【摘要】 目的:探讨青藤碱(SN)对1-甲基-4-苯基吡啶离子(MPP~+)诱导的人神经母细胞瘤SK-N-SH细胞损伤的影响及作用机制,为探讨帕金森病的发病机制寻找线索。方法:用SN干预经MPP~+诱导的SK-N-SH细胞,采用ELISA法检测细胞培养上清液中丙二醛(MDA)和谷胱甘肽(GSH)的水平,流式细胞术检测细胞凋亡,Western blot检测细胞中Bcl-2和Bax蛋白的水平,RT-qPCR检测细胞中长链非编码RNA ANRIL和微小RNA-626(miR-626)的水平。双萤光素酶报告基因实验验证ANRIL和miR-626的调控关系。转染ANRIL小干扰RNA至SK-N-SH细胞,用上述相同方法观察抑制ANRIL表达对MPP~+诱导的SK-N-SH细胞凋亡、Bax和Bcl-2蛋白表达及细胞培养上清液中MDA和GSH水平的影响。结果:MPP~+处理后,SK-N-SH细胞的凋亡率、Bax蛋白水平和ANRIL表达水平升高(P<0.05),Bcl-2蛋白和miR-626表达水平降低(P<0.05),细胞培养液上清中的MDA水平升高(P<0.05),GSH水平降低(P<0.05)。SN处理或抑制ANRIL表达后,MPP~+诱导的SK-N-SH细胞凋亡率、Bax蛋白水平和ANRIL表达水平降低(P<0.05),Bcl-2蛋白和miR-626表达水平升高(P<0.05),细胞培养液上清中的MDA水平降低(P<0.05),GSH水平升高(P<0.05)。结论:SN可能通过调控ANRIL/miR-626通路减轻MPP~+诱导的SK-N-SH细胞损伤。更多还原

【Abstract】 AIM:To investigate the effect of sinomenine(SN)on the damage of human neuroblastoma SK-NSH cells induced by 1-methyl-4-4 phenylpyridine(MPP~+)and its mechanism for exploring the pathogenesis of Parkinson disease.METHODS:SN was used to treat MPP~+-induced SK-N-SH cells. The levels of malondialdehyde(MDA)and glutathione(GSH)in cell culture supernatants were measured by ELISA. The apoptosis was analyzed by flow cytometry.The protein expression levels of Bcl-2 and Bax were determined by Western blot. The expression levels of long noncoding RNA ANRIL and microRNA-626(miR-626)were detected by RT-qPCR. Dual-luciferase reporter assay was used to evaluate the relationship between ANRIL and miR-626. After ANRIL small interfering RNA was transfected into SK-N-SH cells,the effects of ANRIL expression knock-down on MPP~+-induced SK-N-SH cell apoptosis,the protein expression levels of Bcl-2 and Bax,and the levels of MDA and GSH in cell culture supernatants were examined.RESULTS:After treatment with MPP~+,the apoptotic rate,Bax protein level and ANRIL expression in SK-N-SH cells were increased(P<0. 05),and the Bcl-2 protein level and miR-626 expression were decreased(P<0. 05). The level of MDA in cell culture supernatants was increased(P<0. 05),and the level of GSH was decreased(P<0. 05). After SN treatment or ANRIL expression knock-down,decreased apoptotic rate,Bax protein level and ANRIL expression(P<0. 05),and increased Bcl-2 protein level and miR-626 expression in MPP~+-induced SK-N-SH cells were observed(P<0. 05). The level of MDA in the cell culture supernatants was decreased(P<0. 05),and the level of GSH was increased(P<0. 05).CONCLUSION:SN attenuates MPP~+-induced damage in SK-N-SH cells by regulating ANRIL/miR-626 signaling pathway.更多还原