灵芝提取物对MPP^+诱导的Neuro-2a细胞自噬的影响

Ganoderma lucidum extract regulates autophagy induced by MPP^+ in Neuro-2a cells

目的观察灵芝提取物(Ganoderma lucidum extract,GLE)对1-甲基-4-苯基-吡啶离子(1-methyl-4-phenylpyridinium,MPP^+)诱导的Neuro-2a细胞自噬的调节作用。方法采用蛋白印迹检测自噬标志物LC3的转换(LC3-Ⅱ/LC3-Ⅰ)以及荧光显微镜检测LC3点状聚集物的形成。同时Western blotting法检测AMPK/mTOR/ULK1、PINK1/Parkin、NIX/LC3蛋白的表达水平。结果激光共聚焦采集图像结果显示,MPP^+处理组细胞LC3-Ⅱ聚集于自噬体膜上,观察到呈点状聚集状态,而GLE干预组细胞点状聚集状态不明显。以LC3的点状聚集阳性细胞占总细胞的百分比评价自噬水平的高低。MPP^+组呈绿色点状分布的LC3阳性细胞百分比明显升高,GLE组明显降低。Western blotting法检测结果显示GLE组LC3-Ⅱ/LC3-Ⅰ比值降低。同时,经GLE处理后,可以改善MPP^+损伤引起的AMPKα/mTOR/ULK1、PINK1/Parkin通路各蛋白异常表达。结论 GLE能抑制MPP^+诱导的Neuro-2a细胞的自噬反应,调节细胞自噬相关蛋白AMPK/mTOR/ULK1以及PINK1/Parkin的异常表达,表明GLE可能通过调节细胞自噬而发挥神经保护作用。

Objective To investigate the protective effect of Ganoderma lucidum extract( GLE) on autophagy of Neuro-2 a cells induced by 1-methyl-4-phenylpyridinium( MPP^+).Methods Western blotting was used to detect the conversion of autophagy marker LC3(LC3-Ⅱ/LC3-Ⅰ),and fluorescence microscopy was employed to detect the formation of LC3 punctate aggregates.Western blot was used to detect the expression levels of AMPK/mT OR/ULK1,PINK1/Parkin and NIX/LC3 proteins.Results The laser confocal microscopy showed thatLC3-Ⅱ cells in the MPP^+treatment group were clustered on the autophagosome membrane,with a point-like aggregation state observed,while no obvious point aggregation was observed in the GLE intervention group.The level of autophagy was evaluated as the ratio of point-aggregated positive cells of LC3 over the total cells.According to statistics,the percentage of LC3 positive cells with green dot distribution in the MPP^+group was significantly increased.This value was significantly reduced in the GLE group.Western blotting results showed that the ratio ofLC3-Ⅱ/LC3-Ⅰ in GLE group was significantly decreased,and the expression level of NIX protein was significantly increased.After GLE treatment,the abnormal expression of each protein in AMPKα/mT OR/ULK1 and PINK1/Parkin pathway caused by MPP^+injury could be significantly improved.Conclusion GLE inhibited the autophagy response of MPP^+-induced Neuro-2 a cells,regulated the abnormal expression of autophagy regulatory proteins AMPK/mT OR/ULK1 and PINK1/Parkin,suggesting that GLE may play a neuroprotective role by regulating autophagy.