research article baicalin attenuates hypoxia-induced...

Research ArticleBaicalin Attenuates Hypoxia-Induced Pulmonary ArterialHypertension to Improve Hypoxic Cor Pulmonale by Reducingthe Activity of the p38 MAPK Signaling Pathway and MMP-9

Shuangquan Yan1 Yiran Wang2 Panpan Liu3 Ali Chen4 Mayun Chen4 Dan Yao4

Xiaomei Xu4 Liangxing Wang4 and Xiaoying Huang4

1Division of Respiratory Medicine Taizhou Enze Medical Center Enze Hospital Taizhou Zhejiang 318000 China2Division of Equipment The First Affiliated Hospital of Wenzhou Medical University Wenzhou Zhejiang 325035 China3Division of Intensive Care Unit Ningbo Medical Center Lihuili Eastern Hospital Ningbo Zhejiang 315040 China4Division of PulmonaryMedicineThe First AffiliatedHospital ofWenzhouMedical University and Key Laboratory of Heart and LungWenzhou Zhejiang 325035 China

Correspondence should be addressed to Xiaoying Huang zjwzhxy126com

Received 29 April 2016 Revised 7 August 2016 Accepted 11 August 2016

Academic Editor Ki-Wan Oh

Copyright copy 2016 Shuangquan Yan et al 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

Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats but the mechanism of this effect remainsunclear Thus investigating the potential mechanism of this effect was the aim of the present study Model rats that display hypoxicpulmonary hypertension and cor pulmonale under control conditions were successfully generated We measured a series of indi-cators to observe the levels of pulmonary arterial hypertension pulmonary arteriole remodeling and right ventricular remodelingWe assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonarytissue homogenates using immunohistochemistry and western blot analyses respectively The matrix metalloproteinase- (MMP-)9 protein andmRNA levels in the pulmonary arteriole walls weremeasured using immunohistochemistry and in situ hybridizationOur results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissuehomogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls Thisdownregulation was accompanied by the attenuation of pulmonary hypertension arteriole remodeling and right ventricularremodeling These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale which are inducedby chronic hypoxia by downregulating the p38 MAPKMMP-9 pathway

1 Introduction

Pulmonary arterial hypertension (PAH) is characterized bypulmonary vasoconstriction and lung circulation remodel-ing which can gradually elevate pulmonary vascular resis-tance leading to right ventricular hypertrophy dilatationand dysfunction Chronic hypoxic exposure can inducePAH eventually leading to right ventricular hypertrophyand failure Pulmonary arteriole remodeling which includessmooth muscle cell proliferation extracellular matrix (ECM)turnover and collagen fiber accumulation is the key stepin this process [1] Matrix metalloproteinase- (MMP-) 9 can

participate in ECM turnover fibrosis and chronic inflam-mation and MMP-9 promotes the proliferation of smoothmuscle cells in blood vessels and their migration into thevessel wall [2 3] The same process occurs in the smallpulmonary artery

As a member of the mitogen-activated protein kinase(MAPK) family p38MAPK can be activated by the phospho-rylation of its subunits and this activation plays an importantrole in inflammation and cell differentiation and proliferationin arteries [4 5]

Enhanced p38 MAPK activation can upregulate thelevel of MMP-9 by promoting MMP-9 mRNA transcription

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 2546402 9 pageshttpdxdoiorg10115520162546402

2 Evidence-Based Complementary and Alternative Medicine

levels which then leads to a series of biological effects[6]

Baicalin is a flavonoid compound purified from the dryroots of Scutellaria baicalensis and it possesses a varietyof pharmacological activities such as antioxidative anti-inflammatory antitumor and antiapoptotic activities it alsoinhibits smooth muscle cell proliferation [7ndash9] We previ-ously found that baicalin attenuates small pulmonary arteryremodeling in vivo in rats with hypertension [10] but themechanism is complicated and not fully understood Severalstudies have shown that baicalin downregulates the expres-sion of p38MAPK andMMP-9 Baicalin not only inhibits theexpression of p38 MAPK and MMP-9 in new retinal vesselsin amousemodel of oxygen-induced retinopathy [11] but alsoblocks the activation of p38 MAPK which leads to reducedMMP-9 levels and tumorigenicity in MDA-MB-231 cells[12] Thus we speculate that baicalin attenuates pulmonaryarteriole remodeling and hypoxic pulmonary hypertensionfurther improving cor pulmonale by inhibiting the activity ofp38 MAPK and downregulating the expression of MMP-9 inarterioles

2 Materials and Methods

21 Animals Twenty-four male specific-pathogen-free Spra-gue-Dawley rats weighing 220ndash250 g provided by the Labo-ratory Animal Center of Wenzhou Medical University wereused in the present study All rats were housed in controlledconditions and fed a standard diet The animal experimentswere approved by the Laboratory Animal Ethics Committeeof Wenzhou Medical College and performed in strict accor-dance with the guidelines for animal ethics

22 Drugs and Antibodies Baicalin (CAS number 21967-41-9 Product number 572667 purity 95) was purchasedfrom Sigma-Aldrich Co Ltd (USA) A 2 baicalin solutionwas prepared by mixing 1 g of baicalin into 48mL of sterilesaline and titrating the solution under stirring with sodiumhydroxide until reaching a pH of 68ndash70 After the baicalinwas completely dissolved the clear yellow-green solutionwasbrought up to a final volume of 50mL with sterile salineand the final concentration was 20mgmL Then aliquotsof the solution were prepared and stored at minus20∘C Priorto use these aliquots were gradually thawed at 4∘C Therabbit anti-rat p-p38 MAPK polyclonal antibody (4631) wasobtained from Cell Signaling Technology (USA) The sec-ondary horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody and the secondary rabbit anti-goat antibodywere purchased from ZSGB Biotechnology Co Ltd (BeijingChina) The goat anti-rat MMP-9 antibody (sc-6840) wasobtained from Santa Cruz (USA)TheMMP-9 mRNA in situhybridization (ISH) kit (MK1540) was purchased fromBosterBiotechnology Co Ltd (Wuhan China) and the rabbit anti-rat GAPDH antibody was obtained from Goodhere (China)

