research article key molecular mechanisms of...

Research ArticleKey Molecular Mechanisms of Chaiqinchengqi Decoction inAlleviating the Pulmonary Albumin LeakageCaused by Endotoxemia in Severe Acute Pancreatitis Rats

Wei Wu Ruijie Luo Ziqi Lin Qing Xia and Ping Xue

Department of Integrated Traditional Chinese and Western Medicine West China Hospital of Sichuan UniversityChengdu 610041 China

Correspondence should be addressed to Ping Xue weijunhr001163com

Received 19 February 2016 Revised 2 May 2016 Accepted 16 May 2016

Academic Editor Hajime Nakae

Copyright copy 2016 Wei Wu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To reveal the key molecular mechanisms of Chaiqinchengqi decoction (CQCQD) in alleviating the pulmonary albumin leakagecaused by endotoxemia in severe acute pancreatitis (SAP) rats Rats models of SAP endotoxemia-induced acute lung injury wereestablished the studies in vivo provided the important evidences that the therapy of CQCQD significantly ameliorated the increasesin plasma levels of lipopolysaccharide (LPS) sCd14 andLbp the elevation of serumamylase level the enhancements of systemic andpulmonary albumin leakage and the depravation of airways indicators thus improving respiratory dysfunction and also pancreaticand pulmonary histopathological changes According to the analyses of rats pulmonary tissue microarray and protein-proteininteraction network c-Fos c-Src and p85120572 were predicted as the target proteins for CQCQD in alleviating pulmonary albuminleakage To confirm these predictions human umbilical vein endothelial cells were employed in in vitro studies which provide theevidences that (1) LPS-induced paracellular leakage and proinflammatory cytokines release were suppressed by pretreatment withinhibitors of c-Src (PP1) or PI3K (LY294002) or by transfection with siRNAs of c-Fos (2) fortunately CQCQD imitated the actionsof these selective inhibitions agents to inhibit LPS-induced high expressions of p-Src p-p85120572 and c-Fos therefore attenuatingparacellular leakage and proinflammatory cytokines release

1 Introduction

Over the natural course of severe acute pancreatitis (SAP)intestinal permeability has been found to increase within thefirst 72 hours which led to massive endotoxin also knownas the lipopolysaccharide (LPS) aggregates to be uptakeninto blood circulationwhich initiates the occurrence of endo-toxemia [1] Previous study has described that endotoxemiawould initiate the consistent proinflammatory changes ingenic and proteinic expression profile of the immune cellswhich would trigger inflammatory cascade reaction andthen cause the serious dysfunction of capillary endothelium[2] Because pulmonary capillary networks are extremelyenriched in the early stage endotoxemia would initiate thesevere damage to pulmonary capillary endothelial barrier andthen cause acute lung injury (ALI) [3] The analyses of SAP-induced extrapancreatic organs injury indicated that ALI

not only occurs at the earliest stage but also initiates themost serious injury among these extrapancreatic organs [3]The SAP endotoxemia-induced ALI would directly lead toa significant decrease in ventilationperfusion ratio whicheventually result in respiratory failure [4] And the incidenceof ALI in SAP patients is 20 and the mortality rate is ashigh as 15 to 25 [4] Therefore alleviating SAP endo-toxemia-induced ALI could decrease the mortality rate inSAP The physiopathologic mechanisms of the damage topulmonary capillary endothelial barrier mainly include thedisassembly of tight junction (TJ) and adherens junction (AJ)and reorganization of cytoskeleton which would enhancepulmonary albumin leak and then result in the extra-vascularleak of the increased albumin leak-induced edema fluid intopulmonary interstitial and alveolar compartments and even-tually initiate respiratory failure [5 6] Hence pulmonaryalbumin leakage is the key pathophysiological mechanism

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 3265368 14 pageshttpdxdoiorg10115520163265368

2 Evidence-Based Complementary and Alternative Medicine

of ALI Additionally albumin with high molecular weightwould leak across capillary endothelial barrier mainly depen-dent on the paracellular leakage [7] Therefore the enhance-ment to systemic and pulmonary albumin leak depravationof airways indicators respiratory dysfunction and also theadverse change in lung tissue pathology could be consideredas the important features of pulmonary capillary endothelialbarrier injury [8ndash10] Moreover the reversal of the enhancedpulmonary albumin leak should be considered as the thera-peutic goal for ALI caused by endotoxemia in SAP

CQCQD decoction a famous Chinese medicinal for-mula has been used to treat SAP for decades in WestChina Hospital Sichuan University [11] According to theregulations on drug development and approval of SFDAbefore the Chinese medicinal formula is widely used inclinical practice there are four main principles required tobe met by the Chinese medicinal formula (1) action targetarea (2) highly efficient concentration (3) efficacy in animalmodel (4) multicentral random clinical trial The growingstudies evidences of CQCQD indicated that the first threeprinciples mentioned above have been met which will beshown as follows (i) action target area (1) pancreatic tissues(2) gastrointestinal tract (3) lung (4) immune system (5)pancreatic acinar cells (6) alveolar macrophages and (7)peritoneal macrophages [12ndash25] which suggested that thelungs are the important action target area for CQCQD(ii) Highly efficient concentration in clinical practice is asfollows CQCQD consists of Bupleuri Radix (Chai Hu) 15 gScutellariae Radix (Huang Qin) 15 g Magnoliae OfficinalisCortex (Hou Pu) 15 g Aurantii Fructus Immaturus (Zhi Shi)15 g Artemisiae Scopariae Herba (Yin Chen) 15 g GardeniaeFructus (Zhi Zi) 20 g Rhei Radix et Rhizoma (Da Huang)20 g and Natrii Sulfas (Mang Xiao) 20 g [12] according tothe standardized preparation procedure of CQCQD formulathese eight herbs will be decocted to form this decoction [13]since this decoction has been used to treat SAP for decadesand the effective pharmacotherapy to the patients has beencertified with ALI caused by endotoxemia in SAP [14] if thisdecoction is prepared strictly according to CQCQD formulaand the standardized preparation procedure the final con-centration of the prepared decoction can represent the highlyefficient concentrations of CQCQD in clinical application[15] (iii) Efficacy in SAP animal model is as follows (1)effectively ameliorating pancreatic histopathological changesand systemic inflammatory response [16ndash23] (2) effectivelyimproving gastrointestinal motility dysfunction [23] (3)effectively ameliorating alveolar inflammatory response [24](4) effectively alleviating ALI [25] which suggested that thetherapy of CQCQD could effectively alleviate the ALI causedby endotoxemia in SAP animal model Meanwhile accordingto the 4th principle multicentral random clinical trials arebeing conducted in a number of hospitals in China includingWest China Hospital of Sichuan University Chengdu No 1Hospital and Sichuan Hospital of TCM Although the multi-central randomclinical trials have not been completed a largenumber of clinical studies have provided the key evidencesthat (1) the therapy of CQCQD significantly decreased serumlevels of interleukin-6 (IL-6) and IL-1120573 receptor antagonist(IL-1ra) in SAP patients and also the hospitalization time of

SAP patients [12] (2) the therapy of CQCQD could decreasethe serum levels of matrix metalloproteinase-9 (MMP-9)C-reactive protein (CRP) and amyloid A (SAA) to relievethe durations of acute respiratory distress syndrome (ARDS)in patients with SAP [13 14] and the early application ofCQCQD is more effective than late application to shortenthe duration of ARDS [15] which suggested that the therapyof CQCQD could probably effectively alleviate ALI in thepatients with SAP Furthermore there are growing evidencesfor the monomers isolated from the herbs of CQCQDdecoction that (1) saikosaponin A baicalin and chlorogenicacid could inhibit some proinflammatory signal pathwayto ameliorate inflammatory cascade reaction [26ndash28] (2)saikosaponin C and baicalein could inhibit LPS-inducedapoptosis in human umbilical vein endothelial cells [2930] (3) wogonin could inhibit LPS-induced vascular hyper-permeability via inhibiting cytoskeleton reorganization [31]Consequently we speculated that the treatment of CQCQDdecoction in the patients with SAP endotoxemia-inducedALI could potentially ameliorate pulmonary albumin leak viadepressing the inflammatory cascade reaction and alleviatingthe damage to cell-cell junction However the potentialmolecular mechanisms have not been systematically studied

In this study microarray and Western blot and so forthanalyses were employed to reveal the key molecular mech-anisms for CQCQD decoction in alleviating the pulmonaryalbumin leakage caused by endotoxemia in SAP rats

2 Materials and Methods

21 Animals and Cell Culture Adult Sprague-Dawley rats(250ndash300 g) were purchased from the Laboratory AnimalCentre of Huaxi Sichuan University (Chengdu China) Ani-mal experiments performed in this study were approved bythe Institutional Animal Care and Use Committee of SichuanUniversity and the corresponding license number was SCXK(Sichuan) 2014sim09 Human umbilical vein endothelial cells(HUVECs) were obtained from Molecular Biology Lab ofSichuan University (Chengdu China) HUVECs were cul-tured in DMEM with 10 fetal bovine serum (FBS) 100UmL penicillin and 100mgmL streptomycin (Sigma-AldrichMO USA) at 37∘C with 5 CO

2

22 Preparation of CQCQD and Rat CQCQD-ContainingSerum and Control Serum The Chinese drug decoctionpieces in the CQCQD formula (Table 1) were acquiredaccording to the prescribed proportions (ww) through thePharmaceutical Preparation Section West Chinese Hospitalof Sichuan University Chengdu China Each botanical nameof these Chinese drug decoction pieces (Table 1) has beenchecked with httpwwwtheplantlistorg mentioning thedata of accessing that website According to the macroscopicand microscopic means mentioned in Chinese pharma-copoeia the botanical authentication of each Chinese drugdecoction piece has been performed by their manufacturersIn addition the chemical characterization of major activeingredients of these Chinese drug decoction pieces (showedin Figure 1 for Rhei Radix et Rhizoma as an example) wasidentified by HPLC-MS analyses adopting one or more

