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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（30 μmol/L法舒地尔）、法舒地尔+LY294002组（30 μmol/L法舒地尔+25 μmol/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 of α smooth 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 (30 μmol/L Fasudil) and Fasudil+LY294002 group (30 μmol/L Fasudil+25 μmol/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.