SIRT7对高糖环境下小鼠肾足细胞增殖、凋亡的影响及其机制

Effects of silence information regulator 7 on proliferation and apoptosis of mouse renal podocytes under high glucose environment

目的:探讨沉默信息调节因子7(SIRT7)对高糖环境下小鼠肾足细胞增殖、凋亡的影响及其机制。方法:在体外高糖条件下培养小鼠肾足细胞,构建SIRT7过表达载体(pcDNA3.1-SIRT7)和小干扰RNA-SIRT7(siRNA-SIRT7)干扰载体并转染小鼠肾足细胞,依据干预因素分组设计为空白对照组(Control)、高糖组(HG)、SIRT7过表达+高糖组(SIRT7 OE+HG)、SIRT7过表达阴性对照+高糖组(SIRT7 OE-NC+HG)、siRNA-SIRT7干扰+高糖组(siRNA-SIRT7+HG)、siRNA-SIRT7干扰阴性对照+高糖组(siRNA-SIRT7-NC+HG)。实时荧光定量RT-qPCR和免疫印迹验证SIRT7过表达和干扰效率。采用CCK-8法检测细胞的增殖活性,流式细胞术检测细胞凋亡,实时荧光定量RT-qPCR分析SIRT7的mRNA表达水平,免疫印迹检测小鼠肾足细胞功能蛋白Nephrin和Wnt/β-catenin信号通路相关蛋白的表达。结果:CCK-8检测结果显示,与Control组比较,HG组细胞的增殖活性下降(P<0.05),与HG组比较,siRNA-SIRT7+HG组细胞的增殖活性显著降低(P<0.05),而SIRT7 OE+HG组细胞的增殖活性增强(P<0.05);流式细胞术检测结果显示,与Control组比较,HG组细胞的凋亡率升高(P<0.05),与HG组比较,siRNA-SIRT7+HG组细胞的凋亡率显著升高(P<0.05),而SIRT7 OE+HG组细胞的凋亡率下降(P<0.05)。免疫印迹检测结果显示,与Control组比较,HG组Nephrin、Wnt5a和β-catenin蛋白表达下调(P均<0.05),与HG组比较,siRNA-SIRT7+HG组Nephrin、Wnt5a和β-catenin蛋白表达显著下降(P均<0.05),而SIRT7 OE+HG组Nephrin、Wnt5a和β-catenin蛋白表达显著升高(P均<0.05)。结论:高糖环境可抑制小鼠肾足细胞增殖并诱导凋亡、过表达SIRT7可逆转该作用,其机制可能与调控Wnt/β-catenin信号通路的活性有关。

Objective:To investigate the effects and its mechanisms of silence information regulator 7(SIRT7)on mouse renal podocytes proliferation and apoptosis under high glucose environment.Methods:Mouse renal podocytes cultured with high glucose and treated with different methods were divided into the following groups:control group(Control),high glucose group(HG),high glucose+transfecting with SIRT7 overexpression vetor(pcDNA3.1-SIRT7)group(SIRT7 OE+HG),high glucose+transfecting with the negative control vetor(pcDNA3.1)group(SIRT7 OE-NC+HG),high glucose+transfecting with small interfering RNA-SIRT7(siRNA-SIRT7)group(siRNA-SIRT7+HG),high glucose+transfecting with siRNA-SIRT7 control group(siRNA-SIRT7-NC+HG).Viability of proliferation was examined by CCK-8 method.Rate of apoptosis was detected by flow cytometry.The level of SIRT7 mRNA expression was measured by qRT-PCR.Western blot was performed to detect the protein expression of Nephrin and key factors of Wnt/β-catenin signaling pathway.Results:The CCK-8 result showed that,compared with control group,the proliferative activity of mouse renal podocytes in HG group was decreased(P<0.05).After transfected with SIRT7 overexpression vetor or small interfering RNA-SIRT7,compared to HG group,the cell proliferation activity was further decreased in siRNA-SIRT7 group(P<0.05),but it was enhanced in SIRT7 OE+HG group(P<0.05).The results of flow cytometry showed that compared with the control group,the apoptosis rate of cells in the HG group was increased(P<0.05).Compared with the HG group,the apoptosis rate of cells in the siRNA SIRT7+HG group was increased significantly(P<0.05),while that in the SIRT7 OE+HG group was decreased(P<0.05).Compared with control group,the expressions of Nephrin,Wnt5a andβ-catenin were inhibited in HG group(P<0.05).compared to HG group,siRNA-SIRT7 could down-regulate the expression levels of Nephrin,Wnt5a andβ-catenin in siRNA-SIRT7 group(P<0.05),SIRT7 overexpression could up-regulate the expression levels of Nephrin,Wnt5a andβ-catenin in SIRT7 OE+HG group(P<0.05).Conclusion:The findings suggest that high glucose environment is an important factor to inhibit the proliferation and induce apoptosis of mouse renal podocytes.Overexpression of SIRT7 can reverse the effects by activating Wnt/β-catenin signaling pathway and up-regulatingβ-catenin expression.