Evidence-Based Complementary and Alternative Medicine 3

Table1Com

ponentso

fChinese

drug

decoctionpieces

intheC

QCQ

Dform

ulaa

ndtheirp

rodu

ctionlotn

umbersandther

elativec

ontentso

fmajor

activ

eing

redients

Pharmaceuticalnamea

ndCh

inesep

honetic

name

Botanicaln

amea

ndplantp

art

Com

position(

)Lo

tnum

ber

Quantitativ

eanalysis

ofmajor

activ

eing

redients

Referencem

arkers(H

PLC)

Relativec

ontent

(ww

)

BupleuriRa

dix(C

haiH

u)Bu

pleurum

chinenseDC(Apiaceae)roo

t11111

2014100802

Saikosapon

ina

0383ndash0421

Saikosapon

ind

0381ndash0398

Scutellaria

eRadix(H

uang

Qin)

Scutellariabaica

lensis

Georgi(Lamiaceae)root

11111

2014081003

Baicalin

1233

8ndash1432

1

RheiRa

dixetRh

izom

a(DaH

uang

)Rh

eum

palm

atum

L(Polygon

aceae)

14814

2014080301

Aloe-em

odin

0348ndash

0367

Rhein

1002ndash116

2Em

odin

0513ndash0628

Chrysoph

anol

0661ndash0702

Physcion

0268ndash

0301

Natrii

Sulfas(MangXiao)

Mira

bilite(sulfateminerals)crysta

l14814

2014030506

Thenardite

99812ndash9

9993

Magno

liaeO

fficinalis

Cortex(H

ouPu

)Magnolia

officin

alisRe

hder

ampE

HW

ilson

(Magno

liaceae)rootbranchandste

mbark

11111

201406

0501

Magno

lol

2118

ndash2491

Hon

okiol

2181ndash2228

AurantiiFructusImmaturus

(Zhi

Shi)

Citru

saurantiu

mL(Rutaceae)immaturefruit

11111

2014053101

Syneph

rine

0612ndash064

1Hesperid

in12

31ndash1445

Artem

isiae

Scop

ariaeH

erba

(Yin

Chen)

ArtemisiascopariaWaldstampKitam(As

teraceae)

aeria

lpart

11111

20140306

01Ch

lorogenica

cid

0598ndash

0693

Escoparone

0598ndash

06341

Gardeniae

Fructus(Zh

iZi)

Gardeniajasm

inoides

JEllis

(Rub

iaceae)rip

efruit

14814

2014110

301

Geniposide

5989ndash

6959


minus50

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35

(mAU

)

(min)

HPLC-MS analyses of Rhei Radix et Rhizoma

1

2

3

4

5

Positive ion mode

Figure 1 Chromatographic fingerprint of Rhei Radix et Rhizoma

reference marker compounds to engender characteristicqualitative profiles (fingerprints) The assays of the qualitycontrol data of each Chinese drug decoction piece (heavymetal pesticide residues mycotoxins impurity ash watercontent extract and the quantitative analysis of major activeingredients) were performed according to official pharma-copoeial methods and have been determined to be consistentwith or better than pharmacopoeial standards as describedpreviously

Number in the bracket is the relative retention of the peakto themarker peak (1) 065 Aloe-emodin (2) 070 Rhein (3)100 Emodin (4) 134 Chrysophanol and (5) 149 Physcion

Except Natrii Sulfas the other seven dried componentsof Chinese drug decoction pieces were immersed in coldwater for 60 minutes and then individually milled ExceptRhei Radix et Rhizoma the other six milled components ofthe Chinese drug decoction pieces were admixed in the pre-scribed proportion (ww) as shown inTable 1 and thenwereextracted in 10-fold (wv) of purified water by the decoctingmethod for two times (40 minuteseach time) according togood manufacturing practices (GMP) at the College of thePharmacy of Sichuan University At the last ten minutes inthe second decocting extraction the milled component ofRhei Radix et Rhizoma drug decoction pieces was added tothe extract liquids according to the prescribed proportion (ww) as shown in Table 1 for further decocting extractionAfterwards the mixing extracts of the seven Chinese drugdecoction pieces and the pure product of Natrii Sulfas wereadmixed according to the prescribed proportion ( ww) asshown in Table 1 The extracts were filtered concentratedspray-dried and lyophilized to obtain a powder (2844 yieldww based on the raw materials of Chinese drug decoctionpieces)The aliquots weremanufactured into clinical productthrough the addition of water-soluble sodium carboxymethylstarch (excipient) according to the proportion of 1 1 (ww)Before administration to animals the lyophilized powder ofCQCQD was prepared to a concentration of 24 gmL ofcrude herbsaqua pro injectione Rat medicated serums wereprepared according to published protocols [32] Briefly 10rats were randomly divided into blank group and CQCQDgroup Rats in CQCQD group were treated with CQCQD(01mL10 g bw times 1 time2 hrs 6 times) by intragastric

administration while rats in blank group only received thesame amount of normal saline After the last administrationblood from the rats was collected and centrifuged to prepareCQCQD-containing serum and control serum Finally theserums were stored at minus20∘Cprior to useThe previous reportindicated that CQCQD-containing serum at the concentra-tion of 10 was more effective than that at 5 in improvingthe viability of pancreatic acinar cells in SAP rats [33] It canbe concluded that highly efficient concentration of CQCQD-containing serum would probably be considered as 10Therefore the CQCQD-containing serum or control serumwas diluted to the concentration of 10 by using DMEMmedium respectively

23 Experimental Design For in vivo trial rats were ran-domly divided into sham-operation (SO) SAP and CQCQDgroup (119899 = 20group) In this paper ldquoSAP grouprdquo was usedto represent ldquoSAP endotoxemia-induced ALI model grouprdquoSAP endotoxemia-induced ALI model was induced byretrograde main pancreatic duct injection of 5 sodiumtaurocholate (1mLkg bw) combined with cecal ligation-perforation (CLP) At 1 hr after operation rats in CQCQDgroup were treated with CQCQD (01mL10 g bw times 1time2 hrs 6 times) by intragastric administration while ratsin the other two groups only received the same amount ofsaline At 1 hr before the end of experiment 5 rats fromeach group received intravenous injection of 2 Evans blue(EB) (2mLkg) At 12 hours after operation blood sampleswere collectedThenusing Infusion Pump (FreseniusVial SAFrance) the blood in the pulmonary circulation was clearedaway After that the tissues on the edge of 05 cm in thelungs of rats in each group were collected For in vitro trialaccording to our research targets HUVECs were divided intothe following six groups (1) Normal group (pretreated withnormal rat serums for 1 hr and then added DMEM mediumwith 10 FBS and coincubated for 12 hrs) (2) LPS group(pretreated with normal rat serums for 1 hr and then added1 120583M LPS and coincubated for 12 hrs) (3) CQCQD group(pretreated with CQCQDmedicated serums for 1 hr and thenadded 1 120583MLPS and coincubated for 12 hrs) (4)ndash(6) selectiveinhibition agents groups (pretreated with normal rat serums+ inhibitors of c-Src (PP1 10 120583M) or PI3K (LY294002 30 120583M)or transfected with siRNAs of c-Fos and then added 1 120583MLPSand coincubated for 12 hrs)

24 Gene Expression Profiling and Data Analysis The SOgroup SAP group and CQCQD group were represented bythe letters N S and C respectively The gene expressionprofiles of lung tissue in each sample were determined usingRat OneArray Plus Microarray (Phalanx Biotech Taiwan)The data of Rat OneArray Plus were analyzed using RosettaResolver System (Rosetta Biosoftware) Annotation ROA2release 10 (databases NCBI RefSeq release 65 and Ensemblerelease 76 cDNA sequences Rnor 50 annotations) and R303 software (httpwwwr-projectorg) DEGswere identi-fied as119875 lt 005 and | log 2(Ratio)| ≧ 0585 by unpaired t-testThe functional enrichment analyses of KEGG pathways asso-ciated with the lists of genes were generated by WebGestalt(httpwwwwebgestaltorg) (119875 lt 005 and entitles ge3)


25 Protein-Protein Interaction (PPI) Network ConstructionandHubProteins Identification The list of theDEGs selectedaccording to the study objective was input into NetworkAn-alyst software (Hancock Lab Canada)The proteins encodedby the prioritized and training DEGs were employed as theinitial seed proteins to interact with other essential proteinsto construct the PPI network The nodes with high degreesand betweenness would be considered as the hub nodes

26 Measurement of Plasma LPS sCd14 (Soluble-MonocyteDifferentiation Antigen CD14) and Lbp (Lipopolysaccharide-Binding Protein) Plasma LPS were measured by LimulusAmebocyte Lysate assay (Lonza MD USA) according to themanufacturerrsquos protocol and the sCd14 and Lbp levels inplasma were measured using ELISA kits (RampD MinnesotaUSA)

27 Assays of Amylase and Albumin in Sera as well asPaO2 PaCO2 and SpO2 The amylase and albumin in serawere measured by the fully automatic biochemical analyzer(TBA-120FR ToshibaMedical Systems Corporation TochigiJapan) PaO

2 PaCO

2 and SpO

2were detected by the blood-

gas analyzer (RAPIDPOINT 500 Siemens Healthcare Diag-nostics Inc New York USA)

28 Determination of the Total Protein Levels Total CellCounts and the Concentration of TNF-120572 IL-6 and IL-1120573 inthe Bronchoalveolar Lavage Fluid (BALF) The left lungs werelavaged via the bronchuswith 15mLPBS for three times afterthat the BALF was collected for detecting total protein levels(Bicinchoninic Acid Kit for Protein Determination Sigma-Aldrich MO USA) and total cell counts (Wright-Giemsastaining Shanghai Haling Biotechnology Co Ltd ShanghaiChina) as well as the concentrations of TNF-120572 IL-6 andIL-1120573 (ELISA kits RampD Systems Minneapolis MN USA)according to the instructions of manufacturers

