血管紧张素Ⅱ与血管平滑肌细胞中自噬水平的关系及机制研究

Study on the relationship and mechanism of autophagy in vascular smooth machine cells and AngⅡ

目的研究血管紧张素Ⅱ(AngⅡ)是否能够引起血管平滑肌细胞(VSMC)自噬,探讨其相关作用机制。方法以VSMC为研究对象,设立组别分别为空白组、AngⅡ组、自噬抑制剂(3 MA)组、自噬促进剂雷帕霉素(Rap)组、血管紧张素Ⅱ(AT1)受体抑制剂(ARB)组、3 MA+AngⅡ组、Rap+AngⅡ组、ARB+AngⅡ组,采用四唑盐(MTT)比色法检测细胞存活率,蛋白质免疫印迹法检测自噬相关蛋白脂化和膜相关蛋白(LC3Ⅱ)表达的变化,免疫荧光法检测自噬小体数目的变化。结果实验中空白组吸光度为(0.159±0.001),AngⅡ组吸光度为(0.151±0.003),相比空白组,加入AngⅡ药物对细胞存活率变化不明显,差异无统计学意义(P>0.05)。蛋白质免疫印迹结果显示加入AngⅡ促进VSMC的LC3 Ⅱ表达量增多,呈现时间依赖性、浓度依赖性,表明AngⅡ促进VSMC自噬发生。3 MA对AngⅡ(10-7 mol/L,24 h)引起的LC 3Ⅱ表达量增多起到抑制作用,Rap对AngⅡ(10-7 mol/L,24 h)引起的LC 3Ⅱ表达量增多起到叠加作用。ARB对AngⅡ(10-7 mol/L,24 h)引起的LC 3Ⅱ表达量增多起到抑制作用,表明自噬是由AngⅡ通过ATI受体引起的。结论AngⅡ通过AT1受体引起血管平滑肌细胞发生自噬。

Objective To study if angiotensinⅡ(AngⅡ)is able to cause autophagy in vascular smooth muscle cells(VSMC)and the related mechanisms of autophagy.Methods The VSMC was adopted as the research object.The establishment of groups were the control group,the AngⅡgroup,the 3 MA+AngⅡgroup,the Rap+AngⅡgroup,the ARB+AngⅡgroup,the 3 MA(autophagy inhibitor)group,the rapamycin(autophagy accelerator)group,the AngiotensinⅡtype 1 receptor blocker(ARB,AT1 receptor inhibitors)group.MTT detected the cell viability,western blot detected the changes of the expression of autophagy related proteins LC3 Ⅱ,immunofluorescence detected the changes of the number of autophagic bodies.Results MTT results showed that compared to the control group,adding AngⅡdrugs have little effect on cell survival,which suggested that AngⅡdid not affect cell survival.Western blot showed that adding AngⅡcould increase VSMC expression of LC3 Ⅱ,exhibiting a time dependent,concentration dependent manner,which suggested that AngⅡcan promote VSMC autophagy.3 MA play an inhibitory effect on AngⅡ(10-7 mol/L,24 h)caused increased expression of LC 3Ⅱ.Rapamycin play an overlay effect on ANGⅡ(10-7 mol/L,24 h)caused increased expression of LC 3Ⅱ.ARB play an inhibitory effect on AngⅡ(10-7 mol/L,24 h)caused increased expression of LC 3Ⅱ,suggesting that AngⅡinduced autophagy through ATI receptor.Conclusion AngⅡinduces autophagy in vascular smooth muscle cells via AT1 receptor.