1-s2.0-S0378874113005710-main

Paeoniflorin,the main active constituent of Paeonia lactiflora roots,
attenuates bleomycin-induced pulmonaryfibrosis in mice by
suppressing the synthesis of type I collagen
Yu Ji a,Ting Wang a,Zhi-feng Wei a,Guo-xun Lu a,Si-de Jiang b,Yu-feng Xia b,n,Yue Dai a,nn
a Department of Pharmacology of Chinese Materia Medica,China Pharmaceutical University,24Tong Jia Xiang,Nanjing210009,China
b Department of Chinese Materia Medica Analysis,China Pharmaceutical University,24Tong Jia Xiang,Nanjing210009,China
a r t i c l e i n f o
Article history:
Received21March2013
Received in revised form
1July2013
Accepted6August2013
Available online22August2013
Keywords:
Paeoniflorin
Pulmonaryfibrosis
Extracellular matrix
Type I collagen
TGF-β/Smad pathway
a b s t r a c t
Ethnopharmacological relevance:In the theory of traditional Chinese medicine,pulmonaryfibrosis(PF)
belongs to pulmonary arthralgia,which means blood stasis in lung tissue.The roots of Paeonia lactiflora
Pall are usually used to relieve the symptoms of this disease by promoting blood circulation and
removing blood stasis.Paeoniflorin,the main active ingredient of ctiflora,may have anti-PF potential.
Aim of study:This study aimed to investigate the effects and underlying mechanisms of paeoniflorin on
bleomycin(BLM)-induced PF in mice.
Materials and Methods:The PF model was established in mice by an intratracheal instillation of BLM.
Paeoniflorin(25,50,100mg/kg)and prednisone(6mg/kg),as a positive control,were orally adminis-
tered for consecutive21days.Histopathological changes were evaluated by hematoxylin and eosin stain
and Masson's trichrome stain.The content of hydroxyproline was detected by using kits.The contents of
type I collagen,TGF-β1and IFN-γwere detected by ELISA.The levels ofα-SMA,Smad4,Smad7and the
phosphorylations of Smad2/3were detected by western blot.The mRNA expressions of MMP-1and
TIMP-1were detected by RT-PCR.
Results:In mice treated with BLM,paeoniflorin(50mg/kg)significantly prolonged the survival periods,
attenuated infiltration of inflammatory cells,interstitialfibrosis,and deposition of extracellular matrix in
lung tissues.It also decreased the contents of hydroxyproline(a marker of collagens),type I collagen and
α-SMA(an indicator of myofibroblasts)in lung tissues of mice.Paeoniflorin down-regulated the
expressions of TGF-β1,Smad4and the phosphorylations of Smad2/3,while up-regulated the expression
of Smad7in lung tissues.Moreover,paeoniflorin increased the content of IFN-γ.But,it only slightly
affected mRNA expressions of MMP-1and TIMP-1in lung tissues of mice.
Conclusions:Paeoniflorin attenuates PF by suppressing type I collagen synthesis via inhibiting the
activation of TGF-β/Smad pathway and increasing the expression of IFN-γ.
&2013Elsevier Ireland Ltd.All rights reserved.
1.Introduction
Pulmonaryfibrosis(PF)is a devastating and fatal lung disease,
and its incidence is about8.5per100,000people every year.In
addition,the expected survival time of patient with PF is likely to
be only2.5–3.5years(Ley et al.,2011).Cigarette smoking(Oh
et al.,2012),metal and wood dust are considered as the most
important environmental risk factors which contribute to PF.Up to
date,there is no anti-PF drug that has been approved by US Food
and Drug Administration(FDA).Prifenidone,a novel anti-fibrotic
drug,has shown its considerable ability of attenuating PF in
clinical trials(Gan et al.,2011).However,it has multiple side
effects such as nausea,dyspepsia,photosensitivity and rash.