29 Measurement of Albumin Transcapillary Escape Rate(TER) EB would bind tightly to sera albumin and act asa hypersensitive marker for albumin leak [34] After theoperation immediately fromeach group 5 ratswere randomlyselected and received caudal vein injection of EB (1mgmL)according to the concentration of 02mgkg Before theinjection of EB blood samples of 1mL were drawn andcentrifuged for 10min (3000 rpm) to remove the supernatantafter that the absorbance of blank plasma at 620 nm and740 nm was detected by a spectrophotometer (Beijing Anal-ysis Instrument Co Ltd Beijing China) to establish thestandard curve of relationship between EB concentration andEB620corr (a correction value of plasmatic absorbance at620 nm by the EB) Then after injection of EB as well as6 hrs after operation and also at the end of the experimentthe plasmatic absorbance at 620 nmand 740 nm inEB-treatedrats was assayed to calculate TER value according to the atten-uation law of EB620coor of samples which was based on thestandard curve

210 Measurement of Pulmonary EB Content The EB con-tents in tissues are positively correlated with the quantity ofthe albumin leak [34]The lung tissues from each group weresoaked in NN-dimethylformamide Homogenate was incu-bated and centrifuged for detecting the absorbance at 620 nmand the EB concentration was measured based on a standardcurve of EB-NN-dimethylformamide solutions

211 Measurement of Pulmonary Water Content From eachgroup 5 rats were randomly selected and the right lungtissues of these rats were obtained and then we measuredrespectively the wet and dry weight of the lungs to calculatethe pulmonary water content according to the research by Xuet al [35]

212 HE Staining The pancreatic and pulmonary sampleswere fixed in 4 paraformaldehyde and subjected to thestaining of haematoxylin and eosin For eachHE stained slice10 visual fields under a high-power microscope (times400) wererandomly selected and scored by one specific pathologist

213 Transient Transfection with siRNAs The small inter-fering RNA (Stealth siRNA) for Human c-Fos (HSS103799HSS103800 and HSS177475) and scrambled siRNA werefrom Thermo Fisher Scientific Inc (MA USA) Transienttransfection of siRNAs was performed according to themanufacturerrsquos instructions

214 Transwell Assays HUVECs were seeded on 24mmTranswell with 04 120583m Pore Polyester Membrane InsertSterile (Corning MA USA) and grown until confluencyHUVECs monolayers of TEER were measured by Millicellelectrical resistance apparatus (Millipore FL USA) andTEER values were calculated in accordance with the formulaas described previously [36] To further measure paracel-lular permeability FITC-dextran (50 gmL) was added tothe upper chambers of the Transwell and coincubated withcell monolayer at 37∘C for 60min and then samples wereextracted from both the upper and lower chambers forfluorescence determination at 535 nm furthermore the per-meability to dextran was calculated as described previously[37]

215 Determination of the Concentrations of TNF-120572 IL-6 andIL-1120573 in the Supernatant of HUVECs After centrifugationthe supernatant of HUVECs was collected for detectingthe concentrations of TNF-120572 IL-6 and IL-1120573 (ELISA kitsRampD Systems Minneapolis MN USA) according to theinstructions of manufacturers

216 Western Blot Analysis Total protein in the three groupsof HUVECs was separated by 10 acrylamide SDS-PAGEand transferred onto ImmobilonPVDFmembranes (Sigma-Aldrich MO USA) Then the membranes were blocked andincubated with specific primary antibodies in 4∘C overnight(1 1000 of dilution) followed by incubation with a sec-ondary antibody Membranes-bound antibodies were testedby Amersham ECL (Sigma-Aldrich MO USA)


Table 2 KEGG pathway enrichments analyses (closely related to albumin leakage) in the SN and CS DEGs cluster

Pathway Total Hits 119875 value FDRToll-like receptor signaling pathway 96 39 896E minus 22 318E minus 20Tight junction 122 48 147E minus 19 313E minus 18Adherens junction 71 27 197E minus 15 175E minus 14Regulation of actin cytoskeleton 183 62 174E minus 08 686E minus 08Apoptosis 82 25 607E minus 08 223E minus 07

(a)

N_H

001

N_H

002

N_H

003

S_H

004

S_H

005

S_H

006

C_H

007

C_H

008

C_H

009

Low High

Fos

CLASS

Pik3r1

Src

(b)

Figure 2 Predicting target proteins for CQCQD to attenuate pulmonary albumin leakage based on the analyses of rats pulmonary tissuemicroarray and also the construction of PPI network (a)The proteins encoded by these prioritized hub nodes including Fos Pik3r1 and Srcwere predicted as the key target proteins (red nodes = upregulated DEGs green nodes = downregulated DEGs grey nodes = other interactivegenes the size of nodes = the betweenness of the node) (b)The expressions of these 3 prioritized hub nodes in each sample within each group(red represents upregulation and green represents downregulation)

217 Statistical Analysis Data are shown as mean plusmn standarddeviation (SD) Dunnettrsquos pairwise multiple comparison t-test was used to examine the significant differences by SPSS190 software Statistical difference was accepted at 119875 lt 005

3 Results

31 Identification of DEGs in the SN and CS DEGs ClustersAccording to the Rosetta Biosoftware out of 20715 geneprobes on the Rat OneArray Plus Microarray 3190 gens(1678 genes upregulated and 1512 genes downregulated) inSN DEGs cluster and 4138 genes (2404 genes upregulatedand 1734 genes downregulated) in CS DEGs cluster weredifferentially expressed (119875 lt 005 | log 2(Ratio)| ≧ 0585)

32 Predictions of the Target Proteins for CQCQD to AttenuatePulmonary Albumin Leakage Based on the Analyses of RatsPulmonaryTissueMicroarray andAlso theConstruction of PPINetwork The KEGG pathway enrichment analyses closelyrelated to paracellular leakage-dependent albumin leakageprovided the important evidences that some of the DEGs inCS and SN DEGs clusters were significantly enriched in

these 5 pathways including inflammatory response (Toll-likereceptor signaling pathway) paracellular leakage (adherensjunction tight junction and regulation of actin cytoskele-ton) and apoptosis (apoptosis) (119875 lt 005 entitles ≧ 3Table 2) And then the DEGs (CS DEGs cluster) enrichedin these KEGG pathways were inputted into NetworkAn-alyst database to construct a PPI network (nodes = 623edges = 868 and seed proteins = 72) The top three nodeswith the highest degree and betweenness (degree gt40 andbetweenness gt1000) were Fos (protooncogene c-Fos) Pik3r1(phosphatidylinositol 3-kinase regulatory subunit alpha) andSrc (protooncogene tyrosine-protein kinase Src) respectively(Figure 2(a))Therefore the 3 proteins including c-Fos c-Srcand p85120572 encoded by these 3 genes were considered as theprioritized hub proteins and also predicted as the key targetproteins for CQCQD to attenuate pulmonary albumin leak-age Furthermore these 3 genes displayed the expression pat-terns of CS reversing SN (119875 lt 005 | log 2(Ratio)| ≧ 0585)which were shown in the heatmap (Figure 2(b))

33 Effect of CQCQD on the Characteristics Indexes CloselyRelated to SAP Complicated by Endotoxemia As shown inTable 3 the blood concentrations of LPS sCd14 Lbp and


Table 3 The blood concentrations of LPS sCd14 Lbp and amylase

Indexes SO group SAP group CQCQD groupLPS (ngmL) 013 plusmn 004 052 plusmn 006998771998771 036 plusmn 009

LBP (ugmL) 246 plusmn 035 912 plusmn 061998771998771 748 plusmn 049

sCD14 (ugmL) 223 plusmn 124 708 plusmn 537998771998771 431 plusmn 402

Serum amylase (IUL) 148076 plusmn 42246 258396 plusmn 29751998771998771 203923 plusmn 14199998771998771

119875 lt 001 versus SO 119875 lt 005 versus SAP and 119875 lt 001 versus SAP

Table 4 The characteristics indexes closely related to systemic and pulmonary albumin leakage

Indexes SO SAP CQCQDTER (h) 856 plusmn 114 2473 plusmn 219998771998771 1642 plusmn 145

Serum albumin (gL) 2303 plusmn 031 1326 plusmn 063998771998771 1849 plusmn 038

Pulmonary EB content (ugg) 3729 plusmn 309 7714 plusmn 924998771998771 6103 plusmn 438

Pulmonary water content () 7123 plusmn 107 8035 plusmn 137998771998771 7501 plusmn 133998771998771


Table 5 The airways indicators and respiratory function

Indexes SO SAP CQCQDTotal protein levels in BALF (mgmL) 158 plusmn 016 934 plusmn 102998771998771 684 plusmn 072

Total cell counts in BALF (times106L) 289 plusmn 006 982 plusmn 085998771998771 533 plusmn 021

TNF-120572 levels in BALF (ngL) 12407 plusmn 1735 18961 plusmn 1022998771998771 16421 plusmn 788

IL-6 levels in BALF (ngL) 14923 plusmn 1547 21727 plusmn 982998771998771 18875 plusmn 1149


PaCO2

(mmHg) 3002 plusmn 098 4716 plusmn 132998771998771 4107 plusmn 108

PaO2


SaO2

() 9866 plusmn 093 8907 plusmn 074998771998771 9782 plusmn 069998771998771


amylase in SAP group were remarkably higher than that inSO group (998771998771119875 lt 001) strikingly the blood concentrationsof LPS sCd14 Lbp and amylase in CQCQD group wereobviously decreased compared with that in SAP group (119875 lt005 or 119875 lt 001)

