瞬时受体电位通道7参与匹鲁卡品诱导的癫痫体外模型凋亡的调控

Role of transient receptor potential melastatin 7 in the modulation of apoptosis in epilepsy in an in vitro model induced by pilocarpine

目的探讨瞬时受体电位通道7(TRPM7)在癫痫中与凋亡的关系。方法采用CCK-8法检测匹鲁卡品(PILO)对Neuro-2A细胞活性的影响;设置对照组和PILO模型组,采用蛋白质印迹法检测凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶3(caspase-3)、Bcl-2相关X蛋白(Bax)和细胞色素C蛋白(Cyt C)的表达;采用实时荧光定量PCR法检测TRPM7 mRNA的表达;设置对照组(Neuro-2A细胞转染无序siRNA序列)、PILO模型组(Neuro-2A细胞转染无序siRNA序列后经PILO处理24 h)和PILO+siRNA组(Neuro-2A细胞转染TRPM7 siRNA序列后经PILO处理24 h),采用蛋白质印迹法检测凋亡相关蛋白表达;采用TUNEL染色观察细胞凋亡情况。结果Neuro-2A细胞活性随PILO浓度升高而降低;与正常对照组相比,模型组凋亡相关蛋白caspase-3、Bax、Cyt C和TRPM7 mRNA在24 h时表达明显增加(均P<0.05);与PILO模型组相比,PILO+siRNA组凋亡相关蛋白caspase-3、Bax和Cyt C表达和细胞凋亡率明显减少(均P<0.05)。结论TRPM7参与PILO诱导的癫痫体外模型的凋亡。

Objective To investigate the relationship between transient receptor potential melastatin 7(TRPM7)and apoptosis in epilepsy.Methods Neuro-2A cell viability was assessed by the cell counting kit-8(CCK-8)assay.The expressions of caspase-3 protein(caspase-3),Bcl-2 related X protein(Bax),and cytochrome C protein(Cyt C)were determined by Western blotting in the control group and model group.The mRNA expression level of TRPM7 was detected by real-time PCR.Additionally,the protein expressions of caspase-3,Bax,and Cyt C were determined by Western blotting in the control group,pilocarpine(PILO)model group,and PILO+siRNA group.The apoptotic rate was detected by TUNEL staining in the control group,PILO model group,and PILO+TRPM7 siRNA group.Results The results showed that the viability of Neuro-2A cells decreased with increasing PILO concentrations.Moreover,the protein expressions of caspase-3,Bax,and Cyt C and mRNA expression of TRPM7 increased in the PILO group compared with the control group(all P<0.05).Meanwhile,the protein expressions of caspase-3,Bax,and Cyt C and the apoptotic rate decreased in the PILO+siRNA group when compared with those of the PILO model group(all P<0.05).Conclusion TRPM7 was involved in the modulation of apoptosis in epilepsy in an in vitro model induced by PILO.