The precise pathogenesis of PF is not yet completely under-
stood,but the imbalance of synthesis and degradation of collagen
in lung tissues has been recognized as a crucial reason for the
excessive deposition of extracellular matrix(ECM),which leads to
scar and destruction of the lung architecture(Polyakova et al.,
2011).Multiple factors are involved in the regulation for synthesis
and degradation of collagen.Transforming growth factor(TGF)-β1,
afibrogenic cytokine,plays a critical role in the production of
collagen in PF.It is able to up-regulate mRNA expression of type I
collagen,promote the proliferation offibroblasts and drive the
differentiation offibroblasts to myofibroblasts,the main resource
cells of collagen(Bataller and Brenner,2005).On the contrary,
interferon(IFN)-γsuppresses the synthesis of collagen through
activating JAK/STAT pathway(Du et al.,2011a,2011b).On the other
Contents lists available at ScienceDirect
journal homepage:/locate/jep
Journal of Ethnopharmacology
0378-8741/$-see front matter&2013Elsevier Ireland Ltd.All rights reserved.
/10.1016/j.jep.2013.08.017
n Corresponding author.
nn Corresponding author.Tel.:þ862583271400;fax:þ862585301528.
E-mail addresses:yfxiacpu@(Y.-f.Xia),
yuedaicpu@(Y.Dai).
Journal of Ethnopharmacology149(2013)825–832
hand,matrix metalloproteinases (MMPs)are key enzymes respon-sible for the degradation of ECM,and tissue inhibitors of metallo-proteinases (TIMPs)can inhibit the activity of MMPs by forming 1:1complexes with MMPs (Sundararajan et al.,2012).
Paeonia lacti flora Pall root,a famous traditional Chinese med-icine (TCM),has been used for more than 1200years because of its anti-in flammatory and immune-regulatory properties.Paeoni-florin (Fig.1),the principal bioactive ingredient in cti flora ,has been previously reported to attenuate liver fibrosis induced by Schistosoma japonicum egg in mice (Li et al.,2009).It can also inhibit the expressions of ICAM-1,MCP-1,IL-6and TNF-αin endothelial cells stimulated by lysophosphatidylcholine (Li et al.,2013).This study was designed to investigate the effects of paeoni florin on PF induced by bleomycin (BLM)in mice.
2.Materials and methods 2.1.Chemicals and reagents
Paeoni florin (purity 495%)was purchased from Nanjing ZeLang Medical Technology Co.,Ltd.(Nanjing,China);prednisone acetate was purchased from Zhejiang Xianju Pharmaceutical Co.,Ltd.(Taiz-hou,China);BLM hydrochloride was purchased from Nippon Kayaku (Tokyo,Japan);Tween 20,bovine serum albumin (BSA),sodium dodecyl sulfate (SDS),dithiothreitol (DTT)and phenylmethylsulfonyl fluoride (PMSF)were purchased from Sigma Chemical Co.(St.Louis,MO,USA);hydroxyproline assay kits were purchased from Nanjing Jiancheng Bio-engineering Instute (Nanjing,China);TGF-β1and IFN-γenzyme linked immunosorbent assay (ELISA)kits were purchased from R&D system (MN,USA);Type I collagen ELISA kits were purchased from Abcam (Cambridge,UK);rabbit anti-α-SMA mono-clonal antibody was purchased from Epitomics (Burlingame,Califor-nia,USA);rabbit anti-p-Smad3,anti-p-Smad2,anti-Smad2/3,anti-Smad4and anti-Smad7antibodies were purchased from cell signal-ing technology (Boston,MA,USA);GAPDH monoclonal antibody was purchased from Kangchen Biotech (Shanghai,China);TRIzol reagent,M-MLV reverse transcriptase system and Taq polymerase were purchased from TransGen Biotech (Beijing,China).Other chemicals and reagents used were of analytical grade.2.2.Animals
Male ICR mice,weighing 20–25g,were purchased from Com-parative Medicine Center of Yangzhou University (SCXK 2012-0004).The animals were acclimated to the laboratory for at least 7days before being tested at a constant temperature (23721C).Mice were maintained on a standard diet and water was made
available freely.The animal care and use was complied with the Provisions and General Recommendation of the Chinese Experi-mental Animals Administration Legislation.And the study protocol was approved by the Institutional Ethical Committee of China Pharmaceutical University.2.3.BLM-induced PF in mice
PF was established in mice by an intratracheal instillation of BLM hydrochloride (5mg/kg in saline).After instillation,mice were swung for 2min to make sure that BLM was well-distributed in lungs.On day 1,mice were divided into normal group,model group,paeoni florin (25,50and 100mg/kg)-treated groups,and prednisone (6mg/kg)-treated group.Paeoni florin and prednisone were orally administrated for consecutive 21days.Mice in normal and model groups were orally given an equal volume of vehicle (water)in the same schedule.