23 Animal Grouping and Administration The rats wererandomized into the following three groups with eight ratsper group the control group the hypoxia group and thebaicalin + hypoxia groupThe rats in the hypoxia group were

kept in a normobaric hypoxic chamber for 4 weeks with anO2concentration of 8ndash10 and a CO

2concentration that was

below 5 for 6 days per week and 8 hours per dayThe rats inthe baicalin + hypoxia group were intraperitoneally injectedwith 2 baicalin (30mgkg) 30min before being placed intothe normobaric hypoxic chamber The rats in the controlgroup were kept under normobaric and normoxic condi-tions

24 Mean Pulmonary Artery Pressure (mPAP) and Mean Sys-temic Artery Pressure (mSAP) Measurements After 4 weeksthe rats were weighed and anesthetized by an intraperitonealinjection of 5 chloral hydrate (40mgkg of body weight)The specific methods used have been previously reported byour team A small skin incision was made in the neck of eachmouse Then the right external jugular vein was isolatedand a polyethylene catheter (inner diameter 09mm outerdiameter 11mm) with a slightly curved front was graduallyinserted into the right atrium and then the right ventricle(RV) After the recorder entered the RV the large vibrationsof the ventricular waveform could be documented Then thetube was further inserted by approximately 05 cm After theheart rate became normal and the waveform stabilized thetube was rotated clockwise to the lower part of the marksuch that the tube head was facing upward thereby enablingit to move forward 1-2 cm and enter the pulmonary arteryAt this point the effects of breathing on the pulmonaryartery waveform were visible After moving the tube forwardan additional 05ndash1 cm stable pulmonary artery waveformscould be recorded and the mPAP was recorded with apiezoelectric pressure transducer and the PowerLab system(AD Instruments Australia)

25 Pulmonary Arteriole RemodelingMeasurement Lung tis-sue sections were subjected to hematoxylin and eosin (HampE)staining following standard histological procedures For eachsection five vessels near the pulmonary alveoli with diame-ters of approximately 50ndash200120583mwere randomly selected formeasurement First the pulmonary arteriole wall area andtotal area (microscope magnification 40 times 10 = 400x) weremeasured to calculate the ratio of vessel wall area to total area(WATA) Second each arteriole wall thickness (WT) andradius (R) weremeasured at 0∘ 90∘ 180∘ and 270∘ by drawinga line across the vessel and its wall The WT and R valuesacquired from the four angles were then averaged and usedto calculate the WTR ratio Finally the number of nuclei inthe arteriole wall was counted and used to calculate both theratio of the number of nuclei to the vesselWA and the nucleardensity of the wall All measurements were performed usingImage-Pro Plus software (Media Cybernetics Bethesda MDUSA)

26 Right Ventricular Hypertrophy Measurements The leftventricle plus the interventricular septum (LV + S) and theRV were first collected by cutting along the edge of theRV and the interventricular septum then these sampleswere weighed The mass ratios of the RV to the LV + Sand rat body weight (BW) expressed as RV(LV + S) and

Evidence-Based Complementary and Alternative Medicine 3

RVBW respectively were used to reflect the degree of rightventricular hypertrophy Each RV was then immediatelyplaced in 4 formalin where it was kept for 48 hours beforebeing embedded in a paraffin block Subsequently 3 120583m thicksections were prepared and stained with HampE to identify themyocardial tissue

27 Immunohistochemical Analysis of p38 MAPK Phos-phorylation The sections were deparaffinized dehydratedimmunostained with the primary rabbit anti-rat p-p38MAPK polyclonal antibody (1 25) and then incubated inthe secondary HRP-conjugated goat anti-rabbit antibody(1 10000) Negative controls were incubated in phosphate-buffered saline (PBS) in place of the specific primary anti-body All antibodies used for immunohistochemistry werediluted in PBS Immunoreactivity was visualized using 331015840-diaminobenzidine (DAB) and immunohistological imageswere obtained using an intelligent biological microscope(Leica DM1000 Germany) Five fields of viewwere randomlyselected from each section for quantitative analyses Theintegrated optical density (IOD) of the positive products inthe pulmonary arteriole and the arteriole area were bothmeasured using Image-Pro Plus software then the ratio ofthe IOD to the arteriole area was calculated to reflect theexpression of positive products

28 Immunohistochemical Analysis of MMP-9 ExpressionAfter being blocked the sections were incubated with theprimary goat anti-rat MMP-9 antibody (1 50) and then withthe secondary rabbit anti-goat antibody (1 10000) Negativecontrols were prepared by incubating sections in PBS insteadof the specific primary antibody Immunoreactivity wasvisualized using DAB All antibodies used for immunohis-tochemistry were diluted in PBS and five fields of view wererandomly selected from each section for quantitative analysesby Image-Pro Plus software

29 ISH for MMP-9 mRNA Measurements Paraffin-embed-ded sections were deparaffinized and dehydrated through agraded ethanol series and they were then processed usinga commercially available MMP-9 mRNA ISH kit (MK1540)in accordance with the manufacturerrsquos instructions Thesections were then subjected to a chromogenic reagent Thereaction was visually monitored and stopped by placingthe slides in water The slides were counterstained with ared fixed-nucleus reagent to visualize the nuclei then theslides were washed with water dehydrated vitrified withdimethylbenzene mounted in neutral balata and coveredwith coverslips Negative controls were prepared by beinghybridized without probes in parallel with the experimentalreactions Images were captured and saved using an intel-ligent biological microscope and five fields of view wererandomly selected from each section for quantitative analysisby Image-Pro Plus software