34 Effect of CQCQD on the Characteristics Indexes CloselyRelated to Systemic and Pulmonary Albumin Leakage Asshown in Table 4 the TER as well as pulmonary EB and watercontents in SAP group were remarkably higher than thosein SO group meanwhile the serum albumin in SAP groupwas significantly lower than that in SO group (998771998771119875 lt 001)fortunately the treatment of CQCQD had significantlyreversed the changes in these characteristics indexes com-pared with that in SAP group (119875 lt 005 or 119875 lt 001)

35 Effect of CQCQD on the Airways Indicators and Respi-ratory Function As shown in Table 5 acting as the airwaysindicators the total protein levels and total cell counts aswell as the levels of TNF-120572 IL-6 and IL-1120573 of BALF in SAPgroup were remarkably higher than that in SO group (998771998771119875 lt001) acting as the characteristics indexes of respiratoryfunction PaCO

2in SAP group was markedly higher than

that in SO group meanwhile PaO2and SaO

2in SAP group

were evidently lower than those in SO group (998771998771119875 lt001) fortunately the treatment of CQCQD had signifi-cantly reversed the changes in these characteristics indexescomparedwith that in SAP group (119875 lt 005 or119875 lt 001)

36 Effect of CQCQD on the Pancreatic and PulmonaryHistopathological Changes The pancreatic and pulmonaryhistopathological changes were shown as follows (1) theedema hemorrhage massive inflammatory cells infiltrationand a large number of pancreatic acinar cellsrsquo necrosis wereseen in the pancreatic tissue of SAP rats (Figure 3(a)) (2)the broken alveolar structure thickened alveolar wall and theinfiltration of massive inflammatory cell and red blood cellswere shown in SAP rats (Figure 3(b)) (3) nevertheless thetherapy of CQCQD decoction effectively alleviated the pan-creatic and pulmonary histopathological changes comparedwith that in SAP group (Figure 3) (4) according to the abovehistopathological changes the pancreatic and pulmonaryhistopathological scores of rats in SAP group were evidentlyhigher than that in SO group (119875 lt 001) while those inCQCQD group were visibly decreased compared with that inSAP group (119875 lt 005) (Figure 3)


SO SAP CQCQD

(a)

SO SAP CQCQD

(b)

Figure 3 HE staining (a) Pancreatic histopathological changes (times200) (pancreatic histopathological score SO = 032 plusmn 008 SAP = 1685 plusmn289 CQCQD = 887 plusmn 126 SAP versus SO 119875 lt 001 CQCQD versus SAP 119875 lt 001) (b) pulmonary histopathological changes (times400)(pulmonary histopathological score SO = 045 plusmn 012 SAP = 344 plusmn 046 CQCQD = 187 plusmn 035 SAP versus SO 119875 lt 001 CQCQD versusSAP 119875 lt 005)

Table 6 The proinflammatory cytokines release TEER and paracellular leakage in HUVECs

Indexes Normal LPS (1mgL) CQCQD (1mgL) Inhibition agentssiRNA (c-F-os) PP1 (10120583M) LY294002 (30120583M)

TNF-120572 (ngmL) 185 plusmn 035 1246 plusmn 041998771 932 plusmn 058 585 plusmn 028f 678 plusmn 031 518 plusmn 033curren

IL-6 (120588gmL) 3219 plusmn 755 31845 plusmn 947998771 19433 plusmn 861 11218 plusmn 998f 12123 plusmn 1045 9856 plusmn 1010curren


TEER (Ωsdotcm2) 2512 plusmn 166 1861 plusmn 098998771 2123 plusmn 112 2389 plusmn 103f 2264 plusmn 123 2313 plusmn 101curren

Dextran flux (cms) 022 plusmn 012 048 plusmn 029998771 033 plusmn 022 031 plusmn 018f 028 plusmn 016 026 plusmn 014curren998771

119875 lt 005 versus Normal 119875 lt 005 versus LPS f119875 lt 005 versus LPS 119875 lt 005 versus LPS and curren119875 lt 005 versus LPS

37 Effect of the Pretreatment of CQCQD on LPS-MediatedChanges in TEER Paracellular Leakage and Proinflamma-tory Cytokines Release in HUVECs Imitating the Actions ofthe Selective Inhibitions Agents of These Predicted ProteinsAs shown in Table 6 LPS-stimulated HUVECs show theremarkable increase in proinflammatory cytokines releaseand dextran flux (paracellular leakage) compared to that inNormal group (119875 lt 005) meanwhile TEER in LPS groupwas significantly lower than that in Normal group (119875 lt005) According to the analyses results of the PPI net-work constructed by the analyses of ratsrsquo pulmonary tissuemicroarray c-Fos c-Src and p85120572 were predicted as thekey target proteins for CQCQD in alleviating pulmonaryalbumin leakage Additional information p85120572 is encoded byPik3r1 [38] c-Fos is encoded by Fos [39] and c-Src is encodedby Src [40] In order to verify these predictions that firstly

required demonstrating the important involvements of these3 predicted target proteins in LPS-mediated changes inTEERparacellular leakage and proinflammatory cytokines releasein HUVECs HUVECs were pretreated with the inhibitorsof c-Src (PP1) or PI3K (LY294002) or transfected withsiRNAs of c-Fos which provided the important evidencesthat the pretreatment with PP1 or LY294002 as well asthe transfection with siRNAs of c-Fos remarkably reversedLPS-induced increases in proinflammatory cytokines releaseand paracellular leakage and also the decrease in TEER(119875 lt 005 Table 6) Strikingly the pretreatment ofCQCQD imitated the actions of the selective inhibitionsagents of these predicted proteins to significantly reverseLPS-mediated increase in paracellular leakage and proin-flammatory cytokines release as well as the decrease in TEER(119875 lt 005 Table 6)


minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771

PPI (10120583M)CQCQD (1mgL)

LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771

CQCQD (1mgL)LPS (1mgL)

siRNAs (c-Fos)CQCQD (1mgL)

LPS (1mgL)

(c)

Figure 4The expression changes in these 3 predicted proteins in HUVECs 998771998771119875 lt 001 versus Normal group 119875 lt 001 versus LPS group

119875 lt 005 versus LPS group ff119875 lt 001 versus LPS group and f119875 lt 005 versus LPS group

38 Effects of the Pretreatment of CQCQD on the LPS-Induced Expression Changes in These 3 Predicted Proteins inHUVECs Imitating the Actions of the Selective InhibitionsAgents of These Predicted Target Proteins Since the selectiveinhibitions agents of these predicted target proteins show thesignificant alleviating effects on LPS-mediated paracellularleakage and proinflammatory cytokines release in HUVECsthe analyses of the expression of these predicted targetproteins were required to reveal the molecular mechanisms

HUVECs were treated as described previously and thenWestern blot analyses were performed This study providedthe important evidences that (1) LPS observably induced thehigh expressions of p-Src p-p85120572 and c-Fos compared withthat in Normal group (119875 lt 005 Figure 4) but did not inducethe significant changes in expression of nonphosphorylatedSrc and p85120572 compared with that in Normal group(119875 gt 005 Figures 4(a) and 4(b)) (2) the pretreatment withthe inhibitors of c-Src (PP1) remarkably inhibited


LPS-induced high expression of p-Src (119875 lt 005 Figure 4(a))but did not induce the significant change in expression ofnonphosphorylated Src compared with that in Normal orLPS group (119875 gt 005 Figure 4(a)) (3) the pretreatmentwith the inhibitors of PI3K (LY294002) markedly suppressedLPS-induced high expression of p-p85120572 (119875 lt 005Figure 4(b)) but did not trigger the significant change inexpression of nonphosphorylated p85120572 compared withthat in Normal or LPS group (119875 gt 005 Figure 4(b)) (4)the transfection with siRNAs of c-Fos evidently inhibitedLPS-induced overexpression of c-Fos (119875 lt 005 Figure 4(c))(5) strikingly the pretreatment of CQCQD on HUVECssuccessfully imitated the actions of the selective inhibitionsagents in the same way to inhibit LPS-mediated highexpressions of p-Src p-p85120572 and c-Fos (119875 lt 005 Figure 4)and likewise did not induce the significant changes inexpressions of nonphosphorylated Src and p85120572 comparedwith that in Normal or LPS group (119875 gt 005 Figures 4(a)and 4(b)) Therefore the inhibition effects of LPS-mediatedhigh expressions of p-Src p-p85120572 and c-Fos were thekey molecular mechanisms for CQCQD or the selectiveinhibitions agents acting on these 3 predicted target proteins

39 Correlations among the Expressions of p-Src p-p85120572 andc-Fos and LPS-Induced Paracellular Leakage and the Changein TEER as well as LPS-Induced Proinflammatory CytokinesRelease in HUVECs According to the benchmark logarith-mic scale of 10 the line charts had been formed and then thecorrelational analyses were performed among the expressionsof p-Src p-p85120572 and c-Fos and LPS-induced paracellularleakage and the change in TEER as well as LPS-inducedproinflammatory cytokines release in HUVECs The 120588 valueof correlation coefficient is calculated based on the originaldata of these parameters but not the logarithmic data Thisstudy provided the first evidences that (1) the expressions ofp-Src p-p85120572 and c-Fos were positively correlated with LPS-induced paracellular leakage and proinflammatory cytokinesrelease (120588 gt 098) and negatively correlated with LPS-induced changes in TEER (120588 lt minus098) which demon-strated the very highly linear regression correlation (Figures5(a) 5(b) and 5(c)) (2) there were also the remarkablypositive correlations between LPS-induced proinflammatorycytokines release and LPS-induced paracellular leakage (120588 gt098) and also the notably negative correlations between LPS-induced proinflammatory cytokines release and LPS-inducedchanges in TEER (120588 lt minus098) which show the highly linearregression correlation (Figure 5(d))

