血管紧张素II1型受体自身抗体通过上调自噬诱导INS-1胰岛β细胞凋亡

摘要

目的:本研究旨在探究血管紧张素II 1型受体自身抗体(AT1-AA)能否引起胰岛β细胞的凋亡,自噬是否参与其中以及其所发挥的作用.方法:10 -6mol/L AT1-AA处理INS-1细胞24 h后,用流式细胞术、Wes-tern blot及Hoechst 33258染色检测细胞的凋亡水平;Western blot 检测自噬相关蛋白LC3和beclin 1的蛋白水平.使用血管紧张素Ⅱ1型受体拮抗剂替米沙坦以及经典的自噬抑制剂3-甲基腺嘌呤(3-MA)预处理细胞1 h之后再加入AT1-AA处理24 h,检测细胞凋亡、自噬及存活率的变化情况.结果:10-6mol/L AT1-AA处理INS-1细胞24 h可明显降低细胞存活率(P<0.05).与阴性IgG对照组相比,AT1-AA分别处理细胞12 h、24 h和36 h后细胞凋亡水平明显增高(P<0.05);此外,LC3及beclin 1的蛋白水平也随处理时间的延长逐渐升高,且凋亡和自噬水平的升高均可被替米沙坦所阻滞.使用自噬抑制剂3-MA预处理之后,细胞的凋亡率较单独给予AT1-AA处理组明显降低(P<0.05).结论:AT1-AA可通过血管紧张素II 1型受体上调自噬来诱导INS-1胰岛β细胞凋亡.%AIM:To explore whether the angiotensin II type 1 receptor autoantibodies(AT1-AA)induces islet β-cell apoptosis and whether autophagy is involved in the process.METHODS:The INS-1 cells treated with AT1-AA at 10-6mol/L for 24 h,and then the apoptosis was analyzed by flow cytometry,Western blot and Hoechst 33258 staining.In addition,the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot.The effects of AT1-AA on the apoptosis,autophagy and viability of INS-1 cells with or without 3-methyladenine(3-MA;a com-mon autophagy inhibitor)or telmisartan(an angiotensin Ⅱ type 1 receptor blocker)pretreatment, were detected by flow cytometry,Western blot and CCK-8 assay.RESULTS: Treatment with AT1-AA at 10 -6mol/L for 24 h significantly re-duced the cell viability(P<0.05).Compared with the negative IgG control group,the apoptotic cells increased after incu-bation with AT1-AA for 12 h,24 h and 36 h,respectively(P<0.05).Moreover,the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time,and the elevation of apoptosis and autophagy were blocked by telmisartan.After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone.CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet βcells by upregulating autophagy via the angiotensin Ⅱtype 1 receptor pathway.