210 Western Blot Analysis of p38 MAPK PhosphorylationThe protein concentration of the fresh lung tissue wasmeasured using the Bradford method equal amounts of

protein were separated by electrophoresis and transferredonto a polyvinylidene fluoride membrane (Millipore MA)Nonspecific antibody binding was blocked by incubatingthe membrane with 5 bovine serum albumin in Tris-buffered saline with Tween-20 (TBST) for 1 h Subsequentlythe membrane was incubated overnight at 4∘C with specificrabbit anti-rat p-p38 MAPK polyclonal (1 1000) and rabbitanti-rat GAPDH antibodies (1 1000) After it was washedwith fresh TBST the membrane was incubated with HRP-conjugated affinity-purified anti-rabbit IgG (01 120583gmL)Immunoreactive bands were visualized using an enhancedchemiluminescence (ECL) kit (Pierce USA) and an ECLImaging System (Bio-Rad USA)The signal intensities of thecorresponding bands were quantified using Image-Pro Plussoftware

211 Statistical Analysis All values were tested for normaldistribution and are expressed as the mean plusmn SD The datawere analyzed by one-way analysis of variance followedby a least significant difference test 119875 values lt 005 wereconsidered significant

3 Results

31 Baicalin Decreased the mPAP in Rats with HypoxicPulmonary Hypertension The mSAP values of the controlgroup and the hypoxia group were 12235 plusmn 2115 and11340 plusmn 2986mmHg respectively and the mean value afterthe baicalin treatment was 10903 plusmn 1873mmHg Howeverthere were no significant differences among the three groups(Figures 1(b) and 1(d)) The mPAP was significantly higherin the hypoxia group than in the control group (2512 plusmn074mmHg versus 1694 plusmn 107mmHg 119875 lt 001) and thebaicalin treatment remarkably reduced the mPAP to 1750 plusmn148mmHg (119875 lt 001) (Figures 1(a) and 1(c))

32 Baicalin Attenuated the Pulmonary Arteriole and RightVentricular Remodeling As shown in Figures 2(a) and 2(b)the WATA of the hypoxia group was greater than that ofthe control group (7775 plusmn 679 versus 6100 plusmn 686119875 lt 001) and baicalin remarkably decreased the WATAto 6772 plusmn 676 (119875 lt 005) Similarly the WTR in thecontrol group was 4861 plusmn 1092 but was 6321 plusmn 819in the hypoxia group (119875 lt 001) baicalin attenuated thiseffect as demonstrated by the reducedWTR of 5221plusmn786(119875 lt 005) In contrast the vessel wall nuclear density inthe control group was 00127 plusmn 00006120583m2 but was 00077 plusmn00004120583m2 in the hypoxia group (119875 lt 001) baicalin led toan increased nuclear density of 00108 plusmn 00017120583m2 (119875 lt001) (Figure 2(c)) Hypoxic pulmonary arteriole remodelingwas remarkably inhibited by baicalin as visualized by HampEstaining (Figure 2(d)) The RVLV + S was 266 plusmn 08 inthe control group and 342 plusmn 24 (119875 lt 001) in thehypoxia group but this parameter was considerably reducedto 314 plusmn 27 (versus the hypoxia group 119875 lt 005) after thebaicalin treatment (Figure 3(a)) As indicated in Figure 3(b)chronic hypoxia increased the RVBW from 0071 plusmn 0008in the control group to 0107 plusmn 0019 (119875 lt 001) and


Hypoxia Hypoxia + baicalinControl

30

20

10

0

mPA

P (m

mH

g)

lowastlowast

(a)


200

150

100

50

0

mSA

P (m

mH

g)

(b)

PAP

wav

efor

m

Control Hypoxia Hypoxia + baicalin40

30

20

10

40

30

20

10

40

30

20

10

(c)

SAP

wav

efor

m


120

100

80

140

120

100

80

140

120

100

80

(d)

Figure 1 Effect of baicalin on pulmonary artery pressure in rats subjected to chronic hypoxia (a c) The mPAP was significantly increasedin the hypoxia group but was decreased by baicalin (119899 = 8group) indicates 119875 lt 001 compared to the control group and lowastlowast indicates119875 lt 001 compared to the hypoxia group (b d) No significant difference existed among the three groups

baicalin decreased the RVBW to 0087 plusmn 0009 (119875 lt 001)Myocardial hypertrophywas present in the hypoxia group butwas ameliorated by baicalin (Figure 3(c))

33 Baicalin Downregulated p38 MAPK Activity Levels ofthe main active product of p38 MAPK that is p-p38MAPK were detected in the arteriole walls and lung tissuehomogenates by immunohistochemistry and western blotanalysis respectively In the hypoxia group p-p38MAPKwassignificantly increased in the arteriole walls as indicated bythe increase in OD from 0122 plusmn 0008 to 0143 plusmn 0016 (119875 lt001) The administration of baicalin significantly decreasedthis value to 0127 plusmn 0013 (119875 lt 005) (Figures 4(a) and4(b)) The analysis of p-p38 MAPK expression in the lungtissue homogenates showed the same trend Compared with

the control group the hypoxia group showed increased p-p38 MAPK expression (03213 plusmn 00371 vs 07474 plusmn 01130119875 lt 001) and baicalin significantly decreased this value to04518 plusmn 01316 (119875 lt 001) (Figures 4(c) and 4(d))

