research article beneficial effects of qili qiangxin...

Research ArticleBeneficial Effects of Qili Qiangxin Capsule onLung Structural Remodeling in Ischemic Heart Failure viaTGF-1205731Smad3 Pathway

Yaoyao He1 Bai Du1 Huiting Fan1 Jian Cao1 Zi Wang Liu2 Yonglie Zhao2

Mingjing Zhao3 Yizhou Zhao3 Xin Zhao1 and Xiangning Cui1

1Department of Cardiology Guangrsquoanmen Hospital China Academy of Chinese Medical Sciences Beijing 100053 China2Beijing University of Chinese Medicine Third Affiliated Hospital Beijing 100029 China3The Key Laboratory of Chinese Internal Medicine of the Ministry of Education Dongzhimen HospitalBeijing University of Chinese Medicine Beijing 100700 China

Correspondence should be addressed to Xiangning Cui cuixiangning126com

Received 25 May 2015 Revised 21 September 2015 Accepted 27 September 2015

Academic Editor Se Eun Byeon

Copyright copy 2015 Yaoyao He et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Qili qiangxin (QL) capsule is a traditional Chinese medicine that is widely used for the treatment of patients with chronicheart failure (CHF) of all etiologies although the exact mechanisms of action remain unclear CHF leads to pulmonary vascularremodelling and thickening of the alveolar-capillary barrier that may be important mechanisms in the poor clinical outcomein patients with end-stage heart failure We examined whether QL could improve lung injury in ischemic CHF by reducinglung remodeling Rats with myocardial infarct received QL (10 gkgday) for 4 weeks Echocardiographic and morphometricmeasurements were obtained followed by echocardiography histological staining and immunohistochemical analysis of lungsections CHF caused significant lung structural remodeling evidenced by collagen deposition and thickening of the alveolar septaafter myocardial infarct that were greatly improved by QL Lung weight increased after infarct with no evidence of pulmonaryedema and was normalized by QL QL also reduced lung transforming growth factor-1205731 (TGF-1205731) p-Smad3 tumor necrosisfactor-120572 (TNF-120572) and Toll-like receptor-4 (TLR4) expression Thus QL reduces lung remodeling associated with CHF mainlyby suppressing the TGF-1205731Smad3 signaling pathway The mechanism may also involve inhibition of TLR4 intracellular signaling

1 Introduction

Pulmonary hypertension (PH) is a frequent complicationof chronic heart failure (CHF) It is predicted that 60 ofpatients with severe left ventricular (LV) systolic dysfunctionand 70 of patients with LV diastolic dysfunction maydevelop PH [1] PH secondary to chronic LV failure reducesexercise capacity and represents an important independentprognostic factor in sufferers especially when associated withright ventricular (RV) dysfunction [2ndash4] In view of thegrowing prevalence of CHF the clinical importance of PHassociated with CHF is becoming more prominent

The pathophysiology of CHF-associated lung injury isincompletely understood and few effective therapeuticoptions are presently available Although fluid overload and

pulmonary edema no doubt contribute to the process themechanisms responsible for the pulmonarymanifestations ofchronic CHF involve both pulmonary vascular and alveolarsepta structural remodeling [5 6] Therefore novel therapiesspecifically targeting the lung structural remodeling pro-cesses may potentially be utilized in the future

Research has provided strong evidence implicating in-flammatory activation as an important pathway in the pro-gression of CHF Elevated plasma levels of cytokines suchas tumor necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573)and interleukin-6 (IL-6) have been found in CHF patients[7 8] and subsequent animal experiments have suggestedthat certain anti-inflammatory therapies may be beneficialVarious experimental studies have also shown that increasedexpression of inflammatory cytokines is associated with

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 298631 10 pageshttpdxdoiorg1011552015298631

2 Evidence-Based Complementary and Alternative Medicine

tissue and organ fibrosis remodeling in the heart lungand kidney [9ndash11] Anti-inflammatory therapies can preventpathological fibrosis in these organs Animal experimentshave also demonstrated that lung injury associated withCHF is characterized by excessive collagen deposition in thealveoli inside the vessels and in the vascular walls of largevessels accompanied by the accumulation of leukocytes andmacrophages and increased levels of transforming growthfactor-1205731 (TGF-1205731) TNF-120572 and Toll-like receptor-4 (TLR4)mRNA or protein content in the lungs [12] Thus control-ling excessive inflammation could potentially be of greattherapeutic benefit for inhibiting the progressive pulmonarystructural fibrosis remolding in CHF

Qili qiangxin (QL) capsule is a traditional Chinese med-icine that is widely used for the treatment of patients withCHF of all etiologies including hypertension coronary heartdisease and chronic obstructive pulmonary disease [13] Ourlaboratory and others have reported that QL can reducecardiac fibrosis remolding and improve cardiac function [14ndash16] The use of QL in CHF also has an immunomodulatoryeffect by decreasing the proinflammatory cytokine TNF-120572and increasing the anti-inflammatory cytokine IL-10 [17]The use of QL in patients with CHF caused by chronicobstructive pulmonary disease has shown positive effects onpulmonary function by improving gas diffusion and exercisecapacity [18] suggesting that QL could also have beneficialeffects on lung fibrosis and inflammation in CHF In thepresent study we used a postmyocardial infarction heartfailure model to determine whether QL can improve lungstructural remodeling associated with CHF by inhibitinglung inflammation and fibrosis and to elucidate the possiblemechanisms underlying the antifibrotic effect of QL Theeffects of QL were also compared with Valsartan an Ang-II receptor antagonist commonly used in clinical practicewhich is known to have heart-lung protective effects in thetreatment of CHF [6]

