哺乳类雷帕霉素靶蛋白信号通路对柯萨奇病毒B3诱导的心肌成纤维细胞Smad3蛋白和Ⅰ型胶原表达调控的实验研究

Effects of mTOR inhibitor rapamycin on Snmd 3 protein and collagen typeⅠ expression in rat myocardial fibroblasts infected with coxsackie virus B 3

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B3（CVB3）诱导的心肌成纤维细胞Smad3蛋白（Smad3）和Ⅰ型胶原表达的调控作用。方法CVB3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Westernblot方法检测细胞Smad3和Ⅰ型胶原表达。结果（1）雷帕霉素干预心肌成纤维细胞48h，RT—PCR检测mTOR／β-肌动蛋白（actin）光密度值分别为1nmol/L组0．381±0．022、10nmol/L组0．282±0．014、100nmol/L组0．263±0．012，均低于对照组1．45±0．04，10nmol/L组和100nmol/L组低于1nmol/L组，差异均有统计学意义（P均〈0．05）。10nmol/L雷帕霉素干预心肌成纤维细胞0h（对照组）、24h．48h、72h，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Westernblot测得对照组、CVB3组、CVB3＋雷帕霉素组心肌成纤维细胞Smad3／β-actin光密度值分别为0．63±0．06、1．18±0．03、0．77±0．08，Smad3／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／B—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CVB3组光密度值／灰度值高于对照组，CVB3＋雷帕霉素组低于CVB3组，差异有统计学意义（P〈0．05）。结论雷帕霉素抑制CVB3诱导的心肌成纤维细胞Smad3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CVB3诱导的心肌纤维化过程中起重要作用。

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 infected with coxsackievirus 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-dependently reduced （1nmol/L, 0. 381 ± 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-dependently 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, all P 〈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 eardial fibroblasts and this increase could be significantly attenuated by rapamyein （control, 0.63 ±0.06; CVB 3, 1.18 ±0.03; CVB 3 ＋ Rapamyein, 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 rapamyein （ 1.13 ± 0. 06, 1. 303± 0. 012, 0. 82 ± 0.03 by RT-PCR）. Conclusion Rapamycin 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.