34 Baicalin Downregulated MMP-9 Protein and mRNAExpression Immunohistochemistry was used to detectMMP-9 protein expression in paraffin-embedded sectionsChronic hypoxia increased theODvalue from 0188plusmn0019 inthe control group to 0223plusmn0010 (119875 lt 001) and the baicalintreatment decreased this value to 0196 plusmn 0016 (119875 lt 005)(Figures 5(a) and 5(c)) MMP-9 mRNA expression wasdetected by ISH The rat arteriole MMP-9 mRNA ODvalue was significantly elevated from 02073 plusmn 00413 to02689 plusmn 00482 (119875 lt 001) after exposure to hypoxia for


Hypoxia Hypoxia +baicalin

Control

WA

TA

()

lowast

100

80

60

40

20

0

(a)


Control

WT

R (

)

lowast

80

60

40

20

0

(b)


Control

lowastlowast

0015

0010

0005

0000

Nuc

leus

num

ber120583m

2

(c)


25 120583m25 120583m25 120583m

(d)

Figure 2 Effect of baicalin on pulmonary arteriole remodeling in rats subjected to chronic hypoxia (a b) The WATA and WTR ratioswere significantly increased in rats subjected to continuous hypoxia but baicalin attenuated this trend (119899 = 8group) indicates 119875 lt 001compared to the control group and lowast indicates 119875 lt 005 compared to the hypoxia group (c)The nuclei density significantly decreased undercontinuous hypoxia and the trend was partially attenuated by baicalin (119899 = 8group) indicates 119875 lt 001 compared to the control groupand lowastlowast indicates 119875 lt 001 compared to the hypoxia group (d) Baicalin inhibited hypoxic pulmonary arteriole remodeling as indicated byHE staining

4 weeks and this increase was significantly inhibited by thebaicalin treatment as indicated by the OD value reductionto 02206 plusmn 00423 (119875 lt 005) (Figures 5(b) and 5(d))

4 Discussion

Hypoxic pulmonary hypertension is a disease characterizedby continuous increases in pulmonary vascular resistance andPAP Pulmonary arteriole remodeling caused by a continuousstate of hypoxia is the pathological basis of PAH and theremodeling mechanism is complicated ECM accumulationfibroblast proliferation and myofibroblast transformationas well as smooth muscle cell proliferation migration andphenotypic differentiation play key roles in this remodelingprocess [13]

Baicalin a notable extract from Scutellaria baicalensis hasgained the attention of many scholars due to its antioxidativeanti-inflammatory antitumor antiapoptotic antiprolifera-tive and antifibrotic activities In previous studies we haveshown that baicalin attenuates hypoxia-induced pulmonaryarteriole remodeling However this mechanism has yet to befully elucidated

The vascular ECM plays an important role in the forma-tion of pulmonary arteries and contributes to the structuraland functional integrity of the vasculature by providing

the vessels with mechanical strength elasticity and com-pressibility The ECM provides a microenvironment for cell-cell and cell-ECM communication to regulate vascular cellproliferation migration and differentiation and to sustainvascular homeostasis As such maintaining the compositionof the ECM is important The ECM mainly includes elastincollagens proteoglycans structural glycoproteins and adhe-sive glycoproteins and the turnover of these componentscan cause an imbalance that eventually leads to vascularremodeling [14 15] MMP-9 is a member of the MMPfamily that plays a critical role in maintaining the balanceof the ECM A study using a hypoxia-induced pulmonaryhypertension model in Sprague-Dawley rats showed that theenhanced expression of MMP-9 disturbed this balance andled to pulmonary remodeling [16]

As a response to hypoxic stimulation pulmonary arterysmooth muscle cells (PASMCs) proliferate and synthesizematrix material which regulates the phenotype and prolif-eration of vascular smooth muscle cells (VSMCs) leadingto increased vessel WT and abnormal muscularization ofthe normally nonmuscular distal pulmonary arteries VSMCmigration begins with lesion establishment and persiststhroughout the process of vascular remodeling In additionVSMC migration plays a significant role in neointima for-mation and the muscularization of distal pulmonary arteries



RV(

LV+S

) (

)

lowast

40

30

20

10

0

(a)


RVB

W (

)

lowastlowast

015

010

005

000

(b)


Myc

ardi

a HE

stai

ning

400x 400x400x

(c)

Figure 3 Effect of baicalin on myocardial remodeling in rats subjected to chronic hypoxia (a b) The RV(LV + S) and RVBW ratios weresignificantly increased in the hypoxia group but baicalin attenuated this trend (119899 = 8group) indicates 119875 lt 001 compared to the controlgrouplowast indicates119875 lt 005 compared to the hypoxia group andlowastlowast indicates119875 lt 001 compared to the hypoxia group (c)HE staining showedthat chronic hypoxia increased right ventricular myocardial hypertrophy and cardiac muscle disorder but that baicalin partially attenuatedthese effects

