+

minus

minus

minus

minus

lowast

(a)Ki

dney

mRN

A (

GA

PDH

)

0

30

60

90

120

E-selectinP-selectin

LPSAS-IV +

+ +

+

minus

minus

minus

minus

lowast

lowast

(b)

Kidn

ey m

RNA

( o

f GA

PDH

)

0

30

60

90

120

lowastlowast

lowast

MCP-1

IL-6

LPSAS-IV +

+ +

+

minus

minus

minus

minus

TNF120572

(c)

Figure 6 AS-IV inhibits LPS-induced inflammatory gene expression in mouse kidney Mice were treated as described in the legend ofFigure 2 Total RNA was isolated from kidney and inflammatory gene expression was quantified as described in the legend of Figure 2 Datashown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

(iv) therapy Upon iv injection of AS-IV at 075mgkg inrats and 05mgkg in dogs the maximum plasma concentra-tions reached were 379 120583gmL and 439 120583gmL respectivelyand the elimination half-life (119905

12) was 98min and 60min

respectively [44 45] The highest concentration was foundin lung and liver tissues (28ndash29 120583gg) after iv injection of15mgkg AS-IV whereas heart muscle skin and kidneycontained moderate amounts (016ndash10 120583gg) [45] As Astra-galus and AS-IV are now widely used in Chinese medicineand are also available in Europe and the US as dietary

supplements chemical modifications to improve the absolutebioavailability of AS-IV while maintaining its biologicalactivity could be a focus of future research

4 Conclusion

In conclusion our data provide new evidence that AS-IVinhibits LPS-induced acute inflammatory responses in vivoby modulating the NF-120581B and AP-1 signaling pathways Ourresults might lead to the identification of AS-IV as a natural

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 9

00

03

06

09

12

ControlAS-IV

LPS

Lung Heart

NF-120581

B (p65

) act

ivity

(OD450

nm)

lowast

lowast

LPS + AS-IV

(a)

00

01

02

03

Lung Heart

lowast

lowast

ControlAS-IV

LPSLPS + AS-IV

AP-1

(c-fo

s) ac

tivity

(OD450

nm)

(b)

Figure 7 AS-IV inhibits LPS-inducedNF-120581B (a) and AP-1 (b) DNA-binding activity inmouse lung and heart Mice were treated as describedin the legend of Figure 2 Nuclear extracts were isolated from lung and heart DNA-binding activity of NF-120581B (p65) and AP-1 (c-fos) wasquantified by ELISA Data shown are means plusmn SEM of 5 animals per group lowast119875 lt 005 compared to animals treated with LPS only

compound or chemically derived drug that may be usefulfor the prevention or treatment of inflammatory diseases Webelieve that the molecular basis for its therapeutic efficacy isintriguing and warrants further investigation

Abbreviations

AP-1 Activator protein-1AS-IV Astragaloside IVbw Body weightGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1ip Intraperitonealiv IntravenousIKK I120581B kinaseIL InterleukinLPS LipopolysaccharideMCP-1 Monocyte chemoattractant protein-1MPO MyeloperoxidaseNF-120581B Nuclear factor 120581BPMN Polymorphonuclear neutrophilsRedox Reduction-oxidationROS Reactive oxygen speciessICAM-1 Soluble intercellular adhesion molecule-1sVCAM-1 Soluble vascular cell adhesion molecule-1TLR4 Toll-like receptor-4TNF120572 Tumor necrosis factor 120572VCAM-1 Vascular cell adhesion molecule-1

Disclaimer

The content of this work is solely the responsibility of theauthors and does not necessarily represent the official viewsof the NIH

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Wei-Jian Zhang designed and performed experiments Wei-Jian Zhang and Balz Frei analyzed data and wrote andapproved the final paper

Acknowledgments

This work was supported by NCCAM Center of ExcellenceGrant P01 AT002034 from the National Institutes of HealthThe authors thank Stephen Lawson for carefully proofreadingthe paper

References

[1] W C Aird ldquoThe role of the endothelium in severe sepsis andmultiple organ dysfunction syndromerdquo Blood vol 101 no 10pp 3765ndash3777 2003

[2] J E Parrillo ldquoPathogenetic mechanisms of septic shockrdquo TheNew England Journal of Medicine vol 328 no 20 pp 1471ndash14771993

[3] D G Remick ldquoPathophysiology of sepsisrdquo The AmericanJournal of Pathology vol 170 no 5 pp 1435ndash1444 2007

[4] T Collins M A Read A S Neish M Z Whitley D Thanosand T Maniatis ldquoTranscriptional regulation of endothelial celladhesionmolecules NF-120581B and cytokine-inducible enhancersrdquoThe FASEB Journal vol 9 no 10 pp 899ndash909 1995

[5] S F Liu and A B Malik ldquoNF-120581B activation as a pathologicalmechanism of septic shock and inflammationrdquo American Jour-nal of Physiology Lung Cellular and Molecular Physiology vol290 no 4 pp L622ndashL645 2006