The body weights of mice were measured per day,and the time of death was recorded.On day 21,mice were sacri ficed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were taken.2.4.Histopathological examination
The lower lobes of left lung tissues were fixed in 10%formalin and embedded in paraf fin,sectioned,and then selected for hematoxylin and eosin (H&E)stain and Masson's trichrome stain.The results of H&E stain were graded according to epithelial proliferation,alveolitis,edema,in flammatory cell in filtration and interstitial fibrosis,and the results of Masson's trichrome stain were graded according to the levels of ECM.Criteria for grading were as follows:grade 0,normal;grade 0.5,slight;grade 1,mild;grade 2,moderate;and grade 3,severe.2.5.Hydroxyproline analysis
The upper lobes of left lungs of mice were collected.About 40mg of lung tissues were mixed with hydrolysate (1ml)and hydrolyzed in water at 951C for 20min.The contents of hydro-xyproline were detected by using kits according to the manufac-turer's instructions.2.6.ELISA
The lower lobes of right lung tissues were prepared for ELISA.About 40mg of lung tissues were homogenated with 400μl PBS.After centrifugation of the homogenate for 15min at a speed of 3000rpm,the supernatants were obtained and stored in a freezer (À701C).The contents of type I collagen,TGF-β1and INF-γin lung tissues were detected by ELISA according to the manufacturers'instructions.2.7.Western blot analysis
The lower lobes of right lung tissues were prepared.The levels of α-SMA,p-Smad2,p-Smad3,Smad2/3,Smad4and Smad7in lung tissues were detected by western blot.Proteins were extracted from 40mg of lung tissues with lysis buffer (50mM Tris –HCl,150mM NaCl,0.02%NaN 3,1%NP40)for 30min on ice.The supernatants were harvested and centrifuged for 10min at a speed of 12,000rpm.The protein contents were quanti fied by the Bradford assay.Protein samples were separated on a 12%SDS-PAGE gel and transferred to PVDF membranes.Membranes were blocked for 2h at room temperature with 10%nonfat milk in TBST (TBS-Tween-20)and incubated with the corresponding antibodies.The membranes were incubated with ECL reagent for 2–10min and exposed to X-ray fi
lm.
Fig.1.Chemical structure of paeoni florin.
Y.Ji et al./Journal of Ethnopharmacology 149(2013)825–832
826
2.8.Reverse transcriptase-polymerase chain reaction(RT-PCR)
analysis
The mRNA levels of MMP-1and TIMP-1in middle lobes of right
lung tissues were detected by RT-PCR.Total RNA were extracted
from40mg lung tissues of BLM-treated mice with TRIzol(1ml).
The concentrations were determined by optical density measure-
ment at260nm on a spectrophotometer.2μg RNA was used for reverse transcriptase according to the manufacturer's instructions.
2μl cDNA mixed with reaction buffer,dNTPs,Taq polymerase in 50μl reaction mixture were transferred to different PCRs.PCR products were determined by using1.5%agarose gel electrophor-esis and GoldView staining.Images of the gels were analyzed using the Quantity One software(Bio-Rad,Hercules,CA,USA).The
primers used in RT-PCR were as follows:MMP-1:(Forward Primer:
5′-TGA ATG GCA AGG AGA TGA TGG-3′;Reverse Primer:5′-TGG
AAG GAG AGC ACA ATA TCG-3′);TIMP-1:(Forward Primer:5′-CGC
AGA TAT CCG GTA CGC CTA-3′;Reverse Primer:5′-CAC AAG CCT
GGA TTC CGT GG-3′);β-actin:(Forward Primer:5′-AGC CAT GTA CGT AGC CAT CC-3′;Reverse Primer:5′-CTC TCA GCA GTG GTG
GTG AA-3′).
2.9.Statistical analysis
All data were expressed as means7S.D.Survival rates between
the groups were evaluated by log-rank test.The scores of
alveolitis
Fig.2.Effects of paeoniflorin(Pae)and prednisone(Pred)on the survival rates and histopathological changes in lung tissues of bleomycin(BLM)-treated mice.On day21 after BLM instillation,mice were sacrificed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.(A)Survival rates were shown over a21day observation period.(B)The upper lobes of left lung tissues from different groups were taken photos.(C)Histopathological changes of the lower lobes of left lung tissues were examined by H&E stain(magnification200x).(D)The histological scores of all groups were calculated.Data were expressed as means7S.D.,n¼6.##P o0.01vs.normal; n P o0.05,nn P o0.01vs.model.