4 Discussion

The methods of intraperitoneal (IP) or intratracheal (IT)injection of LPS-inducing lung injury are simple but theycannot simulate the natural course of SAP endotoxemia-induced ALI [41] Therefore we used the methods of retro-grade main pancreatic duct injection of 5 sodium tauro-cholate combined with cecal ligation-perforation to establishSAP endotoxemia-induced ALI model After cecal ligationthe feces were accumulated in cecum and then the punctureby using 18 needle was performed on the ligated cecum to

form 3 holes After that the mixed bacteria in the intestinalcontents constantly enter into the abdominal cavity to inducethe infection of the abdominal cavity which would resultin endotoxemia or sepsis and eventually initiate pulmonaryalbumin leakage to trigger ALI Hence the models inducedby retrograde main pancreatic duct injection of sodiumtaurocholate combined with cecal ligation-perforation couldrealistically demonstrate the pathophysiological process ofSAP endotoxemia-induced ALI that is more close to the trueprocess in clinic [41] Strikingly retrograde main pancreaticduct injection of 5 sodium taurocholate combined withcecal ligation-perforation successfully induced SAP com-plicated with endotoxemia (Table 3) the enhancement tosystemic and pulmonary albumin leak (Table 4) deprava-tion of airways indicators (Table 5) respiratory dysfunction(Table 5) and also the adverse change in pancreatic andpulmonary tissue pathology (Figure 3) which suggested thatthe models of ALI caused by endotoxemia in SAP rats weresuccessfully constructed Fortunately the therapy of CQCQDshowed the protective effects on the rats of SAP endotoxemia-induced ALI via significantly ameliorating the changes in theabove characteristics indexes (Tables 3 4 and 5 and Figure 3)

The pathophysiological mechanisms of alleviating para-cellular leakage-dependent albumin leak mainly include (1)inhibiting the excessive reassembly of F-actin and the reorga-nization of cytoskeleton (2) suppressing the disassembly ofTJs AJs and F-actin at cell-cell contact sites (3) reassemblingTJs AJs and F-actin at damaged cell-cell contact sites(4) maintaining AJs TJs and the homeostasis of actin(5) suppressing apoptosis and (6) inhibiting inflammatoryresponse [42] In our study the analyses of rats pulmonarytissues microarray and also the construction of PPI networkprovided the important evidences that (1) some DEGs in CSand SN DEGs clusters were significantly enriched in the 5KEGG pathways closely related to the above pathophysiolog-ical mechanisms (Table 2) (2) there were the 3 prioritizedhub proteins (c-Fos c-Src and p85120572) to be predicted as thekey target proteins for CQCQD in alleviating pulmonaryalbumin leakage and these 3 genes displayed the expressionpatterns of CS reversing SN (Figure 2) In order to verifythese predictions that firstly required demonstrating theimportant involvements of these 3 predicted target proteinsin LPS-mediated changes in TEER paracellular leakage andproinflammatory cytokines release in HUVECs and thenfurther investigate the molecular mechanisms involved ourstudies in vitro provided the several important evidencesthat (1) the selective inhibition agents of these 3 predictedtarget proteins remarkably reversed LPS-induced increases inproinflammatory cytokines release and paracellular leakageas well as the decrease in TEER (Table 6) (2) the inhibitioneffects of LPS-mediated high expressions of p-Src p-p85120572and c-Fos were the key molecular mechanisms for theselective inhibitions agents acting on these predicted targetproteins (Figure 4) (3) the expressions of p-Src p-p85120572 andc-Fos were highly and positively correlated with LPS-inducedparacellular leakage and proinflammatory cytokines releaseand highly and negatively correlated with LPS-inducedchanges in TEER (Figures 5(a) 5(b) and 5(c)) which sug-gested that inhibitions of LPS-mediated high expressions of


0

10

100

1

1000

TNF-120572 levelIL-6 levelIL-1120573 level

TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100


TEERDextran fluxp-p85120572 expression

minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)


TEERDextran fluxc-Fos expression

0

1

10

100

1000

minusminusminus

+minusminus

++minus

+minus+siRNAs (c-Fos)

CQCQDLPS

(c)

minusminusminusminusminus

+minusminusminusminus

++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)

Figure 5 Correlations among the expressions of p-Src p-p85120572 and c-Fos and LPS-induced paracellular leakage and the change in TEER aswell as LPS-induced proinflammatory cytokines release in HUVECs (benchmark logarithmic scale = 10 120588 gt 098 versus dextran flux orthe levels of TNF-120572 or IL-6 or IL-1120573 120588 lt minus098 versus TEER 120588 gt 098 versus dextran flux and 120588 lt minus098 versus TEER)

p-Src p-p85120572 and c-Fos could suppress LPS-induced para-cellular leakage and proinflammatory cytokines release Andthen our in vitro studies successfully confirmed these pre-dictions (1) the pretreatment of CQCQD on LPS-stimulatedHUVECs which imitated the actions of the selective inhibi-tions agents to inhibit LPS-induced high expressions of p-Src p-p85120572 and c-Fos (Figure 4) (2) ultimately significantly

alleviated LPS-mediated increase in paracellular leakage andproinflammatory cytokines release as well as the decrease inTEER (Table 6)The above evidences suggested that CQCQDcould probably attenuate the paracellular leakage-dependentalbumin leak via reversing endotoxemia-triggered expres-sions changes in these predicted target proteinsThe potentialmolecular mechanisms were further analyzed as follows


Alleviating Paracellular Leakage via Inhibiting the Expres-sions of Upstream Trigger Molecules of Toll-Like ReceptorsPathway and Inflammatory Response LPS would activatedistinct pathway to induce inflammatory response via Toll-like receptors cascades and Lbp and Cd14 are key upstreammolecules that are common to all TLRs [43] Fortunatelyour study in vivo demonstrated that the therapy of CQCQDsignificantly decreased the levels of sera amylase and plasmaLPS Lbp and Cd14 compared to that in SAP group (Table 3)which suggested that the therapy of CQCQD could attenuatethe increase in the upstream trigger molecules caused bySAP complicated by endotoxemia The activation of Toll-likereceptors cascades would induce the upregulation of p-Src tophosphorylate p85120572 and induce the activation of Akt (kinaseSerinethreonine-protein) which ultimately leads to the acti-vation of NFKB and MAPK cascades and initiates the exces-sive transcriptions of proinflammatory cytokines [44 45] c-Fos is a key transcription factor in MAPK cascade whoseoverexpression would also mediate the numerous transcrip-tions of inflammatory cytokines [46] These previous reportshave been confirmed by our in vitro studies results that inhi-bitions of LPS-mediated high expressions of p-Src p-p85120572and c-Fos could suppress LPS-induced proinflammatorycytokines release (Figures 4 5(a) 5(b) and 5(c) and Table 6)and the specific analyses have been demonstrated in the firstparagraph of the Discussion Strikingly the pretreatment ofCQCQD imitated the actions of these selective inhibitionsagents to inhibit LPS-mediated high expressions of p-Src p-p85120572 and c-Fos therefore suppressing the proinflammatorycytokines release (Figure 4 and Table 6) It is well known thatthe increase in proinflammatory cytokines release will lead tothe enhancement of the albumin leakage induced by LPSOurstudy in vitro provided the important evidences that therewere also remarkably positive correlations between LPS-induced proinflammatory cytokines release and paracellularleakage (Figure 5(d)) which suggested that the pretreatmentof CQCQD could probably contribute to alleviating LPS-induced paracellular leakage via inhibiting proinflammatorycytokines release (Table 4)

Protections and Maintenance of AJs TJs and the Homeosta-sis of F-Actin LPS-stimulated HUVECs had significantlyinduced the upregulated expressions of p-Src p-p85120572 and c-Fos (Figure 4) which would probably induce the disassemblyof AJs TJs and F-actin at cell-cell contact sites and trigger theexcessive reassembly of F-actin and cytoskeleton reorganiza-tion The molecular mechanisms were described as follows(1) the high expressions of p-Src at Tyr-419 would increasekinase activity to phosphorylate downstreammolecules [47]which would probably induce (a) the high phosphorylationof p85120572 to aggrandize the Akt (Serinethreonine-proteinkinase) activation to mediate the reorganization of cytoskele-ton [48] (b) the Tjp1 Tyr hyperphosphorylation to mediatethe collapse of TJs [49] and (c) the high phosphorylationof Ctnnb1 (Catenin beta-1) and Ctnnd1 (Catenin delta-1) toinduce disassembly of AJs [50 51] (2) the high expressionsof p-p85120572 would enhance Akt activity to induce cytoskeletonreorganization [48] (3) the excessive reassembly of F-actinwould further induce the overexpression and activation

of Fos [52] which would mediate more transcriptions ofinflammatory cytokines thus forming a vicious cycle Theseinferences have been confirmed by the studies in vitro thatLPS-stimulated HUVECs have markedly caused the decreasein TEER as well as the increase in proinflammatory cytokinesrelease and paracellular leak (Table 6) Notably the studies invitro have provided the key evidences that inhibitions of LPS-mediated high expressions of p-Src p-p85120572 and c-Fos couldreverse LPS-induced decrease in TEER and the increase inparacellular leak (Figures 4 5(a) 5(b) and 5(c) and Table 6)and the specific analyses have been demonstrated in the firstparagraph of the Discussion More notably the pretreatmentof CQCQD imitated the actions of these selective inhibitionsagents of these 3 predicted target proteins to inhibit LPS-induced high expressions of p-Src p-p85120572 and c-Fos (Fig-ure 4) Consistent with this the pretreatment of CQCQD evi-dently reversed LPS-induced decrease in TEER as well as theincrease in proinflammatory cytokines release and paracel-lular leak (Table 6) which suggested that the pretreatment ofCQCQDonLPS-stimulatedHUVECs could probably protectand maintain AJs TJs and the homeostasis of F-actin