2 Materials and Methods

21 Vegetal Material QL consists of ginseng Radix Astra-gali Aconite Root Salvia miltiorrhiza Semen LepidiiApetali Cortex Periplocae Sepii Radicis Rhizoma AlismatisCarthamus tinctorius Polygonati odorati rhizoma SeasonedOrange Peel and Ramulus Cinnamomi (Yiling Pharmaceuti-cal Corporation Shijiazhuang China)We dissolved the drugpowder in sterile water at a concentration of 01 gmL QL forthe study Valsartan (batch number X1428) manufactured byBeijing Novartis Pharmaceutical Co Ltd was dissolved insterile water

22 Animal Model and Administration Healthy maleSprague-Dawley rats (body weight 220sim250 g) were providedby Beijing Vital River Laboratory Animal Technology CoLtd (animal license number SCXK (Beijing) 2012-0001)The animals were fed a standard diet and water was providedad libitum The rats were maintained under a 12 h lightand 12 h dark cycle at a temperature of 20 plusmn 2∘C and ata humidity of 50 plusmn 2 All animal experimental protocolswere approved by the Animal Care and Use Committee

Table 1 Echocardiographic ejection fraction level in the studygroups before treatment with qili qiangxin (QL) (119909 plusmn 119904)

Group 119899 EF ()Sham 10 84490 plusmn 73354CHF 12 44708 plusmn 84369lowast

QL 9 47933 plusmn 91211lowast

Valsartan 9 47911 plusmn 91068lowastlowast

119875 gt 005 versus the Sham group

of Beijing University of Chinese Medicine and compliedwith laboratory animal management and use regulationsThe CHF model was induced by myocardial infarctionfollowing ligation of the left anterior descending artery(LAD) A 1 sodium pentobarbital solution (50mgkg) wasadministered by intraperitoneal injection The proceduresperformed included endotracheal intubation positivepressure ventilation using a ventilator preoperative recordingof a 12-lead ECG local skin disinfection chest openingthoracotomy device setup and opening of the pericardiumthe pulmonary cone and the left atrial appendage 2sim3mmfrom the bottom of the left anterior descending coronaryartery ligation For the rats assigned to the Sham groupthe same operation was performed without ligation of theleft coronary artery A 12-lead ECG was recorded after theexperiment Rats with myocardial infarctions (MI) werefed normally for 4 weeks Based on the transthoracic ech-ocardiography results (Table 1) the surviving rats wererandomly assigned to the following groups Model group(CHF 119899 = 12) Sham group (Sham 119899 = 10) QL group(QL 119899 = 9) and Valsartan group (Valsartan 119899 = 9) QL(10 gkg) and Valsartan (10mgkg) were administered bygavage once a day during the 4 weeks An equal volume ofdistilled water was used for the Model and Sham groupsSee Table 1 echocardiographic ejection fraction levels in thestudy groups before treatment with QL

23 Transthoracic Echocardiography Measurements A non-invasive transthoracic echocardiographymethod was used toevaluate the morphology and function of the left ventricleEchocardiography was performed on anesthetized animalsusing a two-dimensional mode that is a time-motion (TM)mode and blood flow was measured using the pulsedDoppler mode

24 Sample Preparation At the end of the 4-week treatmentthe rats were anesthetized The heart lungs and other majororganswere harvested andweighedThe lung tissuewas driedat 58∘C to a constant weightThe relative water content of thelung tissue was calculated The left lung and LV were snap-frozen in liquid nitrogen pending biochemical analysis Theairways of the upper right lobe and hearts were subsequentlyfixed in 4 paraformaldehyde and embedded in paraffin forhistological analysis

25 Histological Staining The relative pulmonary vascularmuscularization was determined using hematoxylin and

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Echocardiographic left ventricular (LV) parameters in the study groups after treatment with qili qiangxin (QL) (119909 plusmn 119904)

Group 119899 EF () FS () LVIDd (mm) LVIDs (mm) EDV (mL) ESV (mL)Sham 10 778 plusmn 71 483 plusmn 82 59 plusmn 07 30 plusmn 06 049 plusmn 014 007 plusmn 007CHF 11 387 plusmn 58998771 279 plusmn 93998771 74 plusmn 12998771 69 plusmn 04998771 092 plusmn 052998771 073 plusmn 013998771

QL 8 542 plusmn 103lowast 343 plusmn 142lowast 65 plusmn 12 58 plusmn 07lowast 073 plusmn 023 051 plusmn 012lowast

Valsartan 8 527 plusmn 48lowast 289 plusmn 107lowast 66 plusmn 09 60 plusmn 07lowast 071 plusmn 037 053 plusmn 014lowast998771

119875 lt 005 versus the Sham group lowast119875 lt 005 versus the CHF group

eosin (HampE) stains Briefly in each mouse 60 intra-acinararteries were examined and categorized as nonmuscular(NM) partially muscular (PM) or fully muscular (FM)The relative percentages of NM PM and FM arteries werecalculated Lung and heart fibrosis areas were stained usingMasson Trichrome Stain Kit from Sigma-Aldrich In addi-tion lung sections were stained with monoclonal antibodiesto identify smooth muscle cells or myofibroblasts (using amouse monoclonal antialpha smooth muscle actin antibodyABcam Inc) Paraffin was removed from 4120583m sections oftissue using xylene endogenous peroxidase was quenchedin 3 H

2O2for 10min and then antigen was recovered in

Tris-EDTA buffer (pH = 90) for 2min 30 s at 140∘C thenwashed in phosphate buffered saline (PBS)The sections wereincubated with monoclonal antialpha smooth muscle actinantibody (1 400) at 37∘C for 1 h followed by biotinylatedsecondary antibody for 20min The sections were stainedwith DAB chromogen and counterstained with hematoxylin

