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目的:Sigma-1受体分子伴侣(Sigma-1 receptor chaperone,Sig-1R)是内质网中主要的伴侣蛋白,参与细胞凋亡等生物学行为,是多种疾病的治疗靶点。本研究旨在探讨Sig-1R基因表达抑制对内质网应激和足细胞损伤的影响,为Sig-1R及内质网应激在肾损伤中的作用提供新视点。方法:体外培养永生性小鼠足细胞系(MPC5),采用LipofectamineTM 2000与Sig-1R siRNA结合形成Sig-1R siRNA-脂质体复合物瞬时转染足细胞,设正常组、阴性对照组和Sig-1R干扰组。采用real-time PCR和Western blot检测Sig-1R的表达水平;Hochest染色法观察凋亡的足细胞核,计数凋亡率;采用Western blot检测足细胞nephrin、desmin、凋亡相关因子bcl-2、bax、caspase 3,8,9,12的表达水平。结果:①与对照组相比,干扰组Sig-1R核酸和蛋白表达均明显下降(P<0.05);②与对照组相比,干扰组足细胞裂孔隔膜蛋白nephrin表达显著下降,肌间蛋白desmin表达显著上升(P<0.05);③与对照组相比,干扰组中凋亡的足细胞数显著增加,抗凋亡蛋白Bcl-2表达明显下降,促凋亡蛋白bax表达明显增加,bcl-2/bax<1∶2(P<0.05),凋亡级联反应执行蛋白caspase-3明显活化(P<0.05);④干扰组中,内质网和线粒体途径相关凋亡蛋白caspase-12,9明显活化(P<0.05),而死亡受体相关凋亡蛋白caspase-8未见明显活化。结论:Sig-1R基因表达抑制可通过诱导内质网应激介导的足细胞凋亡,引起足细胞损伤,提示内质网应激部分参与Sig-1R诱导的足细胞损伤。
AIM: Sigma-1 receptor chaperone (Sig-1R), a major chaperone in the endoplasmic reticulum, is involved in biological behaviors such as apoptosis and is a therapeutic target for many diseases. The purpose of this study was to investigate the effect of Sig-1R gene expression suppression on endoplasmic reticulum stress and podocyte injury and to provide a new perspective for the role of Sig-1R and endoplasmic reticulum stress in renal injury. Methods: The immortalized mouse podocyte line (MPC5) was cultured in vitro. LipofectamineTM 2000 and Sig-1R siRNA were combined to form Sig-1R siRNA-liposome complexes for transient transfection of podocytes. The normal control group, negative control group and Sig -1R interference group. The expression of Sig-1R was detected by real-time PCR and Western blot. The apoptotic podocytes were observed by Hochest staining and the apoptotic rates were counted. Nephrin, desmin, bcl-2, bax , Caspase 3,8,9,12 expression levels. Results: ① Compared with the control group, the expression of Sig-1R mRNA and protein in the interference group was significantly decreased (P <0.05); ②Compared with the control group, the expression of nephrin in the podocyte gap membrane protein significantly decreased, the expression of desmin (P <0.05). ③ Compared with the control group, the number of apoptotic podocytes in the interference group increased significantly, the expression of anti-apoptotic protein Bcl-2 decreased significantly, the expression of pro-apoptotic protein bax increased significantly, the expression of bcl- 2 / bax <1: 2 (P <0.05), and caspase-3, a cascade of caspase-3, was significantly activated (P <0.05) 9 was significantly activated (P <0.05), while death receptor related apoptosis protein caspase-8 showed no significant activation. Conclusion: Inhibition of Sig-1R expression can induce podocyte apoptosis induced by endoplasmic reticulum stress and cause podocyte injury, suggesting that endoplasmic reticulum stress partially participates in Sig-1R-induced podocyte injury.