| As a traditional Chinese medicine,the pharmaceutical value of apocynum venetum is extensive.It has been implicated to posses the preventive and therapeutic effect in a number of cardiovascular diseases,including hypertension, hyperlipemia, atherosclerosis.Flavonoids and flavonols are major chemical constituents of apocynum venetum extract,and they play a protect role against cardiomyopathy.But it is not reported that the influence and prevention of apocynum venetum extract on myocardial fibrosisMyocardial fibrosis is the final common pathway and the major pathological changes in a variety of cardiac disease,characterized with the overproliferation of cardiac fibroblasts,increased of collagen synthesis and expanding of extracelluar matrix (ECM).Transforming growth factor-β(TGF-β), known as yet to be an important cytokine to cause fibrosis, promotes the activation and proliferation of fibroblast, and break the banlace between synthesis and degradation of ECM by its participation in signal tranduction pathway, eventually leads to myocardial fibrosis.TGF-βmediated signaling pathway ,including smad-dependent pathway and Smad-independent pathway, plays an important role in myocardial fibrosis.Along with the growing acknowledgment of its research,the Smad-independent TGF-βsignaling pathway has been developed into a hot spot,among which the ERK signal transduction pathway has drawn more and more interest of researchers.The present study aims to investigate the influence of apocynum venetum extract (AVE) on myocardial fibrosis both in vitro and in vivo.In addition,the mechanisms involved in the regression of AVE on myocardial fibrosis has been preliminar approached in the aspects of TGF-βand ERK signaling pathway.The research of this dissertation mainly includes:①the effects of different conditioned medium on cardiac fibroblast proliferation;②the influnence of AVE on ECM of rat cardiac fibroblasts induced by AngII;③preliminary into the effect of AVE on TGF-βand ERK signaling pathway in rat cardiac fibroblasts stimulated by AngII;④the protective effect of AVE on heart construction in Iso-induced myocardial fibrosis rats;⑤the influnence of AVE on synthesis and degradation of ECM in Iso-induced myocardial fibrosis rat;⑥the inhibition of AVE on TGF-βand ERK signaling pathway in Iso-induced myocardial rat.The following results are made:1 Ang II (0 moLl·-1,10-5 moLl·-1,10-6 molL·-1,10-7 moLl·-1,10-8 molL·-1) could promote the proliferation of rat cardiac fibroblasts,and the culture system preferably for fibroblasts was the DMEM culture fluid containing 10-6 moLl·-1 Ang II after 24 h (P<0.01).Different concentrations of AVE (0.8 moLl·-1,0.4 molL·-1,0.2 moLl·-1) played an inhibitory effect on the proliferation of cardiac fibroblast stimulated by Ang II after treating 24 h (P<0.01).There was defference between defferent ways of administration of AVE (A:pretreat cells with various concentration of AVE for 30 min,and add Ang II into cells for 24h;B:AVE were given for 24h while the cells stimulated by Ang II) on the proliferation in Ang II-induced cardiac fibroblast.When the concentration of AVE was 0.8 molL·-1 (AVE1 group),the defference between A and B was no significant.In contrast,there were markedly defference at the concentration of 0.4 moLl·-1 (AVE2 group) and 0.2 moLl·-1 (AVE3 group),particularly A played a remarkable inhibitory effect on the proliferation of Ang II-induced cardiac fibroblast.2 In the Ang II-induced cardiac fibroblast after treating 24h,the over expression of Col I and Col III, the main components of ECM ,could be inhibited by AVE.Through the measure of Col I and Col III content in the culture supernatants by ELISA,we foud that,compared with Ang II group,various AVE groups had a significant inhibitory effect on Col I (P<0.01),and the expression of Col III in AVE1,AVE2 and AVE3 group was simililar to Col I (P<0.01,P<0.05).It had been implicated by RT-PCR that Col I and Col III mRNA expression in Ang II group were significant higher than that of C group (P<0.05, P<0.01),and Compared to the Ang II group,all AVE groups suppressed the expression of Col I and Col III mRNA (P<0.01).3 Various assay methods showed that,compared with C group,TGF-βwas increased obviously in the Ang II group (P<0.01, P<0.05).All the AVE groups were able to inhibit the increament of TGF-βstimulated by AngII(P<0.01). By the RT-PCR,the components,such as raf,ERK,c-fos, involved in ERK signaling pathway were dectected.Compare with C group,these components in the Ang II group were at the state of over expression (P<0.01).The concentration of Raf,ERK,c-fos mRNA in all AVE groups were lower than that of Ang II group (P<0.01).4 HE staining indicated that, Obvious myocardial fibrosis was induced by multipoint subcutaneous injection with Iso(15mg kg-1) once on rats. After the administration different dose of AVE for 3 w, the fibrosis degree was ameliorated.5 Compare to Iso group, Col I and Col III mRNA and proten expression related to various AVE groups were decreased obviously.The levels of Col I and Col III in the serum,determined by Elisa,showed that,Iso can promote the synthesis of Col I and Col III significantly (P<0.05).Compared with the Iso group,the Col I proten in AVE1 and AVE3 Group were reduced obviously (P<0.05, P<0.01),and Col III proten in AVE1 and AVE2 Group were inhibited significantly (P<0.05).By immunohistochemistry and RT-PCR, we confirmed that,the Col I and Col III of Iso group were in a condition of over expression (P<0.01),and all the AVE groups played an inhibitory effect on the increase expression of Col I and Col III (P<0.01).6 After the administration of AVE for 3 w,there was a decline in the expression of raf,TGF-βmRNA in the Iso-induced myocardial fibrosis rats. TGF-βmRNA in AVE groups was lower than that of Iso group (P<0.01).Compare to Iso group,raf and ERK mRNA expression in AVEl group were decresed obviously (P<0.01),and raf mRNA expression in AVEh group and AVEm group was reduced significantly (P<0.05, P<0.01),while as ERK mRNA expression in the two groups was no significant difference.Based on the results,we can draw the following conclusions:Firstly,in the Ang II-induced myocardial fibrosis,Ang II elevates the proliferation ability of cardiac fibroblasts,break the balance between synthesis and degradation of ECM,and accelerates myocardial fibrosis.Secondly,AVE can preprotect the morphous and structure of heart and dela the progression on myocardial fibrosis probably by decreasing syntheses and inhibiting deposition cardiac tissue collagen.In addition,AVE can reduce the expression of mRNA,protein on the related singalling molecules of TGF-βand ERK pathway,and lighten myocardial fibrosis.The innovations in the dissertation as bellow:1 we found that apocynum venetum can alleviate myocardial fibrosis.2 we investigated the mechanism based on the signal pathway.
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