26 Western Blot Analysis All animals were euthanizedafter 4 weeks of drug administration and their heartsand lungs were immediately harvested and stored in liquidnitrogen until western blot analyses were performed Thefollowing antibodies were used mouse monoclonal anti-TGF beta 1 (1 500 ABcam Inc) rabbit monoclonal anti-Smad3 (phosphor C25A9) (1 500 Cell Signaling TechnologyInc) rabbit polyclonal anti-TNF alpha (1 1000 ABcamInc) and mouse monoclonal anti-TLR4 (1 2000 ABcamInc) Proteins were separated using 10 SDS-PAGE andtransferred to nitrocellulose membranes which were thenincubated with antibodies at 4∘C The membranes werefurther incubated with horseradish peroxidase-conjugatedanti-rabbit IgG (1 2000) for 2 h at room temperature ECLvisualization was performed and the Gene Gnome Gel Imag-ing System (Syngene Co) was used to capture the resultingimages Image J (NIH image Bethesda MD) was used toanalyze the gel images

27 Statistical Methods All experimental data are presentedas the mean plusmn the standard deviation (SD) single factoranalysis of variance (ANOVA) was performed using SPSSversion 170 statistical software Dunnettrsquos T3 was used forunequal variances and a probability of 119875 lt 005 wasconsidered statistically significant

3 Results

31 Effects of QL on LV Function and LV RemodelingLV echocardiographic parameters are presented in Table 2

Comparedwith theCHF group (119899 = 11) the ejection fractionand fractional shortening measurements were elevated in theQL group (119899 = 8) and the Valsartan group (119899 = 8) (119875 lt 005)while the end-systolic volume and LV end-systolic dimen-sion measurements were lower (119875 lt 005) Although themeasurements obtained for end-diastolic volume (EDV) andLV end-diastolic dimension (LVIDd) displayed a decreasingtrend in both the QL and the Valsartan groups versus theCHF group the difference was not statistically significant(119875 gt 005) Compared with those of the Sham group theEF and FS measurements obtained from the CHF group theQL group and the Valsartan group were reduced (119875 lt 005)while the ESV and LVIDs measurements were increased(119875 lt 005) (Figure 1) Histological study revealed that theLV collagen fractional area (Figure 2) was highly increasedin CHF versus Sham rats (119875 lt 005) and was reducedby QL and Valsartan treatments These parameters of LVremodeling and dysfunction were significantly modified bythe QL treatment

32 Effects of QL on Pulmonary Structural Remodeling Thewet lungbody weight ratio increased after MI (119875 lt 005)and QL and Valsartan markedly improved this ratio (119875 lt005 Figure 3(a)) Similarly the dry lungbody weight ratioincreased afterMI (119875 lt 005) providing evidence of substan-tial pulmonary remodeling treatment with QL and Valsartanreversed this increased ratio (119875 lt 005 Figure 3(b)) Thedrywet lung weight ratio was comparable among all groupssuggesting that no significant edema occurred (Figure 3(c))

TheHE staining showed that intact and clear alveoli nor-mal interstitium and few inflammatory cells were observedin the lungs of Sham group However LV dysfunction causedprogressive lung injury as demonstrated by destruction oflung alveoli inflammatory cell infiltration and thickening ofthe lung interstitium QL or Valsartan treatment preventedthese changes in rat lungs with MI LV dysfunction alsocaused an increase in fully muscularized (FM) small arter-ies but the number of FM small arteries was significantlylower among the rats in the QL and Valsartan treatmentgroups compared with those in the CHF group Similarlythe QL treatment group exhibited a significant increase innonmuscularized (NM) small arteries compared with theCHF group (Figure 4) indicating that QL and Valsartan sig-nificantly improved LAD-induced lung vascular remodelingThe Masson Trichrome staining also showed intact alveoliand normal interstitium in the lungs of the rats in the Shamgroup LAD resulted in exacerbated lung alveoli destructioninterstitium thickening and fibroblast diffusion in rat lungsand QL or Valsartan treatment significantly halted these


0

20

40

60

80

100

Sham CHF QL Valsartan

(a) (b)

(c) (d)

EF (

)

0

20

40

60

80

100


FS (

)

0

02

04

06

08

12

1

ESV

(mL)

0

2

4

6

8

10

Sham CHF QL ValsartanSham CHF QL Valsartan

LVID

s (m

m)


lowast

lowast

lowast lowast998771

998771998771

lowast998771

lowast998771

998771

lowast

lowast lowast

998771 998771

998771998771

lowast

lowast998771 lowast

998771

Figure 1 Effects of qili qiangxin (QL) on echocardiographic left ventricular (LV) function in rats with chronic heart failure (CHF) Typicalechocardiography images (upper pictures) and (a) LV ejection fraction (EF) (b) LV fractional shortening (FS) (c) LV end-systolic volume(ESV) and (d) LV end-systolic dimensions (LVIDs) (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versus the CHF group)

changes in rat lungs afterMI (Figure 5) Alpha smoothmuscleactin (120572-SMA) expression has been extensively used as amarker of fibroblast differentiation into its activated statethe myofibroblast Expression of 120572-SMA was increased inthe interalveolar septa of animals with MI when comparedto the Sham group however QL or Valsartan treatmentsignificantly reduced its expression (Figure 6)

The 120572-SMA-positive myofibroblasts actively synthesizeECM components We then measured type I collagen themain collagen isoform produced by fibroblasts in many

fibrotic processes Using western blot we found that typeI collagen deposition was significantly higher in the rats inthe CHF group when compared to those in the Sham grouphowever QL significantly decreased type I collagen proteinexpression in the lung (Figure 6) Considered together theseparameters of lung tissue fibrotic remodeling were signifi-cantly reversed by QL and Valsartan treatment

