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Article(id=1194649189859693389, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1194643387904136153, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.0483.2024.0716, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1680451200000, receivedDateStr=2023-04-03, revisedDate=null, revisedDateStr=null, acceptedDate=1684944000000, acceptedDateStr=2023-05-25, onlineDate=1762756162371, onlineDateStr=2025-11-10, pubDate=1737993600000, pubDateStr=2025-01-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762756162371, onlineIssueDateStr=2025-11-10, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762756162371, creator=13701087609, updateTime=1762756162371, updator=13701087609, issue=Issue{id=1194643387904136153, tenantId=1146029695717560320, journalId=1189873630562394117, year='2025', volume='50', issue='1', pageStart='1', pageEnd='120', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1762754779076, creator=13701087609, updateTime=1762756450259, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1194650397408203370, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1194643387904136153, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1194650397408203371, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1194643387904136153, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=69, endPage=75, ext={EN=ArticleExt(id=1194649190086185811, articleId=1194649189859693389, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Protective effects of paeonol on human neuroblastoma cells in Parkinson's disease model and its underlying mechanism, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the protective effects of paeonol (PAE) on autophagy in human neuroblastoma cells (SH-SY5Y) induced by overexpression of α-synuclein (α-Syn), and to explore its related mechanism. Methods SH-SY5Y cells served as control group, while those induced with A53T-α-Syn mutation were used as model group. Additional groups included PAE (150 μg/ml) group, 3-MA (1 mmol/L) group, and PAE(150 μg/ml)+3-MA (1 mmol/L) group. Cell viability was assessed using CCK-8 method, cell morphology was observed under an optical microscope, and protein expressions of α-Syn, LC3-Ⅱ, p62, Beclin-1, phosphorylated c-Jun N-terminal kinase (p-JNK), and p-Bcl-2 were determined by Western blotting. Results Compared with control group, model control exhibited decreased cell survival (P<0.01), increased α-Syn expression (P<0.001), reduced expression of autophagy-related proteins LC3-Ⅱ and Beclin-1 (P<0.01, P<0.05), elevated autophagy substrate protein p62 (P<0.05), and decreased expression of autophagy pathway-related proteins p-JNK and Bcl-2 (P<0.05, P<0.01). Compared with model group, PAE group showed increased cell survival (P<0.01), decreased α‑Syn and p62 protein expression (P<0.01, P<0.05), and increased expression of LC3-Ⅱ, Beclin-1, p-JNK and Bcl-2 (P<0.05). Compared with PAE group, 3-MA+PAE group demonstrated increased α-Syn expression (P<0.05). Conclusions PAE could attenuate the injury of SH-SY5Y cells induced by A53T-α-Syn and eliminate over-expressed α‑Syn by activating autophagy pathway, which may be associated with the upregulation of JNK/Bcl-2 mediated autophagy pathway.

, correspAuthors=Hui-Jie Fan, Zhi Chai, authorNote=null, correspAuthorsNote=
Chai Zhi, E-mail:
Fan Hui-Jie, E-mail:
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目的 探究丹皮酚(PAE)对过表达α-突触核蛋白(α-Syn)诱导的人神经母细胞瘤细胞(SH-SY5Y)损伤中自噬的影响及其相关作用机制。方法 以SH-SY5Y细胞为对照组，含A53T-α-Syn突变的SH-SY5Y细胞为模型组、PAE(150 μg/ml)组、3-甲基腺嘌呤(3-MA，1 mmol/L)组和PAE(150 μg/ml)+3-MA(1 mmol/L)组。采用CCK-8法检测各组细胞存活率，光学显微镜下观察细胞形态，Western blotting检测α-Syn和自噬通路相关蛋白Beclin-1、p62、微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、磷酸化c-Jun氨基末端激酶(p-JNK)、Bcl-2表达水平。结果 与对照组比较，模型组细胞存活率明显降低(P<0.01)，α-Syn蛋白表达水平明显增高(P<0.001)，自噬相关蛋白LC3-Ⅱ、Beclin-1蛋白表达水平明显降低(P<0.01或P<0.05)，自噬底物蛋白p62蛋白表达水平升高(P<0.05)，调节自噬通路相关蛋白p-JNK、Bcl-2蛋白表达水平降低(P<0.05或P<0.01)。与模型组比较，PAE组细胞存活率增高(P<0.01)，α-Syn、p62蛋白表达水平降低(P<0.01或P<0.05)，LC3-Ⅱ、Beclin-1、p-JNK、Bcl-2蛋白表达水平增高(P<0.05)。与PAE组比较，PAE+3-MA组细胞α-Syn蛋白表达水平增高(P<0.05)。结论 PAE可缓解A53T-α-Syn突变诱导的SH-SY5Y细胞损伤，并通过激活自噬途径清除过表达的α-Syn，其机制可能与上调JNK/Bcl-2介导的自噬途径有关。

