内质网蛋白TMEM166对MPP^(+)诱导的SH-SY5Y细胞氧化应激的影响及其机制

EFFECT OF ENDOPLASMIC RETICULUM PROTEIN TMEM166 ON MPP^(+)-INDUCED OXIDATIVE STRESS IN SH-SY5Y CELLS AND ITS MECHANISM

目的借助1-甲基-4-苯基-吡啶离子(MPP^(+))诱导的SH-SY5Y帕金森病(Parkinson’s disease,PD)细胞模型,研究内质网跨膜蛋白166(TMEM166)对PD细胞模型氧化应激的影响及其机制。方法将SH-SY5Y细胞分为A、B、C、D、E、F组,A~C组分别用0、1.0和1.5 mmol/L的MPP^(+)各处理24、48、72 h;D、E组分别转染Myc-TMEM166质粒和Myc空质粒,再用1.0 mmol/L MPP^(+)处理24 h;F组转染Myc-TMEM166质粒后,再用1.0 mmol/L MPP^(+)和1.0 mmol/L 3-甲基腺嘌呤(3-MA)共处理24 h。采用CCK-8法检测细胞相对活力,采用活性氧(ROS)检测试剂盒检测ROS生成情况,采用实时荧光定量PCR(RT-qPCR)法检测细胞TMEM 166 mRNA的表达水平,采用蛋白质印迹(Western blot)实验检测细胞中TMEM166蛋白及自噬相关蛋白p62和LC3Ⅱ/Ⅰ的表达水平。结果B、C组SH-SY5Y细胞在不同时间的相对细胞活力比较具有显著差异(F=58.56、35.81,P<0.05);在同一时间点,各组细胞的细胞相对活力比较也有显著差异(F=21.56~45.16,P<0.05)。MPP^(+)处理24 h后,A、B组SH-SY5Y细胞ROS生成水平比较有显著差异(t=25.94,P<0.05)。随着MPP^(+)处理浓度增高,A~C组细胞中TMEM166蛋白表达水平逐渐下降(F=290.80,P<0.05),而TMEM 166的mRNA水平逐渐升高(F=315.20,P<0.05)。D组和E组相比,MPP^(+)处理导致的SH-SY5Y细胞ROS生成水平、细胞相对活力、p62蛋白水平和LC3Ⅱ/Ⅰ蛋白水平有显著差异(t=2.91~6.69,P<0.05)。与D组相比,F组SH-SY5Y细胞中ROS生成明显减少(t=3.91,P<0.05)。结论TMEM166可能通过增强自噬加重MPP^(+)诱导的SH-SY5Y细胞氧化应激。

Objective To investigate the effect of endoplasmic reticulum transmembrane protein 166(TMEM166)on oxidative stress in a cell model of Parkinson’s disease(PD)and its mechanism using a 1-methyl-4-phenyl-pyridine ion(MPP^(+))-induced SH-SY5Y cell model of PD.Methods SH-SY5Y cells were divided into groups A,B,C,D,E,and F.Groups A-C were treated with 0 mmol/L,1.0 mmol/L,and 1.5 mmol/L MPP^(+)for 24 h,48 h,and 72 h,respectively.Groups D and E were transfected with Myc-TMEM166 plasmid and Myc empty plasmid,respectively,followed by treatment with 1.0 mmol/L MPP^(+)for 24 h.Group F was transfected with Myc-TMEM166 plasmid,followed by co-treatment with 1.0 mmol/L MPP^(+)and 1.0 mmol/L 3-methyladenine for 24 h.Cell Counting Kit-8 assay was used to determine the relative viability of cells.Reactive oxygen species(ROS)assay kits were used to determine the generation of ROS.Real-time quantitative polymerase chain reaction was used to measure the expression level of TMEM166 mRNA in cells.Western blotting was used to measure the expression levels of TMEM166 protein and autophagy-related proteins p62 and LC3Ⅱ/Ⅰ.Results In groups B and C,there was a significant difference in the relative cell viability of SH-SY5Y cells between different time points(F=58.56,35.81,P<0.05),and at the same time point,there was a significant difference in the relative cell viability between the groups(F=21.56-45.16,P<0.05).After 24 h of MPP^(+)treatment,there was a significant difference in the ROS generation level in SH-SY5Y cells between groups A and B(t=25.94,P<0.05).With the increase in MPP^(+)treatment concentration,the TMEM166 protein expression level in cells in groups A-C decreased gradually(F=290.80,P<0.05),while the TMEM166 mRNA level increased gradually(F=315.20,P<0.05).There were significant differences in the ROS generation level,relative cell viability,p62 protein level,and LC3Ⅱ/Ⅰprotein level in the MPP^(+)-treated SH-SY5Y cells between groups D and E(t=2.91-6.69,P<0.05).Compared with group D,group F had a significantly lower ROS generation level in SH-SY5Y cells(t=3.91,P<0.05).Conclusion TMEM166 may aggravate MPP^(+)-induced oxidative stress in SH-SY5Y cells by enhancing autophagy.