miR-185过表达对MPP+诱导帕金森病模型细胞损伤的影响及其机制

Effect of miR-185 overexpression on MPP+-induced cell damage in Parkinson's disease model

目的探讨miR-185过表达对1-甲基-4-苯基吡啶离子(MPP+)诱导帕金森病(PD)模型细胞损伤的影响及其机制。方法体外传代培养PC12细胞。取传3代、对数生长期PC12细胞,随机分为对照组、模型组、NC mimics组、miR-185 mimics组。模型组、NC mimics组、miR-185 mimics组予1 mmol/L MPP+诱导PD细胞模型,对照组不予MPP+诱导。成模24 h,NC mimics组、miR-185 mimics组分别转染NC mimics、miR-185 mimics。取上述各组细胞,采用RT-qPCR法检测miR-185表达;采用CCK-8法检测培养24、48、72 h细胞增殖活性,采用流式细胞术检测细胞凋亡率,采用ELISA法检测谷胱甘肽过氧化物酶(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA),采用Western blotting法检测Bax、Bcl-2蛋白表达。结果与对照组比较,模型组miR-185相对表达量及培养24、48、72 h细胞增殖活性降低,细胞凋亡率升高(P均<0.05);GSH、SOD含量降低,MDA含量升高(P均<0.05);Bcl-2蛋白相对表达量降低,Bax蛋白相对表达量升高(P均<0.05)。与NC mimics组比较,miR-185 mimics组miR-185相对表达量及培养24、48、72 h细胞增殖活性升高,细胞凋亡率降低(P均<0.05);GSH、SOD含量升高,MDA含量降低(P均<0.05);Bcl-2蛋白相对表达量升高,Bax蛋白相对表达量降低(P均<0.05)。而模型组与NC mimics组上述指标比较差异均无统计学意义(P均>0.05)。结论miR-185过表达能够促进PD细胞增殖并抑制其凋亡,具有明显的保护作用;减轻氧化应激损伤以及抑制Bax蛋白表达和促进Bcl-2蛋白表达可能是其作用机制之一。

Objective To investigate the effect of miR-185 overexpression on 1-methyl-4-phenylpyridine ion(MPP+)-induced cell damage in Parkinson's disease(PD)models and its mechanism.Methods PC12 cells were pas-saged in vitro.PC12 cells of the third generation in the logarithmic growth phase were randomly divided into the control group,the model group,NC mimics group,and miR-185 mimics group,respectively.Cells in the model group,the NC mimics group,and the miR-185 mimics group were induced with 1 mmol/L MPP+to construct the PD cell models,while cells in the control group were not induced with MPP+.After 24 h of modeling,cells in the NC mimics group and miR-185 mimics group were transfected with NC mimics and miR-185 mimics,respectively.Cells from each group were collected.The expression of miR-185 was detected using RT-qPCR,the proliferation ability of cells cultured for 24,48,and 72 h was detected by CCK-8,the apoptosis rate was detected using flow cytometry,the intracellular glutathione peroxidase(GSH),superoxide dismutase(SOD),and malondialdehyde(MDA)were detected by ELISA,and the expression levels of Bax and Bcl-2 proteins were detected by Western blotting.Results Compared with the control group,the relative expression level of miR-185 and the proliferation activity of cells in the model group decreased after 24,48,and 72 h of culture,while the apoptosis rate increased(all P<0.05);the intracellular GSH and SOD content decreased,while the MDA content increased(both P<0.05);the relative expression level of Bcl-2 protein decreased,while the relative expres-sion level of Bax protein increased(both P<0.05).Compared with the NC mimics group,the relative expression level of miR-185 and cell proliferation activity after 24,48,and 72 h of culture increased,and apoptosis rate decreased in the miR-185 mimics group(all P<0.05);the intracellular GSH and SOD content increased,while the MDA content decreased(both P<0.05);the relative expression level of Bcl-2 protein increased,while the relative expression level of Bax protein decreased(both P<0.05).However,there were no statistically significant differences in the above indicators between the model group and the NC mimics group(all P>0.05).Conclusion Overexpression of miR-185 can promote the prolifera-tion and inhibit apoptosis of PD cells,and has a significant protective effect;alleviating oxidative stress damage,inhibit-ing Bax protein expression,and promoting Bcl-2 protein expression may be one of the mechanisms.