33 QL Treatment Decreased TGF-1205731Smad3 Signaling Path-way Expression in Rat Lungs after CHF CHF evidently


QL Valsartan0

5

10

15

20

25

30

Sham CHF

Sham CHF

QL Valsartan

LV co

llage

n de

posit

ion

area

() lowast

lowast

998771

Figure 2 Effects of qili qiangxin (QL) on myocardial fibrosis in rats with chronic heart failure (CHF) Representative Masson Trichrome-stained left ventricular (LV) areas are shown Blue areas indicate fibrotic staining Fibrosis was measured in the whole LV section and 5sections for each heart were calculated (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versus the CHF group)

0

005

01

015


Wet

lung

bod

y w

eigh

t (

)

lowast

lowast

998771

(a)

0

0005

001

0015

002

0025


Dry

lung

bod

y w

eigh

t (

)

lowastlowast

998771

(b)

0

5

10

15

20

25


Dry

wet

lung

wei

ght (

)

(c)

Figure 3 Effect of qili qiangxin (QL) on (a) wet lungbody weights (b) dry lungbody weights and (c) drywet lung weights ratios in ratswith chronic heart failure (CHF) (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versus the CHF group)


Sham CHFQL QLValsartan Valsartan0

10

20

30

40

50

60

70

80

90

100

Lung

vas

cula

r mus

cula

rizat

ion

()

lowast

lowast

lowast

lowast

998771

998771

Nonmuscular arteriolesPartially muscularized arteriolesFully muscularized arterioles

Sham CHF

Figure 4 Effects of qili qiangxin (QL) on lung vascular muscularization in rats with chronic heart failure (CHF) Histological lung sectionwith hematoxylin and eosin staining and quantitative analysis of the distribution of nonmuscular partiallymuscular fullymuscularized smallarteries (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versus the CHF group)

0

5

10

15

20


Lung

colla

gen

frac

tiona

l are

a (

) lowast

lowast

998771

QL Valsartan

Sham CHF

Figure 5 Effects of qili qiangxin (QL) on pulmonary structural remodeling assessed by Masson Trichrome staining for collagen in blue andby quantitative analysis for lung collagen deposition (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versus the chronic heart failure group)

increased the protein levels of TGF-1205731 in rat lungs comparedwith the Sham group at the endpoint of 28 days however QLand Valsartan treatments significantly reduced its expressioncompared with the CHF group (Figure 7) Similarly CHFevidently increased the protein level of p-Smad3 in ratlungs compared with the Sham group and QL or Valsartantreatment significantly reduced its expression compared withthe CHF group (Figure 7)

34 QL Treatment Decreased TLR4 and TNF-120572 Expressionin Rat Lungs after CHF The protein expression of theproinflammatory cytokines tumor necrosis factor-120572 (TNF-120572) and Toll-like receptor 4 (TLR4) was all increased in lungtissue from the rats in the CHF groups and these increases

were markedly reversed in the QL or the Valsartan treatmentgroup rats compared with the CHF group rats (Figure 8)

4 Discussion

Adverse cardiac remodeling after MI leads to progressiveheart failure In the present study we found that QL improvescardiac structure and function in rats with MI After MI ratsdeveloped CHF with important lung structural remodelingcharacterized by alveolar wall collagen deposition a dramaticincrease in the percentage of FM lung vessels and increasesin TGF-1205731 TNF-120572 and TLR4 proteins expression in lungtissues Therapy with QL and Valsartan reduced lung tissuecollagen deposition and the percentage of FM lung vessels



120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)

Figure 6 Effects of qili qiangxin (QL) on expression of proteins related to lung fibrosis in rats with chronic heart failure (CHF) ((a) (b))Expression of smooth muscle 120572-actin (120572-SMA) ((a) (c)) Expression of type I collagen (d) Immunohistochemistry for 120572-SMA (998771119875 lt 005versus the Sham group lowast119875 lt 005 versus the CHF group)

mechanistically QL reduced lung TGF-1205731 TNF-120572 and TLR4protein expression These data suggest some direct beneficialeffects of QL on pulmonary structure fibrosis remoldingand inflammation response in rats with ischemic heartfailure

The lungs are the primary organs affected in CHFCHF caused by all etiologies leads to impairment of LVfilling with subsequent congestion of the pulmonary venouscirculation and pulmonary alveolar-capillary stress failureresulting in cycles of alveolar wall injury and repair Thereparative process causes the proliferation of myofibroblastswith fibrosis and extracellular matrix deposition resultingin thickening of the pulmonary vascular and alveolar wallsAlthough the resultant reduction in vascular permeability

is initially protective against pulmonary edema the processbecomes maladaptive causing a restrictive lung syndromewith impaired gas exchange This pathological process mayalso contribute to type 2 PH (PH secondary to chronic LVfailure is classified as type 2 PH) The structural fibrosisremodeling of the lung may be an important mechanism toexplain the poor clinical outcome in patients with end-stageheart failure Thus the effective treatment of LV end-stageheart failuremay also require additional efforts to reduce lungfibrosis and the inflammatory response

QL capsule has been proven to be effective and safe for thetreatment of CHF after MI [13 19] The compounds include11 Chinese herbs in which Radix Astragali and ginsengcomprise the main active constituents which have effects on



GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)

Figure 7 Effects of qili qiangxin (QL) on lung TGF-1205731Smad3 signaling pathway expression inCHF rats ((a) (b)) Expression of transforminggrowth factor TGF-1205731 ((a) (c)) expression of p-Smad3 (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versus the CHF group)

invigorating the heart qi and lung qi Salvia miltiorrhiza andCarthamus tinctorius accelerate blood circulation and relievecongestion Semen Lepidii Apetali can relieve asthma anddisperse lung edema The use of these herbs demonstratedthat QL has protective effects on the heart and lungs Phar-macological studies have found that QL contains a numberof active substances such as ginseng saponin astragalussaponin flavonoids cardenolide and phenolic acid whichhave been proven to have positive inotropic vasodilationanti-inflammation and antifibrosis effects In this study wefound excessive collagen deposition and upregulated 120572-SMAprotein expression in the lungs of rats with CHF both werereversed by QL and Valsartan therapy suggesting that QLmay also have pulmonary protective effects on ischemic heartfailure