Y.Ji et al./Journal of Ethnopharmacology149(2013)825–832827
and fibrosis between the groups were evaluated by Mann –Whitney U test.Other statistical signi ficance was evaluated by one-way analysis of variance (ANOVA)followed by post hoc Tukey's test.P values less than 0.05(P o 0.05)were accepted as a signi ficant difference.
3.Results
3.1.Effect of paeoni florin on PF induced by BLM in mice
First of all,we investigated the impacts of paeoni florin on the survival rates and histopathological changes of lung tissues in mice with PF induced by BLM.As shown in Fig.2A,intratracheal instillation of BLM reduced the survival rates of mice to 50%.Paeoni florin,at the dose of 50mg/kg,signi ficantly increased the survival rates of mice to 87.5%.However,prednisone did not affect the survival rates of BLM-treated mice.
On day 21after BLM instillation,mice were sacri ficed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.The upper lobes of left lungs were taken photos,and the histopathological changes were detected by H&E stain.Results showed that the lungs of mice in normal group were soft with white color.However,the lungs of mice in model group were hard and shrunken in size with pale color.Paeoni florin (50and 100mg/kg)and prednisone (6mg/kg)treatments resulted in the lungs of mice softer with pink color.Moreover,the lung tissues of mice in model group presented severe epithelial proliferation,alveolitis,edema,in flammatory cell in filtration and interstitial fibrosis.Both paeoni florin (100mg/kg)and prednisone (6mg/kg)treatments signi ficantly reduced the histopathological changes in lung tissues of mice induce by BLM (Fig.2B and C).It was suggested that paeoni florin could signi ficantly alleviate BLM-induced PF in mice.3.2.Effect of paeoni florin on ECM deposition in lung tissues of mice induced by BLM
At the late stage of PF,excessive deposition of ECM leads to progressive loss of lung function.Type I collagen constitutes the
large part of ECM (Raghu et al.,1985;Crouch,1990).Hydroxypro-line,a speci fic marker of collagen,can be used to re flect the content of total collagen.
In the present study,BLM instillation resulted in excess production of ECM in mouse lung tissues as evidenced by Masson's trichrome stain.Paeoni florin (50and 100mg/kg)and prednisone (6mg/kg)treatments obviously attenuated the pro-duction of ECM in lung tissues (Fig.3A and B).In consistent with this,paeoni florin (50and 100mg/kg)and prednisone (6
mg/kg)
Fig.3.Effects of paeoni florin (Pae)and prednisone (Pred)on the levels of extracellular matrix in the lung tissues of bleomycin (BLM)-treated mice.On day 21after BLM instillation,mice were sacri ficed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.(A)Masson's trichrome stain (magni fication 200x).(B)The histological scores of all groups were calculated.(C)The contents of hydroxyproline in the upper lobes of left lung tissues were measured by using kits according to manufacturer's instruction.(D)The contents of type I collagen in the upper lobes of right lung tissues were detected by ELISA.Data were expressed as means 7S.D.,n ¼6.#
P o 0.05,##P o 0.01vs.normal;n P o 0.05,nn P o 0.01vs.
model.
Fig.4.Effects of paeoni florin (Pae)and prednisone (Pred)on the levels of α-SMA in the lung tissues of bleomycin (BLM)-treated mice.On day 21after BLM instillation,mice were sacri ficed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.The levels of α-SMA in the lower lobes of lung tissues were detected by western blot.Data were expressed as means 7S.D.,n ¼6.##
P o 0.01vs.normal;n P o 0.05vs.model.
Y.Ji et al./Journal of Ethnopharmacology 149(2013)825–832
828
treatments significantly decreased the contents of hydroxyproline, and the inhibitory percentages were68.1%,72.1%and70.1%, respectively(Fig.3C).The elevated content of type I collagen in lung tissues of BLM-induced mice were also decreased by paeoni-florin(50and100mg/kg)and prednisone(6mg/kg)treatments. Their inhibitory percentages were39.2%,43.7%and53.0%,respec-tively(Fig.3D).