PASMCs in normal adult lung blood vessels are mostlyquiescent [13 17] MMP-9 which is active in the process ofarterial remodeling plays a critical role in promoting VSMCproliferation andmigration as well as intimal thickening [18ndash20]

p38MAPKbelongs to the serinethreonine protein kinasefamily and plays a key role in the activation of proliferation[21 22] Chen et al reported that the downregulation ofp38 MAPK phosphorylation is associated with the effectof AVE0991 in attenuating angiotensin II-induced VSMCproliferation [23] In balloon-injured rat aortas VSMC pro-liferation and intimal thickening are reduced which mightbe associated with the inhibition of the p38 MAPK signal-ing pathway [24] p38 plays a role in attenuating cardiacmuscle remodeling mainly by activating MMP-9 via anNAD(P)H-independent mechanism [6] Cho et al foundthat MMP-9 expression in arterial smooth muscle cells couldbe obviously inhibited by blocking p38 MAPK signaling[25] In recent years some studies have confirmed thatblocking p38MAPK signalingwith specific blocker SB203580can be used to inhibit MMP-9 expression in the vascularwalls of small arteries and the aorta for example coronaryarteries or the thoracic aorta as well as improve vascular

remodeling [26 27] Morin and his colleagues discoveredthat in a monocrotaline-induced pulmonary hypertensionmodel pulmonary artery remodeling might be improvedvia downregulation of the activity of p38 MAPK and theexpression of MMP-9 [28] Li et al found in their study thatchronic hypoxia could stimulate endothelial cell migrationand pulmonary artery smooth muscle cell proliferation topromote the formation of pulmonary hypertension via p38MAPK signal pathway [29]

In our study an experimental hypoxic chamber was usedto simulate the hypoxic pulmonary state that exists in patientswith chronic obstructive pulmonary disease or individualsliving in regionswith thin air Compared to the control groupthe hypoxia group showed significant increases in the mPAPpulmonary arteriole WT and cardiac hypertrophy indicessuch as the RV(LV + S) and RVBW Importantly baicalinpartially alleviated the negative effects induced by hypoxiaIn this study the MMP-9 mRNA and protein expressionlevels were significantly increased in the hypoxia groupcompared to the control group Based on the role of MMP-9in pulmonary arteriole remodeling as confirmed by the liter-ature we concluded that the upregulation of MMP-9 mRNAand protein might also be involved in hypoxia-induced



OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)

Figure 4 Expression of p-p38 in pulmonary arterioles and lung tissue (a b) p-p38 was expressed more abundantly in pulmonary arteriolecell nuclei in the hypoxia group than in the baicalin group and the control group as visualized by immunohistochemistry (119899 = 8group) indicates 119875 lt 001 compared to the control group and lowast indicates 119875 lt 005 compared to the hypoxia group (c d) Western blot analysis ofp-p38 expression in the lung showed that p-p38 expression was higher in the hypoxia group than in the control group and that this increasedexpression was attenuated by baicalin treatment Representative blots are shown (119899 = 8group) indicates 119875 lt 001 compared to the controlgroup and lowastlowast indicates 119875 lt 001 compared to the hypoxia group

pulmonary hypertension Importantly the baicalin treatmentattenuated the hypoxia-induced increases in MMP-9 andsignificantly improved the pulmonary arteriole remodelingAn analysis of the p-p38 MAPK levels in the arterioles andpulmonary tissue homogenates showed a trend similar tothat of MMP-9 as the level was increased in the continuoushypoxia state but was suppressed by baicalin These data alsoshowed that pulmonary hypertension and the hypertrophy

index were suppressed by baicalin thus demonstrating thatbaicalin treatment can attenuate hypoxic cor pulmonale

Baicalin clearly ameliorates pulmonary hypertensionpulmonary arterial remolding and hypoxic cor pulmonaleinduced by hypoxia by inhibiting the p38 MAPK signalingpathway and MMP-9 expression Considering the closerelationship between p38 MAPK and MMP-9 that has beenreported the protective effect of baicalin may be achieved by



OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)

Figure 5 Effect of baicalin on MMP-9 protein and mRNA expression in pulmonary arterioles (a b) Immunohistochemistry and in situhybridization techniques showed that bothMMP-9 protein andmRNA levels were higher in the hypoxia group than in the control group andthat baicalin attenuated this trend (119899 = 8group) indicates 119875 lt 001 compared to the control group and lowast indicates 119875 lt 005 compared tothe hypoxia group (c) MMP-9 expression in pulmonary arterioles as visualized by immunohistochemistry (d) MMP-9 mRNA expressionin pulmonary arterioles as visualized by in situ hybridization

inhibiting the p38 MAPK signaling pathway and suppressingMMP-9 expression

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Shuangquan Yan and Yiran Wang contributed equally to thiswork

Acknowledgments

This study was supported by grants from the Project ofthe Health Department of Zhejiang Province of China(2016DTA005 and 2012ZDA033) and the National NaturalScience Foundation of China (nos 81473406 and 81270110)

References

[1] K R Stenmark K A Fagan andM G Frid ldquoHypoxia-inducedpulmonary vascular remodeling cellular and molecular mech-anismsrdquo Circulation Research vol 99 no 7 pp 675ndash691 2006


[2] S-J Suh J-R Kim U-H Jin et al ldquoDeoxypodophyllotoxinflavolignan from Anthriscus sylvestris Hoffm inhibits migra-tion and MMP-9 via MAPK pathways in TNF-120572-inducedHASMCrdquoVascular Pharmacology vol 51 no 1 pp 13ndash20 2009

[3] S-J Suh U-H Jin S-H Kim et al ldquoOchnaflavone inhibitsTNF-120572-induced human VSMC proliferation via regulation ofcell cycle ERK12 and MMP-9rdquo Journal of Cellular Biochem-istry vol 99 no 5 pp 1298ndash1307 2006

[4] A Radziwon-Balicka M J Santos-Martinez J J Corbalan etal ldquoMechanisms of platelet-stimulated colon cancer invasionrole of clusterin and thrombospondin 1 in regulation of theP38MAPK-MMP-9 pathwayrdquo Carcinogenesis vol 35 no 2 pp324ndash332 2014

[5] V Grossi A Peserico T Tezil and C Simone ldquop38120572 MAPKpathway a key factor in colorectal cancer therapy and chemore-sistancerdquoWorld Journal of Gastroenterology vol 20 no 29 pp9744ndash9758 2014