To elucidate the possible mechanisms underlying theantifibrotic effect of QL in CHF we examined the impact ofQL on the TGF-1205731Smad3 signaling pathway TGF-1205731 is alocally generated cytokine that has been implicated as amajorcontributor to myofibroblast proliferation and tissue fibrosisin various organ systems Several studies have explored thefunctional consequence of TGF-120573 in heart failure (HF) andresults from these studies have shown that TGF-120573 antagonisminhibits fibrotic processes and provides salutary cardiac

effects inHF [20] As amain downstream signal transducer ofTGF-1205731 Smad3 can be phosphorylated by an activated typeI receptor of TGF-1205731 followed by forming a complex withSmad4 and translocation into the nucleus where it acts asa transcription factor and promotes the expression of targetgenes including type I and type III collagen The presentstudy demonstrated that TGF-1205731 expression in lung tissuesof rats with CHF was increased and the level of p-Smad3was also markedly increased both were reversed by QLtherapy These results suggest that the significant activationof TGF-1205731Smad3 signaling pathway in lung tissues afterCHF can lead to myofibroblast proliferation and a markedupregulation of type I collagen expression suggesting thatQLcould suppress the TGF-1205731Smad3 signaling pathway whichmight contribute to its attenuation of lung fibrosis in CHF

We also found that QL can reverse the increased TLR4and TNF-120572 expression in lung tissues of CHF TLR4 a mem-ber of the family of Toll-like receptors (TLRs) an importantreceptor of pattern recognition can induce the secretion ofmany inflammatory cytokines includingTNF-120572 EndogenousTLR4 ligands not only are important in the regulationof innate and adaptive immune responses but also areinvolved in noninfectious inflammatory and fibrotic diseasesof the heart lung and kidney Moreover mice with genetic



TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771

(c)Figure 8 Effect of qili qiangxin (QL) on expression of protein related to lung inflammation in rats with chronic heart failure (CHF) ((a) (b))Expression of Toll-like receptor-4 (TLR4) ((a) (c)) Tumor necrosis factor-120572 (TNF-120572) (998771119875 lt 005 versus the Sham group lowast119875 lt 005 versusthe CHF group)

ablation of TLR4 are protected from experimentally inducedinflammation and fibrogenesis in these organs suggesting animportant functional role of TLR4 in sustaining pathologicalfibrogenesis [20ndash24] Although multiple mechanisms under-lie these profibrotic effects of TLR4 in fibroblasts enhancedSmad signaling which normally limits the intensity andduration of fibrotic responses appears to be prominent [25]TNF-120572 is a proinflammatory cytokine that plays an importantrole in the activation of host defense and is associated withincreased mortality in CHF patients and the developmentof pulmonary fibrosis [26 27] Sullivan et al confirmedthat TNF-120572 controls the expression of TGF-1205731 synthesisthroughout the MEKERK pathway and AP-1 activation [2829] The present study demonstrated that TNF-120572 expressionin lung tissues of CHF increased and was reversed by QLtherapy Thus our results demonstrated that QL may exertits proinflammatory and profibrotic effect by modulationof TLR4 and downstream of TNF-120572 and TGF-1205731Smad3signaling pathway

5 Conclusions

The present study provides evidence that using QL for 4weeks improves pulmonary structure remodeling in rats withpost-MI congestive heart failure Based on our results we

conclude that QL accomplishes its antifibrotic effects mainlyby suppressing the TGF-1205731Smad3 signaling pathway whichmight contribute to its attenuation of myofibroblast prolifer-ation and collagen depositionThe possible mechanisms mayinvolve inhibition of TLR4 intracellular signaling Furtherstudies should explore the effects of QL on myofibroblastproliferation and TLR4 intracellular signaling pathway invitro to ultimately offer new avenues for the prevention andtreatment of this and other related diseases

Conflict of Interests

All authors declare that they have no conflict of interests

Acknowledgment

This work was supported by the National Science Foundationof China (Grant no 81273945)

References

[1] M Guglin and H Khan ldquoPulmonary hypertension in heartfailurerdquo Journal of Cardiac Failure vol 16 no 6 pp 461ndash4742010

[2] S VAbramson J F Burke J J Kelly Jr et al ldquoPulmonary hyper-tension predicts mortality and morbidity in patients with


dilated cardiomyopathyrdquo Annals of Internal Medicine vol 116no 11 pp 888ndash895 1992

[3] J Butler D B Chomsky and J R Wilson ldquoPulmonary hyper-tension and exercise intolerance in patients with heart failurerdquoJournal of the American College of Cardiology vol 34 no 6 pp1802ndash1806 1999

[4] S Ghio A Gavazzi C Campana et al ldquoIndependent and addi-tive prognostic value of right ventricular systolic function andpulmonary artery pressure in patients with chronic heart fail-urerdquo Journal of the American College of Cardiology vol 37 no 1pp 183ndash188 2001

[5] B K Gehlbach and E Geppert ldquoThe pulmonarymanifestationsof left heart failurerdquo Chest vol 125 no 2 pp 669ndash682 2004

[6] J-F Jasmin A Calderone T-K Leung L Villeneuve and JDupuis ldquoLung structural remodeling and pulmonary hyper-tension after myocardial infarction complete reversal withirbesartanrdquo Cardiovascular Research vol 58 no 3 pp 621ndash6312003

[7] M Rauchhaus W Doehner D P Francis et al ldquoPlasma cy-tokine parameters and mortality in patients with chronic heartfailurerdquo Circulation vol 102 no 25 pp 3060ndash3067 2000