To further verify the therapeutical effect of CQCQDon rats with SAP endotoxemia-induced ALI our studyprovided the key evidences that in in vivo study the therapyof CQCQD significantly ameliorated the enhancements ofsystemic and pulmonary albumin leakage as well as thedepravation of airways indicators (Tables 4 and 5) and ulti-mately improved respiratory dysfunction and pancreatic andpulmonary histopathological changes (Table 4 and Figure 3)

In conclusion the therapy of CQCQD could alleviatethe LPS- and cytokine-mediated pulmonary albumin leakagewhich were mainly via inhibiting the upregulation of p-Srcp-p85120572 and c-Fos

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Qing Xia Ping Xue andWeiWu participated in the design ofthe study data analyses and paper preparation Wei Wu ZiqiLin and Ruijie Luo conducted the assays and analyses Allauthors read and approved the final paper

Acknowledgments

This project was supported by National Natural ScienceFoundation of China (no 81573766) Science and TechnologySupport Program of Sichuan (no 2011SZ0291) and Remead-john Technology Co Ltd Chengdu 610044 China

References

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[2] K Kamisoglu B Haimovich S E Calvano et al ldquoHumanmetabolic response to systemic inflammation assessment of theconcordance between experimental endotoxemia and clinicalcases of sepsisSIRSrdquo Critical Care vol 19 article 71 2015

[3] L Zhou H Yang Y Ai Y Xie and Y Fu ldquoProtective effect offorsythiaside A on acute lung injure induced by lipopolysaccha-ride in micerdquo Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi vol 30 no2 pp 151ndash154 2014 (Chinese)

[4] H Lei W Minghao Y Xiaonan X Ping L Ziqi and X QingldquoAcute lung injury in patients with severe acute pancreatitisrdquoTurkish Journal of Gastroenterology vol 24 no 6 pp 502ndash5072013

[5] L-Q Ma C-S Pan N Yang et al ldquoPosttreatment with Ma-Xing-Shi-Gan-Tang a Chinese medicine formula ameliorateslipopolysaccharide-induced lung microvessel hyperpermeabil-ity and inflammatory reaction in ratrdquo Microcirculation vol 21no 7 pp 649ndash663 2014

[6] D F Ben X Y Yu G Y Ji et al ldquoTLR4mediates lung injury andinflammation in intestinal ischemia-reperfusionrdquo Journal ofSurgical Research vol 174 no 2 pp 326ndash333 2012

[7] C-S Pan Y-H Liu Y-Y Liu et al ldquoSalvianolic acid B ame-liorates lipopolysaccharide-induced albumin leakage from ratmesenteric venules through Src-regulated transcelluar pathwayand paracellular pathwayrdquo PLoS ONE vol 10 no 5 Article IDe0126640 2015

[8] D Kondrikov C Gross S M Black and Y Su ldquoNovelpeptide for attenuation of hyperoxia-induced disruption oflung endothelial barrier and pulmonary edema via modulatingperoxynitrite formationrdquo The Journal of Biological Chemistryvol 289 no 48 pp 33355ndash33363 2014

[9] M I Hermanns AMMuller M Tsokos and C J KirkpatrickldquoLPS-induced effects on angiotensin I-converting enzymeexpression and shedding in human pulmonary microvascularendothelial cellsrdquo In Vitro Cellular andDevelopmental BiologymdashAnimal vol 50 no 4 pp 287ndash295 2014

[10] M-W Liu Y-HWang C-Y Qian and H Li ldquoXuebijing exertsprotective effects on lung permeability leakage and lung injuryby upregulating Toll-interacting protein expression in rats withsepsisrdquo International Journal of Molecular Medicine vol 34 no6 pp 1492ndash1504 2014

[11] P Xue Z-W Huang J Guo J-L Zhao Y-H Li and Z-CWang ldquoClinical study of ChaiqinChengqiDecoction in treatingsevere acute biliary pancreatitisrdquoZhongXiYi JieHeXueBao vol3 no 4 pp 263ndash265 2005 (Chinese)

[12] L Wang Y Li Q Ma et al ldquoChaiqin Chengqi Decoctiondecreases IL-6 levels in patients with acute pancreatitisrdquo Journalof Zhejiang University SCIENCE B vol 12 no 12 pp 1034ndash10402011

[13] J Guo P Xue X N Yang et al ldquoThe effect of Chaiqin ChengqiDecoction () on modulating serum matrix metalloproteinase9 in patients with severe acute pancreatitisrdquo Chinese Journal ofIntegrative Medicine vol 19 no 12 pp 913ndash917 2013

[14] W Wu J Guo X-N Yang et al ldquoEffect of Chaiqinchengqidecoction on serum amyloid A in severe acute pancreatitispatientsrdquoAsian Pacific Journal of TropicalMedicine vol 5 no 11pp 901ndash905 2012

[15] Z-C Wang P Xue and Z-W Huang ldquoEffect of an earlyapplication of chaiqin chengqi decoction in treating severeacute pancreatitis complicated with acute respiratory distresssyndromerdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 29 no 4pp 322ndash324 2009 (Chinese)

[16] J Guo T Jin Z Q Lin et al ldquoEffect of Chaiqin Chengqi Decoc-tion on cholecystokinin receptor 1-mediated signal transduc-tion of pancreatic acinar cells in acute necrotizing pancreatitisratsrdquo Chinese Journal of Integrative Medicine vol 21 no 1 pp29ndash35 2015

[17] Z Lin J Guo P Xue et al ldquoChaiqinchengqi decoction regulatesnecrosis-apoptosis via regulating the release of mitochondrialcytochrome c and caspase-3 in rats with acute necrotizingpancreatitisrdquo Journal of Traditional Chinese Medicine vol 34no 2 pp 178ndash183 2014

[18] J Guo H-Y Zhang T Jin et al ldquoEffect of Chai Qin Cheng Qidecoction on serum CCK-8 and calcium overload of pancreaticacinar inmice with acute pancreatitisrdquo SichuanDa Xue Xue BaoYi Xue Ban vol 42 no 5 pp 704ndash706 2011 (Chinese)

[19] P Xue L-HDeng Z-D Zhang et al ldquoEffect of Chaiqinchengqidecoction on sarcoendoplasmic reticulum Ca2+-ATPasemRNA expression of pancreatic tissues in acute pancreatitisratsrdquo World Journal of Gastroenterology vol 14 no 15 pp2343ndash2348 2008

[20] Y H Li Z W Huang P Xue et al ldquoEffects of Chaiqin ChengqiDecoction on activation of nuclear factor-kappaB in pancreasof rats with acute necrotizing pancreatitisrdquo Zhong Xi Yi Jie HeXue Bao vol 6 no 2 pp 180ndash184 2006 (Chinese)

[21] P Xue Z-W Huang H-Y Zhang et al ldquoImpact of Chai QinChengQiDecoction on cholinergic anti-inflammatory pathwayin rats with severe acute pancreatitisrdquo Sichuan Da Xue Xue BaoYi Xue Ban vol 37 no 1 pp 66ndash68 2006 (Chinese)

[22] L-H Deng D-K Xiang P Xue H-Y Zhang L Huang and QXia ldquoEffects of Chai-Qin-Cheng-Qi Decoction on cefotaximein rats with acute necrotizing pancreatitisrdquo World Journal ofGastroenterology vol 15 no 35 pp 4439ndash4443 2009

[23] P Xue J Guo X-N Yang W Huang and Q Xia ldquoChangesof neuronal acetylcholine receptor alpha 7 of peritonealmacrophage in experimental acute pancreatitis treated byChaiqin Chengqi Decoction (fft)rdquo Chinese Journal of IntegrativeMedicine vol 20 no 10 pp 770ndash775 2014

[24] G Jia W Xiaoxiang L Ruijie et al ldquoEffect of Chaiqinchengqidecoction on inositol requiring enzyme 1120572 in alveolarmacrophages of dogs with acute necrotising pancreatitisinduced by sodium taurocholaterdquo Journal of Traditional ChineseMedicine vol 35 no 4 pp 434ndash439 2015

[25] L Huang MH Wang and ZY Cheng ldquoEffects of Chai-Qin-Cheng-Qi decoction () on acute pancreatitis-associated lunginjury in mice with acute necrotizing pancreatitisrdquo Chin J IntegrMed 2012 [PubMed]

[26] J Zhu C Luo P Wang Q He J Zhou and H Peng ldquoSaikos-aponin A mediates the inflammatory response by inhibitingthe MAPK and NF-120581B pathways in LPS-stimulated RAW 2647cellsrdquo Experimental and Therapeutic Medicine vol 5 no 5 pp1345ndash1350 2013

[27] H A Lim E K Lee J M Kim et al ldquoPPAR120574 activation bybaicalin suppresses NF-120581B-mediated inflammation in aged ratkidneyrdquo Biogerontology vol 13 no 2 pp 133ndash145 2012

[28] M D dos Santos M C Almeida N P Lopes and G EP De Souza ldquoEvaluation of the anti-inflammatory analgesicand antipyretic activities of the natural polyphenol chlorogenicacidrdquo Biological and Pharmaceutical Bulletin vol 29 no 11 pp2236ndash2240 2006

[29] T H Lee J Chang and B M Kim ldquoSaikosaponin C inhibitslipopolysaccharide-induced apoptosis by suppressing caspase-3activation and subsequent degradation of focal adhesion kinasein human umbilical vein endothelial cellsrdquo Biochemical and


Biophysical Research Communications vol 445 no 3 pp 615ndash621 2014

[30] Y-L Wu L-H Lian Y Wan and J-X Nan ldquoBaicalein inhibitsnuclear factor-120581B and apoptosis via c-FLIP and MAPK ind-GalNLPS induced acute liver failure in murine modelsrdquoChemico-Biological Interactions vol 188 no 3 pp 526ndash5342010

[31] Y Huang X Luo X Li et al ldquoWogonin inhibits LPS-inducedvascular permeability via suppressing MLCKMLC pathwayrdquoVascular Pharmacology vol 72 pp 43ndash52 2015