[6] P Spallarossa P Altieri S Garibaldi et al ldquoMatrix metallo-proteinase-2 and -9 are induced differently by doxorubicin inH9c2 cells the role of MAP kinases and NAD(P)H oxidaserdquoCardiovascular Research vol 69 no 3 pp 736ndash745 2006

[7] Z Gao K Huang and H Xu ldquoProtective effects of flavonoidsin the roots of Scutellaria baicalensis Georgi against hydrogenperoxide-induced oxidative stress in HS-SY5Y cellsrdquo Pharma-cological Research vol 43 no 2 pp 173ndash178 2001

[8] M Lin L Li L Li et al ldquoThe protective effect of baicalinagainst renal ischemia-reperfusion injury through inhibitionof inflammation and apoptosisrdquo BMC Complementary andAlternative Medicine vol 14 article 19 2014

[9] M Guo N Zhang D Li et al ldquoBaicalin plays an anti-inflammatory role through reducing nuclear factor-120581B and p38phosphorylation in S aureus-induced mastitisrdquo InternationalImmunopharmacology vol 16 no 2 pp 125ndash130 2013

[10] P Liu S Yan M Chen et al ldquoEffects of baicalin on collagen Iand collagen III expression in pulmonary arteries of ratswith hypoxic pulmonary hypertensionrdquo International Journal ofMolecular Medicine vol 35 no 4 pp 901ndash908 2015

[11] H Jo S H Jung H B Yim S J Lee and K D Kang ldquoThe effectof baicalin in a mouse model of retinopathy of prematurityrdquoBMB Reports vol 48 no 5 pp 271ndash276 2015

[12] X-F Wang Q-M Zhou J Du H Zhang Y-Y Lu and S-B Su ldquoBaicalin suppresses migration invasion and metastasisof breast cancer via p38MAPK signaling pathwayrdquo Anti-CancerAgents in Medicinal Chemistry vol 13 no 6 pp 923ndash931 2013

[13] T Tajsic and N W Morrell ldquoSmooth muscle cell hypertrophyproliferation migration and apoptosis in pulmonary hyperten-sionrdquo Comprehensive Physiology vol 1 no 1 pp 295ndash317 2011

[14] J EWagenseil andR PMecham ldquoVascular extracellularmatrixand arterial mechanicsrdquo Physiological Reviews vol 89 no 3 pp957ndash989 2009

[15] P Chelladurai W Seeger and S S Pullamsetti ldquoMatrix metal-loproteinases and their inhibitors in pulmonary hypertensionrdquoEuropean Respiratory Journal vol 40 no 3 pp 766ndash782 2012

[16] N Li D-Z Dai and Y Dai ldquoCPU86017 and its isomers improvehypoxic pulmonary hypertension by attenuating increased ETAreceptor expression and extracellular matrix accumulationrdquoNaunyn-Schmiedebergrsquos Archives of Pharmacology vol 378 no5 pp 541ndash552 2008

[17] U Hedin B A Bottger E Forsberg S Johansson and J Thy-berg ldquoDiverse effects of fibronectin and laminin on phenotypicproperties of cultured arterial smooth muscle cellsrdquo Journal ofCell Biology vol 107 no 1 pp 307ndash319 1988

[18] R D Kenagy C E Hart W G Stetler-Stevenson and A WClowes ldquoPrimate smooth muscle cell migration from aorticexplants is mediated by endogenous platelet-derived growthfactor and basic fibroblast growth factor acting through matrixmetalloproteinases 2 and 9rdquo Circulation vol 96 no 10 pp3555ndash3560 1997

[19] N Zempo N Koyama R D Kenagy H J Lea and A WClowes ldquoRegulation of vascular smooth muscle cell migrationand proliferation in vitro and in injured rat arteries by asynthetic matrix metalloproteinase inhibitorrdquo ArteriosclerosisThrombosis and Vascular Biology vol 16 no 1 pp 28ndash33 1996

[20] H Lepetit S Eddahibi E Fadel et al ldquoSmooth muscle cellmatrix metalloproteinases in idiopathic pulmonary arterialhypertensionrdquo European Respiratory Journal vol 25 no 5 pp834ndash842 2005

[21] L C Platanias ldquoMap kinase signaling pathways and hemato-logic malignanciesrdquo Blood vol 101 no 12 pp 4667ndash4679 2003

[22] M Ricote I Garcıa-Tunon F Bethencourt et al ldquoThe p38transduction pathway in prostatic neoplasiardquo Journal of Pathol-ogy vol 208 no 3 pp 401ndash407 2006

[23] S-L Chen Y-XWuY-YHuang et al ldquoAVE0991 a nonpeptidecompound attenuates angiotensin II-induced vascular smoothmuscle cell proliferation via induction of heme oxygenase-1 anddownregulation of p-38 mapk phosphorylationrdquo InternationalJournal of Hypertension vol 2012 Article ID 958298 8 pages2012

[24] Y Li Q Wang Q Xu et al ldquoValsartan decreases neointimalhyperplasia in balloon-injured rat aortic arteries by upregulat-ing HO-1 and inhibiting angiotensin II type 1 receptorrdquo LifeSciences vol 110 no 2 pp 70ndash76 2014

[25] A Cho J Graves and M A Reidy ldquoMitogen-activated pro-tein kinases mediate matrix metalloproteinase-9 expression invascular smooth muscle cellsrdquo Arteriosclerosis Thrombosis andVascular Biology vol 20 no 12 pp 2527ndash2532 2000

[26] TDodd R Jadhav LWiggins et al ldquoMMPs2 and 9 are essentialfor coronary collateral growth and are prominently regulated byp38 MAPKrdquo Journal of Molecular and Cellular Cardiology vol51 no 6 pp 1015ndash1025 2011

