山莨菪碱通过lncRNA LINC00657/miR-496通路对MPP^(+)诱导SK-N-SH细胞损伤的保护作用研究

Protective Effect of Anisodamine on SK-N-SH Cell Injury Induced by MPP^(+)Through lncRNA LINC00657/miR-496 Pathway

目的:探讨山莨菪碱(Ani)对1-甲基-4-苯基吡啶离子(MPP+)诱导SK-N-SH细胞损伤的作用及分子机制。方法:将SK-N-SH细胞分为Con组,MPP^(+)组,MPP+Ani-L、M、H组,MPP^(+)+si-NC组,MPP^(+)+si-LINC00657组,MPP^(+)+Ani+pcDNA组,MPP^(+)+Ani+pcDNA-LINC00657组。试剂盒检测丙二醛(MDA)含量和谷胱甘肽(GSH)活性;流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测Bcl-2、Bax蛋白表达;实时荧光定量PCR(RT-qPCR)检测LINC00657和miR-496的表达;双荧光素酶报告实验检测LINC00657和miR-496的靶向关系。结果:不同浓度山莨菪碱处理后,MPP+诱导SK-N-SH细胞中MDA含量降低,GSH活性升高,细胞凋亡率降低,Bcl-2表达水平升高,Bax表达水平降低,LINC00657表达水平降低,miR-496表达水平升高,且呈浓度依赖性(P<0.05)。LINC00657靶向调控miR-496的表达;抑制LINC00657表达后,MPP+诱导SK-N-SH细胞中MDA含量降低,GSH活性升高,细胞凋亡率降低,Bcl-2表达水平升高,Bax表达水平降低(P<0.05)。LINC00657过表达逆转了山莨菪碱对MPP^(+)诱导SK-N-SH细胞损伤的作用。结论:山莨菪碱通过调控lncRNA LINC00657/miR-496通路对MPP+诱导SK-N-SH细胞损伤起保护作用。

Objective:To investigate the effect and molecular mechanism of anisodamine(Ani)on MPP^(+)-induced SK-NSH cell damage.Methods:SK-N-SH cells were divided into Con group,MPP^(+)group,MPP^(+)+Ani-L、M、H group,MPP^(+)+si-NC group,MPP^(+)+si-LINC00657 group,MPP^(+)+Ani+pcDNA group,MPP^(+)+Ani+pcDNA-LINC00657 group.Kit to detect malondialdehyde(MDA)content and glutathione(GSH)activity,flow cytometry to detect apoptosis,Western blot method to detect Bcl-2 and Bax protein expression,real-time fluorescence quantitative PCR(RT-qPCR)detection of LINC00657 and miR-496 expression,dual luciferase report test to detect the targeting relationship between LINC00657 and miR-496.Results:After treatment with anisodamine at different concentrations,MDA content was decreased in MPP+-induced SKN-SH cell,GSH activity was increased,apoptosis rate was decreased,Bcl-2 expression was increased,Bax expression was decreased,LINC00657 expression was decreased,expression of miR-496 was increased,in a concentrationdependent manner(P<0.05).LINC00657 regulates the expression of miR-496,after inhibiting the expression of LINC00657,MDA content was decreased in MPP^(+)-induced SK-N-SH cell,GSH activity was increased,apoptosis rate was decreased,Bcl-2 expression was increased,Bax expression was decreased(P<0.05).LINC00657 overexpression reversed the effect of anisodamine on MPP^(+)-induced SK-N-SH cell damage.Conclusion:Anisodamine protects SK-N-SH cells from MPP^(+)-induced injury by regulating the lncRNA LINC00657/miR-496 pathway.