冬凌草甲素通过腺苷酸活化蛋白激酶/沉默信息调节因子1通路介导自噬途径改善高糖诱导足细胞损伤机制的研究

Rubescensine A reduces podocyte damage induced by high glucose through the autophagy pathway mediated by AMPK/SIRT1 pathway

目的探讨冬凌草甲素通过腺苷酸活化蛋白激酶/沉默信息调节因子1(AMPK/SIRT1通路介导的自噬途径,改善高糖(HG)诱导足细胞损伤的机制。方法体外培养人肾小球足细胞,随机分正常对照(Con)组、HG组、羟氯喹(HCQ)组、雷帕霉素(RAP)组。CCK-8检测细胞活力,Western blot法检测细胞凋亡和足细胞损伤相关蛋白表达。以不同浓度冬凌草甲素处理HG诱导足细胞模型并筛选最佳作用浓度。另将人肾小球足细胞随机分Con、HG、冬凌草甲素(Rub A)组、盐酸多索啡(Compound C)组、Rub A+Compound C组,检测各组自噬、AMPK/SIRT1通路相关蛋白表达。结果与Con组比较,HG组结蛋白(Desmin)、Bcl-2相关X蛋白基因(Bax)蛋白表达升高(P<0.05),足细胞活力及突触足蛋白(Synaptopodin)、B细胞淋巴瘤-2基因(Bcl-2)蛋白表达降低(P<0.05)。与HG组比较,RAP组各指标有相应改善,而HCQ组均无改善且相应进展。与Con组比较,HG组Desmin、Bax蛋白表达升高(P<0.05),足细胞活力降低、自噬小体数量、Synaptopodin、Bcl-2、微管相关蛋白轻链3 II(LC3 II)/LC3I、自噬效应蛋白、p-AMPK/AMPK、SIRT1蛋白表达降低(P<0.05)。分别与HG、Rub A+Compound C组比较,Rub A组上述指标均有所改善。Compound C组逆转冬凌草甲素保护作用。结论冬凌草甲素通过激活AMPK/SIRT1通路促进细胞自噬,减轻HG诱导的足细胞损伤。

Objective To investigate the mechanism that Rubescensine A reduces the podocyte damage induced by high glucose(HG)through the autophagy pathway mediated by AMP activated protein kinase/silent information regulator 1(AMPK/SIRT1)pathway.Methods Human glomerular podocytes were cultured in vitro,and randomly divided into Control group(Con),HG group,hydroxychloroquine(HCQ)group,and Rapamycin(RAP)group.CCK-8 was used to detect cell viability.Western blotting was used to detect cell apoptosis and podocyte injury related protein expression in each group.The podocyte model induced by high glucose(HG)was treated with Rubescensine A(Rub A)at different concentrations and the optimal concentration was selected.Then,human glomerular podocytes were randomly divided into Con group,HG group,Rub A group,Compound C group,and Rub A+Compound C group.The expression of autophagy,AMPK/SIRT1 pathway related proteins were detected in each group.Results Compared with Con group,the podocyte viability and the protein expressions of Synaptopodin and Bcl-2 was significantly reduced(P<0.05),while the protein expressions of Desmin and Bax were significantly increased in HG group(P<0.05).Compared with the HG group,all indicators were relieved in RAP group.However,the levels of all indicators were worsened in HCQ group.Compared with Con group,the expression levels of Desminand Bax proteins in podocytes were significantly increased(P<0.05),and the podocyte viability,number of autophagosomes,the expression levels of Synaptopodin,Bcl-2,microtubule associated protein light chain 3(LC3)II/I,Beclin-1,p-AMPK/AMPK and SIRT1 proteins were significantly reduced in HG group(P<0.05).Compared with HG group and Rub A+Compound C group,the above indicators were improved in Rub A group.Compound C group reversed the protective effect of Rub A.Conclusion Rubescensine A can promote autophagy by activating AMPK/SIRT1 pathway,thereby reduce podocyte damage induced by high glucose.