SOX6基因对H2O2诱导的星形胶质细胞损伤的机制

Mechanism of action of SOX6 gene on astrocyte injury induced by H2O2

目的探讨干扰SOX6基因表达对H2O2诱导的星形胶质细胞(AS)凋亡、线粒体膜电位及PI3K/AKT信号通路影响。方法从乳大鼠大脑皮质分离培养AS细胞,将细胞分为阴性对照组(NC组):AS转染无干扰作用的siRNA;H2O2组:20μmol/L的H2O2处理AS细胞24 h;H2O2+NC组:无干扰作用的siRNA转染AS细胞24h后,20μmol/L的H2O2处理细胞24 h;H2O2+si-SOX6组:SOX6特异性siRNA转染AS细胞24 h后,20μmol/L的H2O2处理细胞24 h。通过LDH试剂盒、流式细胞术及JC-1探针分别检测细胞毒性、凋亡率及线粒体膜电位;Western blotting检测SOX6、Cyt.C、caspase3、caspase9、Bcl-2、Bax和p-AKT蛋白表达。结果H2O2可明显上调AS细胞SOX6、Cyt.C、caspase3、caspase9和Bax表达,下调Bcl-2和p-AKT表达,提高细胞毒性,诱导细胞凋亡,而干扰SOX6基因表达可明显减弱H2O2对细胞SOX6、Cyt.C、caspase3、caspase9、Bcl-2、Bax和p-AKT表达及细胞毒性和凋亡影响。结论干扰SOX6基因表达可通过抗凋亡途径保护H2O2诱导的AS损伤,此保护作用与线粒体通路及PI3K/AKT信号通化激活有关。

Objective To investigate the effects of SOX6 gene interference on apoptosis,mitochondrial membrane potential,and the PI3 K/AKT signaling pathway of astrocytes(AS)induced by H2O2.Methods AS cells were isolated from the cerebral cortex of neonatal rats and divided into the following groups:negative control group(NC group):AS transfected with siRNA without interference;H2O2 group:AS cells treated with 20μmol/L H2O2 for 24 h;H2O2+NC group:non-interfering siRNA transfected into AS cells for 24 h,then treatment of cells with 20μmol/L H2O2 for 24 h;and H2O2+si-SOX6 group:SOX6-specific siRNA transfected into AS cells for 24 h,followed by treatment of the cells with 20μmol/L H2O2 for 24 h.The cytotoxicity,apoptosis rate,and mitochondrial membrane potential were determined by LDH kit,flow cytometry,and JC-1 probe.The expression of SOX6,Cyt.C,caspase 3,caspase9,Bcl-2,Bax,and p-AKT protein was determined by Western blotting.Results H2O2 significantly upregulated the expression of SOX6,Cyt.C,caspase3,caspase9,and Bax;downregulated the expression of Bcl-2 and p-AKT;increased cytotoxicity;and induced apoptosis.Interfering with SOX6 gene expression significantly reduced the effects of H2O2 on SOX6,Cyt.C,caspase3,caspase9,Bcl-2,Bax,and p-AKT expression,cytotoxicity,and apoptosis.Conclusions Interference with SOX6 gene expression can protect against AS injury induced by H2O2 through the anti-apoptotic pathway,which is related to the mitochondrial pathway and PI3 K/AKT signaling activation.