[8] A Deswal N J Petersen A M Feldman J B Young B GWhite and D L Mann ldquoCytokines and cytokine receptors inadvanced heart failure an analysis of the cytokine databasefrom the Vesnarinone Trial (VEST)rdquo Circulation vol 103 no16 pp 2055ndash2059 2001

[9] M Nian P Lee N Khaper and P Liu ldquoInflammatory cytokinesand postmyocardial infarction remodelingrdquo Circulation Re-search vol 94 no 12 pp 1543ndash1553 2004

[10] J F Navarro-Gonzalez and C Mora-Fernandez ldquoThe role ofinflammatory cytokines in diabetic nephropathyrdquo Journal of theAmerican Society of Nephrology vol 19 no 3 pp 433ndash442 2008

[11] A Groth B Vrugt M Brock R Speich S Ulrich and L CHuber ldquoInflammatory cytokines in pulmonary hypertensionrdquoRespiratory Research vol 15 no 1 article 47 2014

[12] Y Chen H Guo D Xu et al ldquoLeft ventricular failure producesprofound lung remodeling and pulmonary hypertension inmice Heart failure causes severe lung diseaserdquo hypertensionvol 59 no 6 pp 1170ndash1178 2012

[13] X Li J Zhang J Huang et al ldquoA multicenter randomizeddouble-blind parallel-group placebo-controlled study of theeffects of qili qiangxin capsules in patients with chronic heartfailurerdquo Journal of the American College of Cardiology vol 62no 12 pp 1065ndash1072 2013

[14] W Liu J Chen T Xu et al ldquoQiliqiangxin improves cardiacfunction in spontaneously hypertensive rats through the inhi-bition of cardiac chymaserdquo American Journal of Hypertensionvol 25 no 2 pp 250ndash260 2012

[15] Y Zou L Lin Y Ye et al ldquoQiliqiangxin inhibits the devel-opment of cardiac hypertrophy remodeling and dysfunctionduring 4 weeks of pressure overload in micerdquo Journal ofCardiovascular Pharmacology vol 59 no 3 pp 268ndash280 2012

[16] XN Cui J Zhang Y B Li et al ldquoEffects of qili qiangxin capsuleon AQP2 V2R and AT1R in rats with chronic heart failurerdquoEvidence-Based Complementary and Alternative Medicine vol2015 Article ID 639450 11 pages 2015

[17] H Xiao Y Song Y Li Y-H Liao and J Chen ldquoQiliqiangxinregulates the balance between tumor necrosis factor-120572 andinterleukin-10 and improves cardiac function in rats withmyocardial infarctionrdquo Cellular Immunology vol 260 no 1 pp51ndash55 2009

[18] X M Zhang S Q Yang and A P Hou ldquoEffects of Qili-Qiangxin capsule oncardiac and pulmonary functions in heartfailure patients with chronic pulmonary heart diseaserdquo Int JTrad Chin Med vol 37 no 2 pp 130ndash133 2015

[19] L P Sun and L C Zhang ldquoTherapeutic effect of Qiliqiangxincapsule on the patients with chronic congestive heart failurerdquoChinese Journal of Difficult and Complicated Cases vol 6 no 2pp 71ndash72 2007

[20] M Ikeuchi H Tsutsui T Shiomi et al ldquoInhibition of TGF-betasignaling exacerbates early cardiac dysfunction but prevents lateremodeling after infarctionrdquo Cardiovascular Research vol 64no 3 pp 526ndash535 2004

[21] S Frantz L Kobzik Y-D Kim et al ldquoToll4 (TLR4) expressionin cardiac myocytes in normal and failing myocardiumrdquo TheJournal of Clinical Investigation vol 104 no 3 pp 271ndash280 1999

[22] L Timmers J P G Sluijter J K van Keulen et al ldquoToll-likereceptor 4 mediates maladaptive left ventricular remodelingand impairs cardiac function after myocardial infarctionrdquoCirculation Research vol 102 no 2 pp 257ndash264 2008

[23] E J Birks L E Felkin N R Banner A Khaghani P J RBarton and M H Yacoub ldquoIncreased toll-like receptor 4 inthe myocardium of patients requiring left ventricular assistdevicesrdquo Journal of Heart and Lung Transplantation vol 23 no2 pp 228ndash235 2004

[24] K C Young SM AHussein R Dadiz et al ldquoToll-like receptor4-deficient mice are resistant to chronic hypoxia-induced pul-monary hypertensionrdquo Experimental Lung Research vol 36 no2 pp 111ndash119 2010

[25] E Seki S de Minicis C H Osterreicher et al ldquoTLR4 enhancesTGF-120573 signaling and hepatic fibrosisrdquo Nature Medicine vol 13no 11 pp 1324ndash1332 2007

[26] L A Ortiz J Lasky R F Hamilton Jr et al ldquoExpression of TNFand the necessity of TNF receptors in bleomycin-induced lunginjury in micerdquo Experimental Lung Research vol 24 no 6 pp721ndash743 1998

[27] P F PiguetM A Collart G E Grau Y Kapanci and P VassallildquoTumor necrosis factorcachectin plays a key role in bleomycin-induced pneumopathy and fibrosisrdquo Journal of ExperimentalMedicine vol 170 no 3 pp 655ndash663 1989

[28] D E Sullivan M Ferris H Nguyen E Abboud and AR Brody ldquoTNF-alpha induces TGF-beta1 expression in lungfibroblasts at the transcriptional level via AP-1 activationrdquoJournal of Cellular and Molecular Medicine vol 13 no 8 pp1866ndash1876 2009

[29] D E Sullivan M Ferris D Pociask and A R Brody ldquoTumornecrosis factor-alpha induces transforming growth factor-beta1expression in lung fibroblasts through the extracellular signal-regulated kinase pathwayrdquo American Journal of Respiratory Cellamp Molecular Biology vol 32 no 4 pp 342ndash349 2005

