褪黑素通过抑制SOX2的表达减轻缺氧诱导PC12细胞损伤

Melatonin alleviates hypoxia-induced injury of PC12 cells by inhibiting SOX2 expression

目的探讨褪黑素对缺氧诱导PC12细胞损伤的影响及机制。方法将体外培养的大鼠嗜铬细胞瘤P12细胞分为对照组,缺氧组及缺氧+低、中、高(12.5、25、50μmol/L)剂量褪黑素组,采用MTT法检测细胞活力,流式细胞仪检测细胞凋亡,免疫印迹法检测细胞中剪切的含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved-Caspase-3)和性别决定相关基因簇2(SOX2)蛋白表达,实时荧光定量聚合酶链式反应(PCR)检测细胞中SOX2 mRNA表达,试剂盒检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。筛选出效果最为显著的高剂量褪黑素组作为缺氧+褪黑素组,然后考察缺氧+褪黑素、缺氧+siRNA-NC、缺氧+siRNA-SOX2、缺氧+褪黑素+pcDNA、缺氧+褪黑素+pcDNA-SOX2分别对细胞活力、细胞凋亡、cleaved-Caspase-3蛋白表达水平、SOX2蛋白和mRNA水平、SOD活性和MDA水平的影响。结果与对照组比较,缺氧组细胞存活率和SOD活性降低,细胞凋亡率、cleaved-Caspase-3蛋白表达水平、MDA水平和SOX2 mRNA与蛋白表达水平均显著升高(均P<0.05);低、中、高剂量褪黑素和SOX2低表达均可改善缺氧引起的上述变化(均P<0.05),而SOX2过表达可减轻褪黑素对缺氧诱导的PC12细胞活力、凋亡和氧化应激的影响(均P<0.05)。结论褪黑素可通过抑制SOX2的表达减轻缺氧诱导PC12细胞损伤。

Objective To investigate the effect of melatonin(MT)on hypoxia-induced injury of PC12 cells and its mechanism.Methods PC12 cells cultured in vitro were divided into the control,the hypoxic,and three groups of hypoxic cells combined with different doses(low,medium,and high)of MT at 12.5,25,and 50μmol/L,respectively.Cell viability was measured by MTT.The expression of cysteine-containing aspartate 3(cleaved caspase-3)and SOX2 proteins was detected by Western blot.The expression of SOX2 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction(PCR).The activity of superoxide dismutase(SOD)and content of malondialdehyde(MDA)were detected by the kit.The combined treatment group with the most significant effect was selected as the hypoxia+MT group,and then the effects of hypoxia+MT,hypoxia+siRNA-NC,hypoxia+siRNA-SOX2,hypoxia+MT+pcDNA,and hypoxia+MT+pcDNA-SOX2 on cell viability,apoptosis,protein expression level of cleaved-caspase-3,protein and mRNA levels of SOX2,SOD activity,and MDA levels,respectively,were investigated.Results Compared with the control group,cell survival rate and SOD activity in the hypoxia group were significantly decreased,while cell apoptosis rate,cleaved caspase-3 protein expression level,MDA content,SOX2 mRNA,and protein expression level were significantly increased(all P<0.05).The effects of low,medium,and high levels of MT and the low expression of SOX2 could ameliorate the above changes induced by hypoxia(all P<0.05),while the overexpression of SOX2 could alleviate the effects of MT on the viability,apoptosis,and oxidative stress of hypoxia-induced PC12 cells(all P<0.05).Conclusions MT can reduce hypoxia-induced injury to PC12 cells by inhibiting the expression of SOX2.