[27] G Cheng LWeiWXiurong L Xiangzhen Z Shiguang and FSongbin ldquoIL-17 stimulates migration of carotid artery vascularsmooth muscle cells in an MMP-9 dependent manner via p38MAPK and ERK12-dependent NF-120581B and AP-1 activationrdquoCellular andMolecularNeurobiology vol 29 no 8 pp 1161ndash11682009

[28] C Morin R Hiram E Rousseau P U Blier and S FortinldquoDocosapentaenoic acid monoacylglyceride reduces inflam-mation and vascular remodeling in experimental pulmonaryhypertensionrdquo American Journal of PhysiologymdashHeart and Cir-culatory Physiology vol 307 no 4 pp H574ndashH586 2014

[29] Q Li M Mao Y Qiu et al ldquoKey role of ROS in the pro-cess of 15-lipoxygenase15-hydroxyeicosatetraenoiccid-inducedpulmonary vascular remodeling in hypoxia pulmonary hyper-tensionrdquo PLoS ONE vol 11 no 2 article e0149164 2016

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


levels which then leads to a series of biological effects[6]

Baicalin is a flavonoid compound purified from the dryroots of Scutellaria baicalensis and it possesses a varietyof pharmacological activities such as antioxidative anti-inflammatory antitumor and antiapoptotic activities it alsoinhibits smooth muscle cell proliferation [7ndash9] We previ-ously found that baicalin attenuates small pulmonary arteryremodeling in vivo in rats with hypertension [10] but themechanism is complicated and not fully understood Severalstudies have shown that baicalin downregulates the expres-sion of p38MAPK andMMP-9 Baicalin not only inhibits theexpression of p38 MAPK and MMP-9 in new retinal vesselsin amousemodel of oxygen-induced retinopathy [11] but alsoblocks the activation of p38 MAPK which leads to reducedMMP-9 levels and tumorigenicity in MDA-MB-231 cells[12] Thus we speculate that baicalin attenuates pulmonaryarteriole remodeling and hypoxic pulmonary hypertensionfurther improving cor pulmonale by inhibiting the activity ofp38 MAPK and downregulating the expression of MMP-9 inarterioles

2 Materials and Methods

21 Animals Twenty-four male specific-pathogen-free Spra-gue-Dawley rats weighing 220ndash250 g provided by the Labo-ratory Animal Center of Wenzhou Medical University wereused in the present study All rats were housed in controlledconditions and fed a standard diet The animal experimentswere approved by the Laboratory Animal Ethics Committeeof Wenzhou Medical College and performed in strict accor-dance with the guidelines for animal ethics

22 Drugs and Antibodies Baicalin (CAS number 21967-41-9 Product number 572667 purity 95) was purchasedfrom Sigma-Aldrich Co Ltd (USA) A 2 baicalin solutionwas prepared by mixing 1 g of baicalin into 48mL of sterilesaline and titrating the solution under stirring with sodiumhydroxide until reaching a pH of 68ndash70 After the baicalinwas completely dissolved the clear yellow-green solutionwasbrought up to a final volume of 50mL with sterile salineand the final concentration was 20mgmL Then aliquotsof the solution were prepared and stored at minus20∘C Priorto use these aliquots were gradually thawed at 4∘C Therabbit anti-rat p-p38 MAPK polyclonal antibody (4631) wasobtained from Cell Signaling Technology (USA) The sec-ondary horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody and the secondary rabbit anti-goat antibodywere purchased from ZSGB Biotechnology Co Ltd (BeijingChina) The goat anti-rat MMP-9 antibody (sc-6840) wasobtained from Santa Cruz (USA)TheMMP-9 mRNA in situhybridization (ISH) kit (MK1540) was purchased fromBosterBiotechnology Co Ltd (Wuhan China) and the rabbit anti-rat GAPDH antibody was obtained from Goodhere (China)

23 Animal Grouping and Administration The rats wererandomized into the following three groups with eight ratsper group the control group the hypoxia group and thebaicalin + hypoxia groupThe rats in the hypoxia group were

kept in a normobaric hypoxic chamber for 4 weeks with anO2concentration of 8ndash10 and a CO

2concentration that was

below 5 for 6 days per week and 8 hours per dayThe rats inthe baicalin + hypoxia group were intraperitoneally injectedwith 2 baicalin (30mgkg) 30min before being placed intothe normobaric hypoxic chamber The rats in the controlgroup were kept under normobaric and normoxic condi-tions

24 Mean Pulmonary Artery Pressure (mPAP) and Mean Sys-temic Artery Pressure (mSAP) Measurements After 4 weeksthe rats were weighed and anesthetized by an intraperitonealinjection of 5 chloral hydrate (40mgkg of body weight)The specific methods used have been previously reported byour team A small skin incision was made in the neck of eachmouse Then the right external jugular vein was isolatedand a polyethylene catheter (inner diameter 09mm outerdiameter 11mm) with a slightly curved front was graduallyinserted into the right atrium and then the right ventricle(RV) After the recorder entered the RV the large vibrationsof the ventricular waveform could be documented Then thetube was further inserted by approximately 05 cm After theheart rate became normal and the waveform stabilized thetube was rotated clockwise to the lower part of the marksuch that the tube head was facing upward thereby enablingit to move forward 1-2 cm and enter the pulmonary arteryAt this point the effects of breathing on the pulmonaryartery waveform were visible After moving the tube forwardan additional 05ndash1 cm stable pulmonary artery waveformscould be recorded and the mPAP was recorded with apiezoelectric pressure transducer and the PowerLab system(AD Instruments Australia)