3.3.Effect of paeoniflorin on the level ofα-SMA in lung tissues
of mice induced by BLM
α-SMA is recognized to be the hallmark of myofibroblasts,the main source cells of type I collagen.In the lung tissues of mice induced by BLM,the levels ofα-SMA were significantly elevated. As depicted in Fig.4,paeoniflorin(100mg/kg)and prednisone (6mg/kg)produced marked inhibition ofα-SMA levels in lung tissues of mice,and the inhibitory percentages were72.6%and 91.8%,respectively.
3.4.Effect of paeoniflorin on the activation of TGF-β/Smad pathway in lung tissues of mice induced by BLM
Among all pro-fibrotic cytokines,TGF-β1is crucial in promot-ing proliferation offibroblasts,driving differentiation offibroblasts to myofibroblasts and increasing synthesis of type I collagen.As depicted in Fig.5A,BLM instillation resulted in significant increase of TGF-β1expression in lung tissues of mice.Paeoniflorin(50and 100mg/kg)and prednisone(6mg/kg)treatments obviously decreased the levels of TGF-β1.
TGF-β1functions mainly through the activation of Smads, which locate at the downstream of TGF-βreceptors.Phosphory-lated Smad2/3can combine with Smad4,forming Smad2/3-Smad4 complexes,and translating into the nucleus to regulate the
mRNA
Fig.5.Effects of paeoniflorin(Pae)and prednisone(Pred)on the activation of TGF-β/Smad pathway in the lung tissues of bleomycin(BLM)-treated mice.On day21after BLM instillation,mice were sacrificed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.(A)The contents of TGF-β1in the upper lobes of right lung tissues were analyzed by ELISA.(B)The expressions of p-Smad2,p-Smad3,Smad2/3,Smad4and Smad7in the lower lobes of right lung tissues were detected by western blot.(C)The semi-quantitative analysis of p-Smad2.(D)The semi-quantitative analysis of p-Smad3.(E)The semi-quantitative analysis of Smad4.(F)The semi-quantitative analysis of Smad7.Data were expressed as means7S.D.,n¼6.##P o0.01vs.normal;n P o0.05,nn P o0.01vs.model.
Y.Ji et al./Journal of Ethnopharmacology149(2013)825–832829
expression of type I collagen.Smad7inhibits the phosphorylations of Smad2/3.Therefore,we investigated the effects of paeoni florin on the phosphorylations of Smad2/3and the expressions of Smad4and Smad7in mouse lung tissues induced by BLM.Data showed that BLM instillation resulted in increased phosphorylations of Smad2/3and expression of Smad4,but decreased expression of Smad7.Paeoni florin (100mg/kg)and prednisone (6mg/kg)treat-ments obviously down-regulated Smad2/3phosphorylations and Smad4expression,and conversely up-regulated Smad7expression (Fig.5B –F).These findings implicated that the anti-fibrotic effect of paeoni florin was achieved by suppressing synthesis of type I collagen via regulating TGF-β/Smad pathway.3.5.Effect of paeoni florin on the levels of IFN-γin lung tissues of mice induced by BLM
IFN-γ,an anti-fibrotic cytokine,plays important roles in con-trolling collagen synthesis.It interferes TGF-β1signals by increas-ing the expression of Smad7via activating the JAK/STAT signaling pathway (Ulloa et al.,1999).To recognize whether inhibition of paeoni florin on the synthesis of type I collagen was associated with IFN-γ,the levels of IFN-γin mouse lung tissues were detected by ELISA.As depicted in Fig.6,BLM instillation led to signi ficant decrease of IFN-γlevels in lung tissues,and paeoni florin (50and100mg/kg)and prednisone (6mg/kg)treatments increased IFN-γlevels.
3.6.Effect of paeoni florin on the expressions of MMP-1and TIMP-1mRNA in lung tissues of mice induced by BLM
MMP-1,which functions as collagenase-1,can degrade type I collagen in PF.TIMP-1inhibits the activity of MMP-1through binding with it.As described above,paeoni florin suppressed the synthesis of type I collagen by inhibiting the activation of TGF-β/Smad pathway and increasing the levels of IFN-γin mouse lung tissues induced by BLM.To recognize whether paeoni florin affect the degradation of type I collagen,its effects on the levels of MMP-1and TIMP-1were addressed.As depicted in Fig.7,BLM instilla-tion promoted the expressions of MMP-1and TIMP-1at mRNA levels,and paeoni florin only showed slight inhibition of MMP-1and TIMP-1expressions.On contrast,prednisone (6mg/kg)sig-ni ficantly decreased the mRNA expression of TIMP-1but not MMP-1.It was suggested that paeoni florin had little effect on the degradation of type I collagen.