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Mediators of Inflammation

[6] W-J Zhang P Hufnagl B R Binder and J Wojta ldquoAntiinflam-matory activity of astragaloside IV is mediated by inhibition ofNF-120581B activation and adhesionmolecule expressionrdquoThrombo-sis and Haemostasis vol 90 no 5 pp 904ndash914 2003

[7] C Weber W Erl A Pietsch M Strobel H W L Ziegler-Heitbrock and P C Weber ldquoAntioxidants inhibit monocyteadhesion by suppressing nuclear factor-120581B mobilization andinduction of vascular cell adhesion molecule-1 in endothe-lial cells stimulated to generate radicalsrdquo Arteriosclerosis andThrombosis vol 14 no 10 pp 1665ndash1673 1994

[8] W-J Zhang and B Frei ldquo120572-Lipoic acid inhibits TNF-120572-inducedNF-120581B activation and adhesion molecule expression in humanaortic endothelial cellsrdquo The FASEB Journal vol 15 no 13 pp2423ndash2432 2001

[9] W J Zhang H Wei T Hagen and B Frei ldquo120572-Lipoic acidattenuates LPS-induced inflammatory responses by activatingthe phosphoinositide 3-kinaseAkt signaling pathwayrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 104 no 10 pp 4077ndash4082 2007

[10] C Chu L-W Qi E-H Liu B Li W Gao and P Li ldquoRadixastragali (Astragalus) latest advancements and trends in chem-istry analysis pharmacology and pharmacokineticsrdquo CurrentOrganic Chemistry vol 14 no 16 pp 1792ndash1807 2010

[11] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007

[12] K L Denzler R Waters B L Jacobs Y Rochon and JO Langland ldquoRegulation of inflammatory gene expression inPBMCs by immunostimulatory botanicalsrdquo PloS ONE vol 5no 9 Article ID e12561 2010

[13] X J Chen Z-P Bian S Lu et al ldquoCardiac protective effectof Astragalus on viral myocarditis mice comparison withPerindoprilrdquoTheAmerican Journal of Chinese Medicine vol 34no 3 pp 493ndash502 2006

[14] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-PCheng ldquoCardioprotective effects of Astragali Radix againstisoproterenol-inducedmyocardial injury in rats and its possiblemechanismrdquo Phytotherapy Research vol 22 no 3 pp 389ndash3942008

[15] S Fu J Zhang F Menniti-Ippolito et al ldquoHuangqi injection (atraditional chinese patent medicine) for chronic heart failure asystematic reviewrdquo PLoS ONE vol 6 no 5 Article ID e196042011

[16] M Ryu E H Kim M Chun et al ldquoAstragali Radix elicitsanti-inflammation via activation of MKP-1 concomitant withattenuation of p38 and Erkrdquo Journal of Ethnopharmacology vol115 no 2 pp 184ndash193 2008

[17] X-H Gao X-X Xu R Pan et al ldquoSaponin fraction fromAstra-galus membranaceus roots protects mice against polymicrobialsepsis induced by cecal ligation and puncture by inhibitinginflammation and upregulating protein C pathwayrdquo Journal ofNatural Medicines vol 63 no 4 pp 421ndash429 2009

[18] J B Jiang J D Qiu L H Yang J P He G W Smith andH Q Li ldquoTherapeutic effects of astragalus polysaccharides oninflammation and synovial apoptosis in rats with adjuvant-induced arthritisrdquo International Journal of Rheumatic Diseasesvol 13 no 4 pp 396ndash405 2010

[19] S-Y Gui W Wei H Wang et al ldquoEffects and mechanisms ofcrude astragalosides fraction on liver fibrosis in ratsrdquo Journal ofEthnopharmacology vol 103 no 2 pp 154ndash159 2006

[20] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[21] M Li W Wang J Xue Y Gu and S Lin ldquoMeta-analysisof the clinical value of Astragalus membranaceus in diabeticnephropathyrdquo Journal of Ethnopharmacology vol 133 no 2 pp412ndash419 2011

[22] W C S Cho and K N Leung ldquoIn vitro and in vivo anti-tumoreffects ofAstragalusmembranaceusrdquoCancer Letters vol 252 no1 pp 43ndash54 2007

[23] C Tohda T Tamura S Matsuyama and K Komatsu ldquoPromo-tion of axonal maturation and prevention of memory loss inmice by extracts of Astragalus mongholicusrdquo British Journal ofPharmacology vol 149 no 5 pp 532ndash541 2006

[24] J-Z Song H H W Yiu C-F Qiao Q-B Han and H-X XuldquoChemical comparison and classification of Radix Astragali bydetermination of isoflavonoids and astragalosidesrdquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 399ndash406 2008

[25] I Kitagawa H KWangM Saito A Takagi andM YoshikawaldquoSaponin and sapogenol XXXV Chemical constituents ofastragali radix the root of Astragalus membranaceus Bunge 2Astragalosides I II and IV acetylastragaloside I and isoastra-galoside I and IIrdquo Chemical and Pharmaceutical Bulletin vol31 no 2 pp 698ndash708 1983