25 Pulmonary Arteriole RemodelingMeasurement Lung tis-sue sections were subjected to hematoxylin and eosin (HampE)staining following standard histological procedures For eachsection five vessels near the pulmonary alveoli with diame-ters of approximately 50ndash200120583mwere randomly selected formeasurement First the pulmonary arteriole wall area andtotal area (microscope magnification 40 times 10 = 400x) weremeasured to calculate the ratio of vessel wall area to total area(WATA) Second each arteriole wall thickness (WT) andradius (R) weremeasured at 0∘ 90∘ 180∘ and 270∘ by drawinga line across the vessel and its wall The WT and R valuesacquired from the four angles were then averaged and usedto calculate the WTR ratio Finally the number of nuclei inthe arteriole wall was counted and used to calculate both theratio of the number of nuclei to the vesselWA and the nucleardensity of the wall All measurements were performed usingImage-Pro Plus software (Media Cybernetics Bethesda MDUSA)

26 Right Ventricular Hypertrophy Measurements The leftventricle plus the interventricular septum (LV + S) and theRV were first collected by cutting along the edge of theRV and the interventricular septum then these sampleswere weighed The mass ratios of the RV to the LV + Sand rat body weight (BW) expressed as RV(LV + S) and









3 Results





30

20

10

0

mPA

P (m

mH

g)

lowastlowast

(a)


200

150

100

50

0

mSA

P (m

mH

g)

(b)

PAP

wav

efor

m


30

20

10

40

30

20

10

40

30

20

10

(c)

SAP

wav

efor

m


120

100

80

140

120

100

80

140

120

100

80

(d)








Control

WA

TA

()

lowast

100

80

60

40

20

0

(a)


Control

WT

R (

)

lowast

80

60

40

20

0

(b)


Control

lowastlowast

0015

0010

0005

0000

Nuc

leus

num

ber120583m

2

(c)


25 120583m25 120583m25 120583m

(d)



4 Discussion








RV(

LV+S

) (

)

lowast

40

30

20

10

0

(a)


RVB

W (

)

lowastlowast

015

010

005

000

(b)


Myc

ardi

a HE

stai

ning

400x 400x400x

(c)








OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)







OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



Competing Interests




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results





30

20

10

0

mPA

P (m

mH

g)

lowastlowast

(a)


200

150

100

50

0

mSA

P (m

mH

g)

(b)

PAP

wav

efor

m


30

20

10

40

30

20

10

40

30

20

10

(c)

SAP

wav

efor

m


120

100

80

140

120

100

80

140

120

100

80

(d)








Control

WA

TA

()

lowast

100

80

60

40

20

0

(a)


Control

WT

R (

)

lowast

80

60

40

20

0

(b)


Control

lowastlowast

0015

0010

0005

0000

Nuc

leus

num

ber120583m

2

(c)


25 120583m25 120583m25 120583m

(d)



4 Discussion








RV(

LV+S

) (

)

lowast

40

30

20

10

0

(a)


RVB

W (

)

lowastlowast

015

010

005

000

(b)


Myc

ardi

a HE

stai

ning

400x 400x400x

(c)








OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)







OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



Competing Interests




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















30

20

10

0

mPA

P (m

mH

g)

lowastlowast

(a)


200

150

100

50

0

mSA

P (m

mH

g)

(b)

PAP

wav

efor

m


30

20

10

40

30

20

10

40

30

20

10

(c)

SAP

wav

efor

m


120

100

80

140

120

100

80

140

120

100

80

(d)








Control

WA

TA

()

lowast

100

80

60

40

20

0

(a)


Control

WT

R (

)

lowast

80

60

40

20

0

(b)


Control

lowastlowast

0015

0010

0005

0000

Nuc

leus

num

ber120583m

2

(c)


25 120583m25 120583m25 120583m

(d)



4 Discussion








RV(

LV+S

) (

)

lowast

40

30

20

10

0

(a)


RVB

W (

)

lowastlowast

015

010

005

000

(b)


Myc

ardi

a HE

stai

ning

400x 400x400x

(c)








OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)







OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



Competing Interests




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Control

WA

TA

()

lowast

100

80

60

40

20

0

(a)


Control

WT

R (

)

lowast

80

60

40

20

0

(b)


Control

lowastlowast

0015

0010

0005

0000

Nuc

leus

num

ber120583m

2

(c)


25 120583m25 120583m25 120583m

(d)



4 Discussion








RV(

LV+S

) (

)

lowast

40

30

20

10

0

(a)


RVB

W (

)

lowastlowast

015

010

005

000

(b)


Myc

ardi

a HE

stai

ning

400x 400x400x

(c)








OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)







OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



Competing Interests




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















RV(

LV+S

) (

)

lowast

40

30

20

10

0

(a)


RVB

W (

)

lowastlowast

015

010

005

000

(b)


Myc

ardi

a HE

stai

ning

400x 400x400x

(c)








OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)







OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



Competing Interests




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















OD

val

ue o

f p-p

38 IH

C

020

015

010

005

000

lowast

(a)


p-p3

8 (I

HC)

25 120583m25 120583m25 120583m

(b)


GAPDH

p-p38

(c)


The e

xpre

ssio

n of

p-p

38 in

lung

tiss

ue

10

08

06

04

02

00

lowastlowast

(d)







OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



Competing Interests




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















OD

val

ue o

f MM

P-9

025

020

015

010

005

00

lowast

(a)


OD

val

ue o

f MM

P-9

mRN

A

04

03

02

01

00

lowast

(b)


MM

P-9

IHC

25 120583m25 120583m25 120583m

(c)


MM

P-9

mRN

A IS

H

25 120583m25 120583m25 120583m

(d)



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
















research article baicalin attenuates hypoxia-induced...

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