4.Discussion
PF is a chronic in flammatory progressive and lethal lung disease with few treatments available.Although the precise pathogenesis and etiology of this disease are not yet understood,the main pathological changes have been de fined.Firstly,epithelial cells of lungs are injured and activated by chronic in flamma-tion.Secondly,abundant of proliferative factors,such as TGF-β1,PDGF,TNF-αand others that are produced mainly from the activated epithelial cells (Selman and Pardo,2006),provoke the proliferation of fibroblasts and the differentiation of fibroblasts to myo fibroblasts (Nathan et al.,2011).Thirdly,myo fibroblasts secrete a considerable amount of ECM.The imbalance between synthesis and degradation of ECM results in lung remodeling and dysfunction (Jin and Dong,2011
).
Fig.6.Effects of paeoni florin (Pae)and prednisone (Pred)on IFN-γcontent in the lung tissues of bleomycin (BLM)-treated mice.On day 21after BLM instillation,mice were sacri ficed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.The contents of IFN-γin the upper lobes of lung tissues were detected by ELISA.Data were expressed as means 7S.D.,n ¼6.##P o 0.01vs.normal;n P o 0.05,nn P o 0.01vs.
model.
Fig.7.Effects of paeoni florin (Pae)and prednisone (Pred)on the mRNA expres-sions of MMP-1and TIMP-1in the lung tissues of bleomycin (BLM)-treated mice.On day 21after BLM instillation,mice were sacri ficed with excess chloral hydrate hydrochloride anesthesia,and lung tissues were isolated.(A)The mRNA expres-sions of MMP-1and TIMP-1in the middle lobes of lung tissues were detected by RT-PCR.(B)The semi-quantitative analysis of MMP-1.(C)The semi-quantitative analysis of TIMP-1.Data were expressed as means 7S.D.,n ¼6.##P o 0.01vs.normal;n P o 0.05vs.model.
Y.Ji et al./Journal of Ethnopharmacology 149(2013)825–832
830
Many animal models of PF,induced by BLM,FITC,irradiation
and silica stimulation,have been adopted for experimental studies.
Up to now,BLM-induced PF in mice is considered to be the best
characterized model(Moore and Hogaboam,2008).It well
manifests pathological characteristics of PF,such as injury and
activation of epithelial cells,production of proliferative factors,
and excessive deposition of ECM in lung tissues(Moore and
Hogaboam,2008).Therefore,this model was selected to study
the anti-fibrosis effects of paeoniflorin.
In the development of PF,epithelial cells are initially damaged
and activated.They secrete a large number of proinflammatory
cytokines,which result in pathological changes in lung tissues,
such as epithelial proliferation,alveolitis,edema,and inflamma-
tory cell infiltration(Coward et al.,2010).In our study,paeoniflorin
significantly increased the survival rates of BLM-treated mice,but
prednisone did not show remarkable effects.Furthermore,the
results of H&E stain demonstrated that both paeoniflorin and
prednisone could obviously ameliorate alveolitis,infiltration of
inflammatory cells,and edema in mouse lung tissues induced by
BLM,and maintain the structure of lung tissues.
ECM is mainly composed of collagen,proteoglycan,fibronectin,
elastin and other matrix components.Among them,collagen is the
main component,and the predomination of collagen is type I
collagen(Friedman,2008).In the present study,Masson's tri-
chrome stain showed that paeoniflorin and prednisone could
significantly decrease ECM levels in mouse lung tissues induced
by BLM.Hydroxyproline is a special amino acid that derives from
collagen,and its level can represent the total content of collagen.
Paeoniflorin and prednisone significantly reduced the levels of
hydroxyproline in lung tissues of mice.Furthermore,paeoniflorin
was shown to decrease the content of type I collagen in lung
tissues of mice.These results indicated that both paeoniflorin and
prednisone were able to reduce ECM deposition in mouse lung
tissues induced by BLM.