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


tissue and organ fibrosis remodeling in the heart lungand kidney [9ndash11] Anti-inflammatory therapies can preventpathological fibrosis in these organs Animal experimentshave also demonstrated that lung injury associated withCHF is characterized by excessive collagen deposition in thealveoli inside the vessels and in the vascular walls of largevessels accompanied by the accumulation of leukocytes andmacrophages and increased levels of transforming growthfactor-1205731 (TGF-1205731) TNF-120572 and Toll-like receptor-4 (TLR4)mRNA or protein content in the lungs [12] Thus control-ling excessive inflammation could potentially be of greattherapeutic benefit for inhibiting the progressive pulmonarystructural fibrosis remolding in CHF

Qili qiangxin (QL) capsule is a traditional Chinese med-icine that is widely used for the treatment of patients withCHF of all etiologies including hypertension coronary heartdisease and chronic obstructive pulmonary disease [13] Ourlaboratory and others have reported that QL can reducecardiac fibrosis remolding and improve cardiac function [14ndash16] The use of QL in CHF also has an immunomodulatoryeffect by decreasing the proinflammatory cytokine TNF-120572and increasing the anti-inflammatory cytokine IL-10 [17]The use of QL in patients with CHF caused by chronicobstructive pulmonary disease has shown positive effects onpulmonary function by improving gas diffusion and exercisecapacity [18] suggesting that QL could also have beneficialeffects on lung fibrosis and inflammation in CHF In thepresent study we used a postmyocardial infarction heartfailure model to determine whether QL can improve lungstructural remodeling associated with CHF by inhibitinglung inflammation and fibrosis and to elucidate the possiblemechanisms underlying the antifibrotic effect of QL Theeffects of QL were also compared with Valsartan an Ang-II receptor antagonist commonly used in clinical practicewhich is known to have heart-lung protective effects in thetreatment of CHF [6]

2 Materials and Methods

21 Vegetal Material QL consists of ginseng Radix Astra-gali Aconite Root Salvia miltiorrhiza Semen LepidiiApetali Cortex Periplocae Sepii Radicis Rhizoma AlismatisCarthamus tinctorius Polygonati odorati rhizoma SeasonedOrange Peel and Ramulus Cinnamomi (Yiling Pharmaceuti-cal Corporation Shijiazhuang China)We dissolved the drugpowder in sterile water at a concentration of 01 gmL QL forthe study Valsartan (batch number X1428) manufactured byBeijing Novartis Pharmaceutical Co Ltd was dissolved insterile water

22 Animal Model and Administration Healthy maleSprague-Dawley rats (body weight 220sim250 g) were providedby Beijing Vital River Laboratory Animal Technology CoLtd (animal license number SCXK (Beijing) 2012-0001)The animals were fed a standard diet and water was providedad libitum The rats were maintained under a 12 h lightand 12 h dark cycle at a temperature of 20 plusmn 2∘C and ata humidity of 50 plusmn 2 All animal experimental protocolswere approved by the Animal Care and Use Committee

Table 1 Echocardiographic ejection fraction level in the studygroups before treatment with qili qiangxin (QL) (119909 plusmn 119904)

Group 119899 EF ()Sham 10 84490 plusmn 73354CHF 12 44708 plusmn 84369lowast

QL 9 47933 plusmn 91211lowast

Valsartan 9 47911 plusmn 91068lowastlowast

119875 gt 005 versus the Sham group

of Beijing University of Chinese Medicine and compliedwith laboratory animal management and use regulationsThe CHF model was induced by myocardial infarctionfollowing ligation of the left anterior descending artery(LAD) A 1 sodium pentobarbital solution (50mgkg) wasadministered by intraperitoneal injection The proceduresperformed included endotracheal intubation positivepressure ventilation using a ventilator preoperative recordingof a 12-lead ECG local skin disinfection chest openingthoracotomy device setup and opening of the pericardiumthe pulmonary cone and the left atrial appendage 2sim3mmfrom the bottom of the left anterior descending coronaryartery ligation For the rats assigned to the Sham groupthe same operation was performed without ligation of theleft coronary artery A 12-lead ECG was recorded after theexperiment Rats with myocardial infarctions (MI) werefed normally for 4 weeks Based on the transthoracic ech-ocardiography results (Table 1) the surviving rats wererandomly assigned to the following groups Model group(CHF 119899 = 12) Sham group (Sham 119899 = 10) QL group(QL 119899 = 9) and Valsartan group (Valsartan 119899 = 9) QL(10 gkg) and Valsartan (10mgkg) were administered bygavage once a day during the 4 weeks An equal volume ofdistilled water was used for the Model and Sham groupsSee Table 1 echocardiographic ejection fraction levels in thestudy groups before treatment with QL

23 Transthoracic Echocardiography Measurements A non-invasive transthoracic echocardiographymethod was used toevaluate the morphology and function of the left ventricleEchocardiography was performed on anesthetized animalsusing a two-dimensional mode that is a time-motion (TM)mode and blood flow was measured using the pulsedDoppler mode

24 Sample Preparation At the end of the 4-week treatmentthe rats were anesthetized The heart lungs and other majororganswere harvested andweighedThe lung tissuewas driedat 58∘C to a constant weightThe relative water content of thelung tissue was calculated The left lung and LV were snap-frozen in liquid nitrogen pending biochemical analysis Theairways of the upper right lobe and hearts were subsequentlyfixed in 4 paraformaldehyde and embedded in paraffin forhistological analysis

25 Histological Staining The relative pulmonary vascularmuscularization was determined using hematoxylin and












3 Results






0

20

40

60

80

100


(a) (b)

(c) (d)

EF (

)

0

20

40

60

80

100


FS (

)

0

02

04

06

08

12

1

ESV

(mL)

