灵芝提取物对MPP+诱导的Neuro-2a细胞自噬的影响

doi:10.3969/j.issn.1006-7795.2019.04.019

首都医科大学学报 ›› 2019, Vol. 40 ›› Issue (4): 596-601.doi: 10.3969/j.issn.1006-7795.2019.04.019

• 神经系统退行性疾病的基础研究 • 上一篇下一篇

灵芝提取物对MPP⁺诱导的Neuro-2a细胞自噬的影响

丁晖, 蔡彦宁, 陈彪

首都医科大学宣武医院神经生物室首都医科大学帕金森病临床诊疗与研究中心, 北京 100053

收稿日期:2019-05-15 出版日期:2019-07-21 发布日期:2019-07-19
通讯作者: 陈彪 E-mail:pbchan@hotmail.com
基金资助:
国家重点研发计划（2018YFC1312001，2017YFC0840105），北京市医院管理局"使命"计划专项（SML20150803），北京市科学技术委员会项目（Z171100000117013）。

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

Ding Hui, Cai Yanning, Chen Biao

Department of Neurobiology, Xuanwu Hospital, Capital Medical University;Clinical Center for Parkinson's Disease, Beijing Key Laboratory for Parkinson's Disease, Capital Medical University, Beijing 100053, China

Received:2019-05-15 Online:2019-07-21 Published:2019-07-19
Supported by:
This study was supported by National Key Research and Development Program of China (2018YFC1312001,2017YFC0840105),Beijing Municipal Administration of Hospitals' Mission Plan(SML20150803), Beijing Municipal Science & Technology Commission(Z171100000117013).

摘要/Abstract

摘要： 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。

关键词: 灵芝, 帕金森病, 自噬, 神经保护

Abstract: 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.

Key words: Ganoderma lucidum, Parkinson's disease, autophagy, neuroprotection

中图分类号:

R592

丁晖, 蔡彦宁, 陈彪. 灵芝提取物对MPP⁺诱导的Neuro-2a细胞自噬的影响[J]. 首都医科大学学报, 2019, 40(4): 596-601.

Ding Hui, Cai Yanning, Chen Biao. Ganoderma lucidum extract regulates autophagy induced by MPP⁺ in Neuro-2a cells[J]. Journal of Capital Medical University, 2019, 40(4): 596-601.

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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

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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

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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