[26] W-D Zhang C Zhang X H Wang et al ldquoAstragaloside IVfrom Astragalus membranaceus shows cardioprotection duringmyocardial ischemia in vivo and in vitrordquo PlantaMedica vol 72no 1 pp 4ndash8 2006

[27] X-L Xu H Ji S-Y Gu Q Shao Q-J Huang and Y-P ChengldquoModification of alterations in cardiac function and sarcoplas-mic reticulum by astragaloside IV in myocardial injury in vivordquoEuropean Journal of Pharmacology vol 568 no 1ndash3 pp 203ndash212 2007

[28] W J Zhang J Wojta and B R Binder ldquoRegulation of the fibri-nolytic potential of cultured human umbilical vein endothelialcells astragaloside IV downregulates plasminogen activatorinhibitor-1 and upregulates tissue-type plasminogen activatorexpressionrdquo Journal of Vascular Research vol 34 no 4 pp 273ndash280 1997

[29] L Zhang Q Liu L Lu X Zhao X Gao and Y Wang ldquoAstra-galoside IV stimulates angiogenesis and increases hypoxia-inducible factor-1120572 accumulation via phosphatidylinositol 3-kinaseakt pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 338 no 2 pp 485ndash491 2011

[30] J Zhao P Yang F Li et al ldquoTherapeutic effects of astragalosideIV on myocardial injuries multi-target identification and net-work analysisrdquo PLoS ONE vol 7 no 9 Article ID e44938 2012

[31] W J Zhang HWei and B Frei ldquoGenetic deficiency of NADPHoxidase does not diminish but rather enhances LPS-inducedacute inflammatory responses in vivordquo Free Radical Biology andMedicine vol 46 no 6 pp 791ndash798 2009

[32] S S Awad ldquoState-of-the-art therapy for severe sepsis and mul-tisystem organ dysfunctionrdquo The American Journal of Surgeryvol 186 supplement 1 no 5 pp 23Sndash30S 2003

[33] W-J Zhang H Wei Y-T Tien and B Frei ldquoGenetic ablationof phagocytic NADPH oxidase in mice limits TNF120572-inducedinflammation in the lungs but not other tissuesrdquo Free RadicalBiology and Medicine vol 50 no 11 pp 1517ndash1525 2011

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 11

[34] J E Juskewitch J L Platt B E Knudsen K L KnutsonG J Brunn and J P Grande ldquoDisparate roles of marrow-and parenchymal cell-derived TLR4 signaling in murine LPS-induced systemic inflammationrdquo Scientific Reports vol 2 article918 2012

[35] H Bohrer F Qiu T Zimmermann et al ldquoRole of NF120581B in themortality of sepsisrdquoThe Journal of Clinical Investigation vol 100no 5 pp 972ndash985 1997

[36] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[37] G Schabbauer M Tencati B Pedersen R Pawlinski andN Mackman ldquoPI3K-Akt pathway suppresses coagulation andinflammation in endotoxemic micerdquo Arteriosclerosis Thrombo-sis and Vascular Biology vol 24 no 10 pp 1963ndash1969 2004

[38] D L Williams C Li T Ha et al ldquoModulation of thephosphoinositide 3-kinase pathway alters innate resistance topolymicrobial sepsisrdquo Journal of Immunology vol 172 no 1 pp449ndash456 2004

[39] D Gui Y Guo F Wang et al ldquoAstragaloside IV a novelantioxidant prevents glucose-induced podocyte apoptosis invitro and in vivordquo PLoS ONE vol 7 no 6 Article ID e398242012

[40] Y He M Du Y Gao et al ldquoAstragaloside IV attenuatesexperimental autoimmune encephalomyelitis of mice by coun-teracting oxidative stress at multiple levelsrdquo PLoS ONE vol 8no 10 Article ID e76495 2013

[41] Q Zhang L-L Zhu G-G Chen andY Du ldquoPharmacokineticsof astragalosideiv in beagle dogsrdquo European Journal of DrugMetabolism and Pharmacokinetics vol 32 no 2 pp 75ndash79 2007

[42] Y Du Q Zhang G G Chen P Wei and C Y Tu ldquoPharma-cokinetics of Astragaloside IV in rats by liquid chromatographycoupled with tandem mass spectrometryrdquo European Journal ofDrug Metabolism and Pharmacokinetics vol 30 no 4 pp 269ndash273 2005

[43] C R Huang G J Wang X L Wu et al ldquoAbsorption enhance-ment study of astragaloside IV based on its transport mecha-nism in Caco-2 cellsrdquo European Journal of DrugMetabolism andPharmacokinetics vol 31 no 1 pp 5ndash10 2006

[44] Y Gu GWang G Pan J P Fawcett J A and J Sun ldquoTransportand bioavailability studies of astragaloside IV an active ingre-dient in Radix astragalirdquo Basic and Clinical Pharmacology andToxicology vol 95 no 6 pp 295ndash298 2004

[45] W D Zhang C Zhang R-H Liu et al ldquoPreclinical pharma-cokinetics and tissue distribution of a natural cardioprotectiveagent astragaloside IV in rats and dogsrdquo Life Sciences vol 79no 8 pp 808ndash815 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom