法舒地尔促进平滑肌细胞自噬和凋亡的机制研究

Study on mechanism of Fasudil promoting autophagy and apoptosis of smooth muscle cells

目的探讨法舒地尔对主动脉平滑肌细胞(VSMC)自噬及凋亡的影响以及与磷脂酰肌醇3激酶(PI3K)-蛋白激酶B(Akt)-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的关系。方法用10%胎牛血清+DMEM培养液培养SD大鼠VSMC,并利用免疫细胞化学染色法检测传代细胞α平滑肌肌动蛋白(a-SMA)的表达情况鉴定VSMC。采用血小板源性生长因子(PDGF)诱导VSMC,并将PDGF诱导的VSMC分为空白组、对照组、法舒地尔组(30μmol/L法舒地尔)、法舒地尔+LY294002组(30μmol/L法舒地尔+25μmol/L LY294002)。采用MTT法检测各组细胞增殖情况,流式细胞仪检测细胞凋亡率和凋亡周期,GFP-mRFP-LC3双荧光检测细胞自噬水平。采用蛋白免疫印迹法(WB)检测各组细胞Bax、Bcl-2、LC3、Beclin-1及PI3K、Akt、p-Akt、mTOR、p-mTOR的蛋白表达情况并比较分析。结果镜下观察培养细胞呈长梭形、放射性生长,出现典型的"峰-谷"样结构,a-SMA阳性表达率为99%,确认所培养细胞为高纯度主动脉VSMC。法舒地尔组细胞增殖能力低于对照组(P<0.05),法舒地尔+LY294002组高于法舒地尔组(P<0.05)。与对照组比较,法舒地尔组凋亡率及G_(0)-G_(1)期细胞比例升高(均P<0.05);与法舒地尔组相比,法舒地尔+LY294002组凋亡率及G_(0)-G_(1)期细胞比例回降(均P<0.05)。与对照组比较,法舒地尔组自噬增强(P<0.05),而法舒地尔+LY294002组的自噬则较法舒地尔组有所减弱(P<0.05)。与对照组比较,Bax、Bcl-2、LC3-Ⅱ/LC3-Ⅰ比值、Beclin1水平以及PI3K、Akt、mTOR磷酸化水平升高(均P<0.05);法舒地尔+LY294002组Bax、Bcl-2、LC3-Ⅱ/LC3-Ⅰ比值、Beclin 1水平以及PI3K、Akt、mTOR磷酸化水平较法舒地尔组有所回降(均P<0.05)。结论法舒地尔通过激活PI3K-AKT-mTOR信号通路抑制VSMC增殖,促进自噬及凋亡,改善血管功能。

Objective To investigate the effect of Fasudil on autophagy and apoptosis of aortic smooth muscle cells(VSMC)and its relationship with phosphatidylinositol 3 kinase(PI3K)-protein kinase B(Akt)-mammalian target of rapamycin(mTOR)signaling pathway.Methods The VSMC of SD rat were cultured with 10%fetal bovine serum and DMEM medium,and the expression ofαsmooth muscle actin(a-SMA)was detected by immunocytochemical staining to identify VSMC.Platelet derived growth factor(PDGF)was used to induce VSMC,and PDGF-induced VSMC were divided into blank group,control group,Fasudil group(30μmol/L Fasudil)and Fasudil+LY294002 group(30μmol/L Fasudil+25μmol/L LY294002).MTT assay was used to detect the cell proliferation,flow cytometry was used to detect the apoptosis rate and apoptosis cycle,and GFP-mRFP-LC3 double fluorescence was used to detect the level of autophagy.Western blotting(WB)was used to detect the protein expressions of Bax,Bcl-2,LC3,Beclin-1,PI3K,Akt,p-Akt,mTOR and p-mTOR,and the results were compared and analyzed.Results Under microscope,the cultured cells showed long spindle-shaped,radioactive growth and typical"peak-valley"-like structure.The positive expression rate of a-SMA was 99%,which confirmed that the cultured cells were high-purity aortic VSMC.The results of MTT experiment showed that the cell proliferation capacity of Fasudil group was significantly lower than that of control group(P<0.05),and that of Fasudil+LY294002 group was higher than that of Fasudil group(P<0.05).Flow cytometry showed that compared with the control group,the apoptosis rate and the proportion of G_(0)-G_(1)phase cells in Fasudil group were increased(both P<0.05).Compared with the Fasudil group,the apoptosis rate and the proportion of G_(0)-G_(1)phase cells in the Fasudil+LY294002 group were reduced(both P<0.05).GFP-mRFP-LC3 double fluorescence detection results showed that compared with control group,autophagy was enhanced in Fasudil group(P<0.05).Compared with the Fasudil group,autophagy was decreased in the Fasudil+LY294002 group(P<0.05).WB detection results showed that compared with the control group,the levels of Bax、Bcl-2,LC3-Ⅱ/LC3-Ⅰ,Beclin1 and the phosphorylation levels of PI3K,Akt and mTOR were increased(all P<0.05).Compared with Fasudil group,the levels of Bax、Bcl-2,LC3-Ⅱ/LC3-Ⅰ,Beclin1 and the phosphorylation levels of PI3K,Akt and mTOR in Fasudil+LY294002 group decreased(all P<0.05).Conclusions Fasudil can inhibit VSMC proliferation,promote autophagy and apoptosis,and improve vascular function by activating PI3K-Akt-mTOR signaling pathway.