灵芝提取物对MPP+诱导的Neuro-2a细胞自噬的影响

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。

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

Objective To investigate the protective effect of Ganoderma lucidum extract (GLE) on autophagy of Neuro-2a 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/mTOR/ULK1, PINK1/Parkin and NIX/LC3 proteins. Results The laser confocal microscopy showed that LC3-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 of LC3-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/mTOR/ULK1 and PINK1/Parkin pathway caused by MPP⁺ injury could be significantly improved.Conclusion GLE inhibited the autophagy response of MPP⁺-induced Neuro-2a cells, regulated the abnormal expression of autophagy regulatory proteins AMPK/mTOR/ULK1 and PINK1/Parkin, suggesting that GLE may play a neuroprotective role by regulating autophagy.