The abnormally activated epithelial cells push forward the
initialfibrotic response.They can produce mediators that induce
the proliferation of resident mesenchymal cells,resulting in the
formation offibroblasts and myofibroblasts in lung tissues(King
et al.,2011).Myofibroblasts andfibroblasts are the major effector
cells,contributing to the excessive deposition of ECM.There were
reports demonstrating that type I collagen was mainly derived
from myofibroblasts,which play more important role in the
synthesis of type I collagen thanfibroblasts(Ju et al.,2012;
Sivakumar et al.,2012).Our results showed that paeoniflorin could
decrease the levels ofα-SMA,a specific marker of myofibroblasts, in mouse lung tissues induced by BLM,indicating that paeoniflorin
might function through reducing the numbers of myofibroblasts.
TGF-β1is a typical pro-fibrotic cytokine,which promotes fibroblast proliferation,drives the differentiation offibroblasts to myofibroblasts and increases the synthesis of type I collagen.Its overexpression is believed to contribute to the development of kidneyfibrosis and PF by activating the signal transducers Smads (Weiss et al.,2010;Lan and Chung,2012;Morty et al.,2009), which transmit signals inside cells and regulate the synthesis of type I collagen.Smad2/3,the receptor-regulated Smads(R-Smads), can bind to the common-partner Smad(Smad4),and then trans-locate into the nucleus(Verrecchia et al.,2006).After enters into the cell nucleus,the Smad2/3-Smad4complex binds to Smad-specific DNA sequences,and regulates the gene expression of type I collagen.In Smad3deficiency mice,progressivefibrosis cannot develop(Gauldie et al.,2006).On the other hand,Smad7,as an inhibitory Smad(I-Smad),can inhibit the activation of both TGF-βand bone morphogenetic protein(BMP)signaling pathways. It prevents the phosphorylation of R-Smad and the sub-sequent nucleus translocation of R-Smad/Smad4heterocomplexes. Furthermore,Smad7competitively inhibited the binding of Smad3to TGF-β-RI(Leask and Abraham,2004).The transfer of Smad7 gene could alleviate BLM-induced lungfibrosis in mice(Nakao et al.,1999).In this study,paeoniflorin was shown to obviously reduce the expression of TGF-β1,the phosphorylations of Smad2 and Smad3,and the expression of Smad4in mouse lung tissues induced by BLM.One the contrary,it increased the expression of Smad7.It was suggested that paeoniflorin reduced collagen I deposition in lung tissues through inhibition of TGF-β/Smad pathway.
On contrary,IFN-γ,as a classical anti-fibrotic cytokine,can inhibit TGF-β1expression,fibroblast proliferation,differentiation offibroblasts to myofibroblasts,and collagen synthesis(Aggarwal and Behera,2000).It prevents the signals of TGF-βthrough increasing the expressions of Smad7via activating JAK1and STAT1. In the present study,paeoniflorin and prednisone were shown to dramatically up-regulate IFN-γlevels in mouse lung tissues induced by BLM,suggesting that increase of IFN-γlevel might contribute to the anti-fibrosis effect of paeoniflorin.
Moreover,collagen degradation well contributes to the allevia-tion of ECM deposition.MMPs are a family of matrix-degrading proteinases,which are essential for ECM degradation in PF.MMP-1 (collagenase-1)is responsible for degradation of type I collagen, the predominate collagen of ECM in lung tissues of patients with PF(Gao et al.,2012).TIMP-1,an endogenous inhibitor of MMP1, can inhibit the activity of MMP-1via binding with MMP-1.In mouse lung tissues induced by BLM,the expressions of both MMP-1and TIMP-1mRNA were increased.Paeoniflorin showed little effect on the expressions,suggesting that it might not affect MMPs-mediated degradation of type I collagen.
5.Conclusions
Paeoniflorin can significantly attenuate pulmonaryfibrosis induced by BLM in mice.It acts mainly by suppressing ECM deposition in lung tissues through reducing the synthesis of type I collagen via down-regulating the expression of TGF-β1and activation of related signal pathway.Paeoniflorin has a therapeutic potential for the treatment of pulmonaryfibrosis.
Acknowledgments
This work was supported by the Innovative Training Plan for Graduate Students of Jiangsu Province(No.CXZZ11_0829),the Fundamental Research Funds for the Central Universities(No. JKY2011079),and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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