自噬在高磷诱导的大鼠血管平滑肌细胞钙化过程中的作用研究

Effect of Autophagy on Process of Phosphate Induced Vascular Smooth Muscle Cell Calcification in Experimental Rats

目的:探讨自噬在高磷诱导的大鼠血管平滑肌细胞(VSMC)钙化过程中的作用。方法:采用磷酸盐(3.2 mmol/L Pi,即高磷状态)建立大鼠VSMC钙化模型。实验分为三组:对照组、钙化组(分为3个亚组:3.2 mmol/L Pi 4d组、3.2 mmol/L Pi 6d组、3.2 mmol/L Pi 8d组);钙化+3-甲基腺嘌呤(3-MA)组(3.2mmol/L Pi 8 d+5 mmol/L 3-MA)。分别通过茜素红S染色法和邻甲酚肽络合酮比色法检测各组细胞钙结节形成及钙含量;蛋白免疫印迹法检测各组细胞转录因子蛋白(Runx2)、α-肌动蛋白(α-SMA)和自噬相关蛋白—膜型微管蛋白1轻链3β(LC3Ⅱ)蛋白表达量。透射电子显微镜观察VSMC内自噬小体形成情况。免疫荧光显微镜下观察VSMC中LC3和Runx2定位表达。结果:与对照组相比,3.2 mmol/L Pi 8 d组钙结节、钙含量、Runx2和LC3Ⅱ蛋白表达量及自噬小体均显著增高,α-SMA蛋白表达量降低,差异具有统计学意义(P<0.05)。与3.2 mmol/L Pi 8 d组相比,钙化+3-MA组细胞钙含量增多,LC3荧光分布量降低,Runx2阳性细胞数增多,差异具有统计学意义(P<0.05)。结论:自噬在磷酸盐诱导的VSMC钙化过程中具有保护作用。

Objective：To explore the effect of autophagy on process of high phosphate salt induced vascular smooth muscle cell（VSMC） calcification in experimental rats.Methods： Rats＇ model of VSMC calcification was induced by phosphate incubation. VSMC were divided into 3 groups：① Control group,②Calcification group which included 3 subgroups as 4-day subgroup, the cells were cultured by 3.2 mmol/L phosphate for 4 days, 6-day subgroup and 8-day subgroup,③ Calcification＋ 3-MA（autophagy inhibitor） group, in which the 8-day cells were cultured with 5mmol/L 3-MA. Calcium nodule formation and calcium deposition in VSMC were measured by Alizarin red staining and o-cresolphthaleincomplexone method, protein expressions of Runx2, α-SMA and LC3 Ⅱ were examined by Western blot analysis, autophagosome formation in VSMC was measured by transmission electron microscope and the localization and expression of Runx2 and LC3 Ⅱ in VSMC were observed by immunofluorescent microscope.Results： Compared with Control group, the cells at 8-day subgroup showed more calcium nodules, higher calcium deposition, increased protein expressions of Runx2, LC3 Ⅱ, more autophagosome and decreased α-SMA expression, all P〈0.05. Compared with 8-day subgroup, the cells in Calcification＋3-MA group presented increased calcium deposition, decreased fluorescence distribution of LC3 Ⅱ and more cells with positive Runx2 protein expression, all P〈0.05.Conclusion： Autophagy has the protective effect on process of phosphate induced VSMC calcification in experimental rats.