[32] X Hu H Lu Y-L Deng Q Wan and S-M Yie ldquoEffect ofrat medicated serum containing Zuo Gui Wan andor You GuiWan on the differentiation of stem cells derived from humanfirst trimester umbilical cord into oocyte-like cells in vitrordquoEvidence-Based Complementary and Alternative Medicine vol2015 Article ID 825805 17 pages 2015

[33] L-H Deng X-N Yang and Q Xia ldquoProtective effects ofChaiqin Chengqi Decoction on isolated pancreatic acinar cellsin acute pancreatitis rats and the mechanismsrdquo Zhong Xi Yi JieHe Xue Bao vol 6 no 2 pp 176ndash179 2008 (Chinese)

[34] B Stopa J Rybarska A Drozd et al ldquoAlbumin binds self-assembling dyes as specific polymolecular ligandsrdquo Interna-tional Journal of Biological Macromolecules vol 40 no 1 pp1ndash8 2006

[35] X F Xu H P Guo D Gong et al ldquoDecellularized porcine pul-monary arteries cross-linked by carbodiimiderdquo InternationalJournal of Clinical and Experimental Medicine vol 6 no 7 pp524ndash531 2013

[36] B Srinivasan A R Kolli M B Esch H E Abaci M L Shulerand J J Hickman ldquoTEER measurement techniques for in vitrobarrier model systemsrdquo Journal of Laboratory Automation vol20 no 2 pp 107ndash126 2015

[37] S M Armstrong V Khajoee C Wang et al ldquoCo-regulationof transcellular and paracellular leak across microvascularendothelium by dynamin and RacrdquoAmerican Journal of Pathol-ogy vol 180 no 3 pp 1308ndash1323 2012

[38] K Okkenhaug and B Vanhaesebroeck ldquoNew responsibilitiesfor the PI3K regulatory subunit p85 alphardquo Sciencersquos STKE no65 article pe1 2001

[39] M Gustems A Woellmer U Rothbauer et al ldquoC-Junc-Fosheterodimers regulate cellular genes via a newly identified classof methylated DNA sequence motifsrdquo Nucleic Acids Researchvol 42 no 5 pp 3059ndash3072 2014

[40] A Pfannkuche K Buther J Karthe et al ldquoC-Src is requiredfor complex formation between the hepatitis C virus-encodedproteins NS5A and NS5B a prerequisite for replicationrdquo Hepa-tology vol 53 no 4 pp 1127ndash1136 2011

[41] S E Gill R Taneja M Rohan L Wang and S Mehta ldquoPul-monary microvascular albumin leak is associated with endo-thelial cell death in murine sepsis-induced lung injury in vivordquoPLoS ONE vol 9 no 2 Article ID e88501 2014

[42] I Klaassen C J F Van Noorden and R O SchlingemannldquoMolecular basis of the inner blood-retinal barrier and itsbreakdown in diabetic macular edema and other pathologicalconditionsrdquo Progress in Retinal and Eye Research vol 34 pp 19ndash48 2013

[43] Z-Y Zou Y-R Hu H Ma et al ldquoCoptisine attenuates obesity-related inflammation through LPSTLR-4-mediated signalingpathway in Syrian golden hamstersrdquo Fitoterapia vol 105 pp139ndash146 2015

[44] Y Xiong M Murphy T T Manavalan et al ldquoEndotoxin tol-erance inhibits Lyn and c-Src phosphorylation and association

with toll-like receptor 4 but increases expression and activity ofprotein phosphatasesrdquo Journal of Innate Immunity vol 8 no 2pp 171ndash184 2016

[45] J P Luyendyk G A Schabbauer M Tencati T Holscher RPawlinski and N Mackman ldquoGenetic analysis of the role ofthe PI3K-Akt pathway in lipopolysaccharide-induced cytokineand tissue factor gene expression in monocytesmacrophagesrdquoJournal of Immunology vol 180 no 6 pp 4218ndash4226 2008

[46] J-Y Kim H M Oh S C Kwak et al ldquoPurslane suppressesosteoclast differentiation and bone resorbing activity via inhi-bition of AktGSK3120573-c-Fos-NFATc1 signaling in vitro and pre-vents lipopolysaccharide-induced bone loss in vivordquo Biologicaland Pharmaceutical Bulletin vol 38 no 1 pp 66ndash74 2015

[47] Y Xiong M Murphy T T Manavalan et al ldquoEndotoxin tol-erance inhibits Lyn and c-Src phosphorylation and associationwith toll-like receptor 4 but increases expression and activity ofprotein phosphatasesrdquo Journal of Innate Immunity vol 8 no 22016

[48] G Xue and B A Hemmings ldquoPKBAkt-dependent regulationof cell motilityrdquo Journal of the National Cancer Institute vol 105no 6 pp 393ndash404 2013

[49] X Cao H Lin L Muskhelishvili J Latendresse P Richter andR H Heflich ldquoTight junction disruption by cadmium in an invitro human airway tissue modelrdquo Respiratory Research vol 16article 30 2015

[50] M Zarzycka K Chojnacka D D Mruk et al ldquoFlutamide altersthe distribution of c-Src and affects the N-cadherin-120573-catenincomplex in the seminiferous epithelium of adult ratrdquoAndrologyvol 3 no 3 pp 569ndash581 2015

[51] A Kourtidis S P Ngok P Pulimeno et al ldquoDistinct E-cadherin-based complexes regulate cell behaviour throughmiRNA processing or Src and p120 catenin activityrdquoNature CellBiology vol 17 no 9 pp 1145ndash1157 2015

[52] A Du Y Wang S Zhao et al ldquoThe relationship between c-fosgene and filamentous actin cytoskeleton in MG-63 osteoblastsunder cyclic tensile stressrdquoHua xi Kou Qiang yi Xue za Zhi vol30 no 4 pp 430ndash438 2012 (Chinese)

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of ALI Additionally albumin with high molecular weightwould leak across capillary endothelial barrier mainly depen-dent on the paracellular leakage [7] Therefore the enhance-ment to systemic and pulmonary albumin leak depravationof airways indicators respiratory dysfunction and also theadverse change in lung tissue pathology could be consideredas the important features of pulmonary capillary endothelialbarrier injury [8ndash10] Moreover the reversal of the enhancedpulmonary albumin leak should be considered as the thera-peutic goal for ALI caused by endotoxemia in SAP

CQCQD decoction a famous Chinese medicinal for-mula has been used to treat SAP for decades in WestChina Hospital Sichuan University [11] According to theregulations on drug development and approval of SFDAbefore the Chinese medicinal formula is widely used inclinical practice there are four main principles required tobe met by the Chinese medicinal formula (1) action targetarea (2) highly efficient concentration (3) efficacy in animalmodel (4) multicentral random clinical trial The growingstudies evidences of CQCQD indicated that the first threeprinciples mentioned above have been met which will beshown as follows (i) action target area (1) pancreatic tissues(2) gastrointestinal tract (3) lung (4) immune system (5)pancreatic acinar cells (6) alveolar macrophages and (7)peritoneal macrophages [12ndash25] which suggested that thelungs are the important action target area for CQCQD(ii) Highly efficient concentration in clinical practice is asfollows CQCQD consists of Bupleuri Radix (Chai Hu) 15 gScutellariae Radix (Huang Qin) 15 g Magnoliae OfficinalisCortex (Hou Pu) 15 g Aurantii Fructus Immaturus (Zhi Shi)15 g Artemisiae Scopariae Herba (Yin Chen) 15 g GardeniaeFructus (Zhi Zi) 20 g Rhei Radix et Rhizoma (Da Huang)20 g and Natrii Sulfas (Mang Xiao) 20 g [12] according tothe standardized preparation procedure of CQCQD formulathese eight herbs will be decocted to form this decoction [13]since this decoction has been used to treat SAP for decadesand the effective pharmacotherapy to the patients has beencertified with ALI caused by endotoxemia in SAP [14] if thisdecoction is prepared strictly according to CQCQD formulaand the standardized preparation procedure the final con-centration of the prepared decoction can represent the highlyefficient concentrations of CQCQD in clinical application[15] (iii) Efficacy in SAP animal model is as follows (1)effectively ameliorating pancreatic histopathological changesand systemic inflammatory response [16ndash23] (2) effectivelyimproving gastrointestinal motility dysfunction [23] (3)effectively ameliorating alveolar inflammatory response [24](4) effectively alleviating ALI [25] which suggested that thetherapy of CQCQD could effectively alleviate the ALI causedby endotoxemia in SAP animal model Meanwhile accordingto the 4th principle multicentral random clinical trials arebeing conducted in a number of hospitals in China includingWest China Hospital of Sichuan University Chengdu No 1Hospital and Sichuan Hospital of TCM Although the multi-central randomclinical trials have not been completed a largenumber of clinical studies have provided the key evidencesthat (1) the therapy of CQCQD significantly decreased serumlevels of interleukin-6 (IL-6) and IL-1120573 receptor antagonist(IL-1ra) in SAP patients and also the hospitalization time of

SAP patients [12] (2) the therapy of CQCQD could decreasethe serum levels of matrix metalloproteinase-9 (MMP-9)C-reactive protein (CRP) and amyloid A (SAA) to relievethe durations of acute respiratory distress syndrome (ARDS)in patients with SAP [13 14] and the early application ofCQCQD is more effective than late application to shortenthe duration of ARDS [15] which suggested that the therapyof CQCQD could probably effectively alleviate ALI in thepatients with SAP Furthermore there are growing evidencesfor the monomers isolated from the herbs of CQCQDdecoction that (1) saikosaponin A baicalin and chlorogenicacid could inhibit some proinflammatory signal pathwayto ameliorate inflammatory cascade reaction [26ndash28] (2)saikosaponin C and baicalein could inhibit LPS-inducedapoptosis in human umbilical vein endothelial cells [2930] (3) wogonin could inhibit LPS-induced vascular hyper-permeability via inhibiting cytoskeleton reorganization [31]Consequently we speculated that the treatment of CQCQDdecoction in the patients with SAP endotoxemia-inducedALI could potentially ameliorate pulmonary albumin leak viadepressing the inflammatory cascade reaction and alleviatingthe damage to cell-cell junction However the potentialmolecular mechanisms have not been systematically studied

