Objective Mammalian target of rapamycin(mTOR)plays a central role in controlling cell proliferation.survival and growth.We investigated the role of mTOR signal transduction on viral myocarditis by observing the effect of mTOR inhibitor rapamycin on Smad 3 and collagen type Ⅰ expression in rat myocardial fibroblasts infeeted with coxsaekievirus B 3(CVB 3).Methods Primary cultured myocardial fibroblasts of SD rats infected with CVB 3 were treated with or without rapamycin.The Smad 3 and collagen type Ⅰ expression of the cells were determined by RT-PCR and Western blot.Results (1) mTOR/β-actin ratio was dose-dependenfly reduced(1 nmol/L,0.38 ±0.022:10 nmol/L,0.282±0.014;100 nmol/L,0.263±0.012 vs.control 1.45±0.04．all P＜0.05 vs.control)after 48 hours rapamycin treatments and time-dependendy reduced after 10 nmol/L rapamycin treatment(24 h,0.203±0.021;48 h,0.163±0.022;72 h,0.144±0.013 vs.0 h,0.341±0.022,allP＜0.05 vs.0 h)in CVB 3 infected myocardial fibroblasts.(2)Smad 3/β-actin ratio of myocardiol fibroblasts was significantly increased in CVB 3 infected cardial fibroblasts and this increase could be significantly attenuated by rapamycin(control,0.63±0.06;CVB 3,1.18±0.03;CVB 3+Rapamycin,0.77±0.08 by RT-PCR and 0.89±0.07,2.27±0.13 and 0.131±0.013 by Western blot).Collagen type Ⅰ/β-actin ratio was also significantly increased by CVB 3 and this increase could be reversed by rapamycin(1.13±0.06,1.303±0.012,0.82±0.03 by RT-PCR).Conclusion Rapamyein can inhibit the Smad 3 and collagen type Ⅰ expressions in CVB 3 infected myocardial fibroblasts suggesting that the mTOR signal pathway may play an important role in the pathogenesis of CVB 3 induced myocardial fibrosis.%目的 探讨哺乳类雷帕霉素靶蛋白(mTOR)信号通路对柯萨奇病毒B 3(CVB 3)诱导的心肌成纤维细胞Smad 3蛋白(Smad 3)和Ⅰ型胶原表达的调控作用.方法 CVB 3感染原代培养的SD鼠心肌成纤维细胞,用mTOR的抑制剂-雷帕霉素阻断mTOR信号通路,采用逆转录-聚合酶链反应(RT-PCR)和Western blot方法检测细胞Smad 3和Ⅰ型胶原表达.结果 (1)雷帕霉素干预心肌成纤维细胞48 h,RT-PCR检测mTOR/β-肌动蛋白(actin)光密度值分别为1 nmol/L组0.381±0.022、10 nmol/L组0.282±0.014、100 nmol/L组0.263±0.012,均低于对照组1.45±0.04,10 nmol/L组和100 nmol/L组低于1nmol/L组,差异均有统计学意义(P均＜0.05).10 nmol/L雷帕霉素干预心肌成纤维细胞0 h(对照组)、24 h、48 h、72 h,其mTOR/β-actin光密度值分别为0.341±0.022、0.203±0.021、0.163±0.022、0.144±0.013,光密度值随雷帕霉素干预时间的延长而降低(P＜0.05).(2)RT-PCR和Western blot测得对照组、CVB 3组、CVB 3+雷帕霉素组心肌成纤维细胞Smad 3/β-actin光密度值分别为0.63±0.06、1.18±0.03、0.77±0.08,Smad 3/β-actin灰度值分别为0.89±0.07、2.27±0.13、0.131±0.013.RT-PCR测得上述各组Ⅰ型胶原/β-actin光密度值分别为1.13±0.06、1.303±0.012、0.82±0.03.以上结果显示CVB 3组光密度值/灰度值高于对照组,CVB 3+雷帕霉素组低于CVB 3组,差异有统计学意义(P＜0.05).结论 雷帕霉素抑制CVB 3诱导的心肌成纤维细胞Smad 3和Ⅰ型胶原表达,提示mTOR信号通路可能通过调控Smad及Ⅰ型胶原表达,在CVB.3诱导的心肌纤维化过程中起重要作用.
展开▼