0

2

4

6

8

10


LVID

s (m

m)


lowast

lowast

lowast lowast998771

998771998771

lowast998771

lowast998771

998771

lowast

lowast lowast

998771 998771

998771998771

lowast

lowast998771 lowast

998771







QL Valsartan0

5

10

15

20

25

30

Sham CHF

Sham CHF

QL Valsartan

LV co

llage

n de

posit

ion

area

() lowast

lowast

998771


0

005

01

015


Wet

lung

bod

y w

eigh

t (

)

lowast

lowast

998771

(a)

0

0005

001

0015

002

0025


Dry

lung

bod

y w

eigh

t (

)

lowastlowast

998771

(b)

0

5

10

15

20

25


Dry

wet

lung

wei

ght (

)

(c)




10

20

30

40

50

60

70

80

90

100

Lung

vas

cula

r mus

cula

rizat

ion

()

lowast

lowast

lowast

lowast

998771

998771


Sham CHF


0

5

10

15

20


Lung

colla

gen

frac

tiona

l are

a (

) lowast

lowast

998771

QL Valsartan

Sham CHF





4 Discussion




120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)








GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results






0

20

40

60

80

100


(a) (b)

(c) (d)

EF (

)

0

20

40

60

80

100


FS (

)

0

02

04

06

08

12

1

ESV

(mL)

0

2

4

6

8

10


LVID

s (m

m)


lowast

lowast

lowast lowast998771

998771998771

lowast998771

lowast998771

998771

lowast

lowast lowast

998771 998771

998771998771

lowast

lowast998771 lowast

998771







QL Valsartan0

5

10

15

20

25

30

Sham CHF

Sham CHF

QL Valsartan

LV co

llage

n de

posit

ion

area

() lowast

lowast

998771


0

005

01

015


Wet

lung

bod

y w

eigh

t (

)

lowast

lowast

998771

(a)

0

0005

001

0015

002

0025


Dry

lung

bod

y w

eigh

t (

)

lowastlowast

998771

(b)

0

5

10

15

20

25


Dry

wet

lung

wei

ght (

)

(c)




10

20

30

40

50

60

70

80

90

100

Lung

vas

cula

r mus

cula

rizat

ion

()

lowast

lowast

lowast

lowast

998771

998771


Sham CHF


0

5

10

15

20


Lung

colla

gen

frac

tiona

l are

a (

) lowast

lowast

998771

QL Valsartan

Sham CHF





4 Discussion




120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)








GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100


(a) (b)

(c) (d)

EF (

)

0

20

40

60

80

100


FS (

)

0

02

04

06

08

12

1

ESV

(mL)

0

2

4

6

8

10


LVID

s (m

m)


lowast

lowast

lowast lowast998771

998771998771

lowast998771

lowast998771

998771

lowast

lowast lowast

998771 998771

998771998771

lowast

lowast998771 lowast

998771







QL Valsartan0

5

10

15

20

25

30

Sham CHF

Sham CHF

QL Valsartan

LV co

llage

n de

posit

ion

area

() lowast

lowast

998771


0

005

01

015


Wet

lung

bod

y w

eigh

t (

)

lowast

lowast

998771

(a)

0

0005

001

0015

002

0025


Dry

lung

bod

y w

eigh

t (

)

lowastlowast

998771

(b)

0

5

10

15

20

25


Dry

wet

lung

wei

ght (

)

(c)




10

20

30

40

50

60

70

80

90

100

Lung

vas

cula

r mus

cula

rizat

ion

()

lowast

lowast

lowast

lowast

998771

998771


Sham CHF


0

5

10

15

20


Lung

colla

gen

frac

tiona

l are

a (

) lowast

lowast

998771

QL Valsartan

Sham CHF





4 Discussion




120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)








GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















QL Valsartan0

5

10

15

20

25

30

Sham CHF

Sham CHF

QL Valsartan

LV co

llage

n de

posit

ion

area

() lowast

lowast

998771


0

005

01

015


Wet

lung

bod

y w

eigh

t (

)

lowast

lowast

998771

(a)

0

0005

001

0015

002

0025


Dry

lung

bod

y w

eigh

t (

)

lowastlowast

998771

(b)

0

5

10

15

20

25


Dry

wet

lung

wei

ght (

)

(c)




10

20

30

40

50

60

70

80

90

100

Lung

vas

cula

r mus

cula

rizat

ion

()

lowast

lowast

lowast

lowast

998771

998771


Sham CHF


0

5

10

15

20


Lung

colla

gen

frac

tiona

l are

a (

) lowast

lowast

998771

QL Valsartan

Sham CHF





4 Discussion




120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)








GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10

20

30

40

50

60

70

80

90

100

Lung

vas

cula

r mus

cula

rizat

ion

()

lowast

lowast

lowast

lowast

998771

998771


Sham CHF


0

5

10

15

20


Lung

colla

gen

frac

tiona

l are

a (

) lowast

lowast

998771

QL Valsartan

Sham CHF





4 Discussion




120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)








GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120572-SMA

Collagen I

GAPDH

(a)

0

02

04

06

08

1

12

14

16


120572-S

MA

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


lowast lowast

998771

Col

lage

n I e

xpre

ssio

n

(c)


(d)








GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















GAPDH

TGF-1205731

p-Smad3

(a)

0

04

08

12

16

2


TGF-1205731

expr

essio

n

lowast lowast

998771

(b)

0

04

08

12

16

2


p-Sm

ad3

expr

essio

n lowast lowast

998771

(c)








TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















TLR4

TNF-120572

GAPDH

(a)

0

02

04

06

08

1

12

14

16

18


TLR4

expr

essio

n lowast

lowast

998771

(b)

0

02

04

06

08

1

12


TNF-120572

expr

essio

n lowast lowast

998771



5 Conclusions





Acknowledgment


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 beneficial effects of qili qiangxin...

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