In this study microarray and Western blot and so forthanalyses were employed to reveal the key molecular mech-anisms for CQCQD decoction in alleviating the pulmonaryalbumin leakage caused by endotoxemia in SAP rats

2 Materials and Methods

21 Animals and Cell Culture Adult Sprague-Dawley rats(250ndash300 g) were purchased from the Laboratory AnimalCentre of Huaxi Sichuan University (Chengdu China) Ani-mal experiments performed in this study were approved bythe Institutional Animal Care and Use Committee of SichuanUniversity and the corresponding license number was SCXK(Sichuan) 2014sim09 Human umbilical vein endothelial cells(HUVECs) were obtained from Molecular Biology Lab ofSichuan University (Chengdu China) HUVECs were cul-tured in DMEM with 10 fetal bovine serum (FBS) 100UmL penicillin and 100mgmL streptomycin (Sigma-AldrichMO USA) at 37∘C with 5 CO

2

22 Preparation of CQCQD and Rat CQCQD-ContainingSerum and Control Serum The Chinese drug decoctionpieces in the CQCQD formula (Table 1) were acquiredaccording to the prescribed proportions (ww) through thePharmaceutical Preparation Section West Chinese Hospitalof Sichuan University Chengdu China Each botanical nameof these Chinese drug decoction pieces (Table 1) has beenchecked with httpwwwtheplantlistorg mentioning thedata of accessing that website According to the macroscopicand microscopic means mentioned in Chinese pharma-copoeia the botanical authentication of each Chinese drugdecoction piece has been performed by their manufacturersIn addition the chemical characterization of major activeingredients of these Chinese drug decoction pieces (showedin Figure 1 for Rhei Radix et Rhizoma as an example) wasidentified by HPLC-MS analyses adopting one or more


Table1Com

ponentso

fChinese

drug

decoctionpieces

intheC

QCQ

Dform

ulaa

ndtheirp

rodu

ctionlotn

umbersandther

elativec

ontentso

fmajor

activ

eing

redients

Pharmaceuticalnamea

ndCh

inesep

honetic

name

Botanicaln

amea

ndplantp

art

Com

position(

)Lo

tnum

ber

Quantitativ

eanalysis

ofmajor

activ

eing

redients

Referencem

arkers(H

PLC)

Relativec

ontent

(ww

)

BupleuriRa

dix(C

haiH

u)Bu

pleurum


t11111

2014100802

Saikosapon

ina

0383ndash0421

Saikosapon

ind

0381ndash0398

Scutellaria

eRadix(H

uang

Qin)

Scutellariabaica

lensis


11111

2014081003

Baicalin

1233

8ndash1432

1

RheiRa

dixetRh

izom

a(DaH

uang

)Rh

eum

palm

atum

L(Polygon

aceae)

14814

2014080301

Aloe-em

odin

0348ndash

0367

Rhein

1002ndash116

2Em

odin

0513ndash0628

Chrysoph

anol

0661ndash0702

Physcion

0268ndash

0301

Natrii

Sulfas(MangXiao)

Mira


l14814

2014030506

Thenardite

99812ndash9

9993

Magno

liaeO

fficinalis

Cortex(H

ouPu

)Magnolia

officin

alisRe

hder

ampE

HW

ilson

(Magno


mbark

11111

201406

0501

Magno

lol

2118

ndash2491

Hon

okiol

2181ndash2228


(Zhi

Shi)

Citru

saurantiu


11111

2014053101

Syneph

rine

0612ndash064

1Hesperid

in12

31ndash1445

Artem

isiae

Scop

ariaeH

erba

(Yin

Chen)


teraceae)

aeria

lpart

11111

20140306

01Ch

lorogenica

cid

0598ndash

0693

Escoparone

0598ndash

06341

Gardeniae

Fructus(Zh

iZi)

Gardeniajasm

inoides

JEllis

(Rub

iaceae)rip

efruit

14814

2014110

301

Geniposide

5989ndash

6959


minus50

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35

(mAU

)

(min)


1

2

3

4

5

Positive ion mode












2 PaCO

2 and SpO















(a)

N_H

001

N_H

002

N_H

003

S_H

004

S_H

005

S_H

006

C_H

007

C_H

008

C_H

009

Low High

Fos

CLASS

Pik3r1

Src

(b)



3 Results
























PaCO2


PaO2


SaO2








2in SAP group




SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Com

ponentso

fChinese

drug

decoctionpieces

intheC

QCQ

Dform

ulaa

ndtheirp

rodu

ctionlotn

umbersandther

elativec

ontentso

fmajor

activ

eing

redients

Pharmaceuticalnamea

ndCh

inesep

honetic

name

Botanicaln

amea

ndplantp

art

Com

position(

)Lo

tnum

ber

Quantitativ

eanalysis

ofmajor

activ

eing

redients

Referencem

arkers(H

PLC)

Relativec

ontent

(ww

)

BupleuriRa

dix(C

haiH

u)Bu

pleurum


t11111

2014100802

Saikosapon

ina

0383ndash0421

Saikosapon

ind

0381ndash0398

Scutellaria

eRadix(H

uang

Qin)

Scutellariabaica

lensis


11111

2014081003

Baicalin

1233

8ndash1432

1

RheiRa

dixetRh

izom

a(DaH

uang

)Rh

eum

palm

atum

L(Polygon

aceae)

14814

2014080301

Aloe-em

odin

0348ndash

0367

Rhein

1002ndash116

2Em

odin

0513ndash0628

Chrysoph

anol

0661ndash0702

Physcion

0268ndash

0301

Natrii

Sulfas(MangXiao)

Mira


l14814

2014030506

Thenardite

99812ndash9

9993

Magno

liaeO

fficinalis

Cortex(H

ouPu

)Magnolia

officin

alisRe

hder

ampE

HW

ilson

(Magno


mbark

11111

201406

0501

Magno

lol

2118

ndash2491

Hon

okiol

2181ndash2228


(Zhi

Shi)

Citru

saurantiu


11111

2014053101

Syneph

rine

0612ndash064

1Hesperid

in12

31ndash1445

Artem

isiae

Scop

ariaeH

erba

(Yin

Chen)


teraceae)

aeria

lpart

11111

20140306

01Ch

lorogenica

cid

0598ndash

0693

Escoparone

0598ndash

06341

Gardeniae

Fructus(Zh

iZi)

Gardeniajasm

inoides

JEllis

(Rub

iaceae)rip

efruit

14814

2014110

301

Geniposide

5989ndash

6959


minus50

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35

(mAU

)

(min)


1

2

3

4

5

Positive ion mode












2 PaCO

2 and SpO















(a)

N_H

001

N_H

002

N_H

003

S_H

004

S_H

005

S_H

006

C_H

007

C_H

008

C_H

009

Low High

Fos

CLASS

Pik3r1

Src

(b)



3 Results
























PaCO2


PaO2


SaO2








2in SAP group




SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus50

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35

(mAU

)

(min)


1

2

3

4

5

Positive ion mode












2 PaCO

2 and SpO















(a)

N_H

001

N_H

002

N_H

003

S_H

004

S_H

005

S_H

006

C_H

007

C_H

008

C_H

009

Low High

Fos

CLASS

Pik3r1

Src

(b)



3 Results
























PaCO2


PaO2


SaO2








2in SAP group




SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2 PaCO

2 and SpO















(a)

N_H

001

N_H

002

N_H

003

S_H

004

S_H

005

S_H

006

C_H

007

C_H

008

C_H

009

Low High

Fos

CLASS

Pik3r1

Src

(b)



3 Results
























PaCO2


PaO2


SaO2








2in SAP group




SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(a)

N_H

001

N_H

002

N_H

003

S_H

004

S_H

005

S_H

006

C_H

007

C_H

008

C_H

009

Low High

Fos

CLASS

Pik3r1

Src

(b)



3 Results
























PaCO2


PaO2


SaO2








2in SAP group




SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































PaCO2


PaO2


SaO2








2in SAP group




SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SO SAP CQCQD

(a)

SO SAP CQCQD

(b)













minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

c-Src

p-Src

120573-actin 0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

c-Srcp-c-Src

998771998771


LPS (1mgL)


LPS (1mgL)

(a)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

p85120572

p-p85120572

p85120572p-p85120572

120573-actin0

1

2

3

Relat

ive e

xpre

ssio

n

998771998771

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)

minusminusminus

+minusminus

++minus

+minus+

minusminusminus

+minusminus

++minus

+minus+

siRNAs (c-Fos)

c-Fos

GAPDH0

2

4

6

Relat

ive e

xpre

ssio

n

c-Fos

998771998771



LPS (1mgL)

(c)








4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Discussion





0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

100

1

1000


TEERDextran flux

minusminusminus

+minusminus

++minus

+minus+


LPS (1mgL)

p-Src expression

(a)

0

10

1

1000

100



minusminusminus

+minusminus

++minus

+minus+

LY294002 (30120583M)CQCQD (1mgL)

LPS (1mgL)

(b)



0

1

10

100

1000

minusminusminus

+minusminus

++minus


CQCQDLPS

(c)



++minusminusminus

+minus+minusminus

+minusminus+minus

+minusminusminus+

siRNAs (c-Fos)


⋄

⋄

⋄


PPI (10120583M)LY294002 (30120583M)

TEERDextran flux

0

1

10

100

1000

(d)










Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Competing Interests




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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