从具有潜在神经保护功能的中药中筛选自噬激活剂

Screening of Autophagy Activators from Chinese Medicinals with Potential Neuroprotective Function

目的:研究表明错误折叠蛋白的聚集是神经退行性疾病的病因所在,而自噬溶酶体是清除错误折叠蛋白缓解神经退行性疾病进程的重要途径,本研究旨在从来源丰富多样的具有潜在神经保护功能的中药中筛选自噬激活剂,以为后期中药神经保护剂的开发奠定基础。方法:从《中华本草》中选取记载有对中枢神经系统具保护作用的149味中药,使用75%乙醇回流提取并浓缩干燥后得到中药的总提取物,并以DMSO溶解配置成一定浓度的母液;应用MTT法测定中药总提取物对PC-12细胞的IC50以确定药物的作用浓度;应用pEGFP-LC3质粒稳定转染的Hela细胞研究药物对GFP-LC3斑点形成的影响;应用Western blot检测药物对Hela及PC-12细胞LC3蛋白表达的影响。结果:149味中药中M25(九头狮子草)、M119(远志)、M9(南沙参)、M98(皂荚)、M132(补骨脂)、M57(乌金藤)、M26(桔梗)、M53(猪牙皂)、M47(毛冬青)、M16(常春藤)、M131(益智仁)、M5(回心草)、M77(女贞子)、M40(虎杖)及M46(桑白皮)提取物有强效的自噬诱导作用。其中,M119(远志)、M9(南沙参)、M26(桔梗)、M47(毛冬青)、M5(回心草)和M46(桑白皮)提取物对PC-12细胞的安全性较高。结论:远志、桔梗、南沙参、毛冬青、回心草、桑白皮这6味中药安全性高,其潜在的神经保护功能可能与细胞自噬激活有关。

Objective:Misfolded protein aggregation is the cause of neurodegenerative diseases,and autophagy-lysosomal pathway plays an important role in clearing misfolded proteins and alleviating neurodegenerative diseases. This study aims to screen autophagy activators from the abundant Chinese medicinals with potential neuroprotective function,which is expected to lay a foundation for further development of neuroprotective Chinese medicine. Methods: A total of 149 Chinese medicinals,which can protect the central nervous system and treat related diseases as recorded in Chinese Materia Medica,were screened out. The medicinals were extracted with 75% ethanol by refluxing,concentrated,and dried to yield the total extract. The extract was dissolved in DMSO to prepare the stock solution with certain concentration. MTT assay was used to determine the half-maximal inhibitory concentration( IC50) on PC-12 cells in a bid to confirm the effective concentration. Hela cells which had been transformed with pG FP-LC3 were employed to monitor the effect on GFP-LC3 puncta formation. Western blotting was performed to determine the LC3 protein expression in PC-12 and Hela cells. Results: Among the 149 medicinals,M25 [Jiutoushizicao( 九头狮子草) ],M119 [Yuanzhi( 远志) ],M9 [Nanshashen( 南沙参) ],M98 [Zaojia( 皂荚) ],M132 [Buguzhi( 补骨脂) ],M57[Wujinteng( 乌金藤) ],M26 [Jiegeng( 桔梗) ],M53 [Zhuyazao( 猪牙皂) ],M47[Maodongqing( 毛冬青) ],M16 [Changchunteng( 常春藤) ],M131 [Yizhiren( 益智仁) ],M5 [Huixincao( 回心草) ],M77 [Nvzhenzi( 女贞子) ],M40 [Huzhang( 虎杖) ],and M46 [Sangbaipi( 桑白皮) ]exhibited strong effect in autophagy induction. In addition,the extract of M119 [Yuanzhi( 远志) ],M9 [Nanshashen( 南沙参) ],M26 [Jiegeng( 桔梗) ],M47 [Maodongqing( 毛冬青) ],M5 [Huixincao( 回心草) ],and M46 [Sangbaipi( 桑白皮) ]was safe to PC-12 cells. Conclusion: Yuanzhi( 远志),Jiegeng( 桔梗),Nanshashen( 南沙参),Maodongqing( 毛冬青),Huixincao( 回心草),and Sangbaipi( 桑白皮),which prove safe,have potential neuroprotective effect and the effect may be related to the activation of autophagy.