, correspAuthors=樊慧杰, 柴智, authorNote=null, correspAuthorsNote=
柴智，E-mail：
樊慧杰，E-mail：
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孙胜男，硕士研究生，主要从事神经系统疾病的中西医结合防治研究

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孙胜男，硕士研究生，主要从事神经系统疾病的中西医结合防治研究

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孙胜男，硕士研究生，主要从事神经系统疾病的中西医结合防治研究

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caption=Effects of different concentrations of PAE on the survival rates of A53T-α-Syn cells, figureFileSmall=T+LGblqsgNoH4gU9ELKARQ==, figureFileBig=J/PwDWkVWwfrRBFYdx8GCQ==, tableContent=null), ArticleFig(id=1194661969320973078, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=CN, label=图1, caption=不同浓度PAE对A53T-α-Syn细胞存活率的影响

α-Syn. α-突触核蛋白；PAE. 丹皮酚；与对照组比较，(1)P<0.01；与模型组比较，(2)P<0.05，(3)P<0.01

, figureFileSmall=T+LGblqsgNoH4gU9ELKARQ==, figureFileBig=J/PwDWkVWwfrRBFYdx8GCQ==, tableContent=null), ArticleFig(id=1194661969434219287, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=EN, label=Fig.2, caption=Morphology of SH-SY5Y cells of each group in microscope (40×), figureFileSmall=JwEdg7CLZc2++93b368KzQ==, figureFileBig=TBO17nCvuBptVuFrNDcEdA==, tableContent=null), ArticleFig(id=1194661969518105368, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=CN, label=图2, caption=光学显微镜下各组SH-SY5Y细胞形态(40×)

α-Syn. α-突触核蛋白；PAE. 丹皮酚；Ａ. 对照组；Ｂ. 模型组；Ｃ. PAE组

, figureFileSmall=JwEdg7CLZc2++93b368KzQ==, figureFileBig=TBO17nCvuBptVuFrNDcEdA==, tableContent=null), ArticleFig(id=1194661969593602841, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=EN, label=Fig.3, caption=Comparison of the expression levels of α-synuclein in each group of cells (n=3), figureFileSmall=2tWHNqaO1/mYqYNO7ArpcQ==, figureFileBig=gXE7esLxACCeHnhKh14HpA==, tableContent=null), ArticleFig(id=1194661969669100314, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=CN, label=图3, caption=各组细胞中α-突触核蛋白表达水平比较(n=3)

α-Syn. α-突触核蛋白；PAE. 丹皮酚；与对照组比较，(1)P<0.001；与模型组比较，(2)P<0.01

, figureFileSmall=2tWHNqaO1/mYqYNO7ArpcQ==, figureFileBig=gXE7esLxACCeHnhKh14HpA==, tableContent=null), ArticleFig(id=1194661969740403483, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=EN, label=Fig.4, caption=Comparison of the expression levels of autophagy related proteins Beclin-1, p62, and LC3-Ⅱ in each group of cells (n=3), figureFileSmall=VFwBCgP65vG9Lpb/Uy5iUA==, figureFileBig=Je6yhB0FMp8ers46dqZOEg==, tableContent=null), ArticleFig(id=1194661969815900956, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=CN, label=图4, caption=各组细胞中自噬相关蛋白Beclin-1、p62、LC3-Ⅱ的表达水平比较(n=3)

PAE. 丹皮酚；LC3-Ⅱ. 微管相关蛋白轻链3 Ⅱ；与对照组比较，(1)P<0.01，(2)P<0.05；与模型组比较，(3)P<0.05

, figureFileSmall=VFwBCgP65vG9Lpb/Uy5iUA==, figureFileBig=Je6yhB0FMp8ers46dqZOEg==, tableContent=null), ArticleFig(id=1194661969887204125, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=EN, label=Fig.5, caption=Comparison of the expression level of α‑Syn after autophagy inhibited in each group of cells (n=3), figureFileSmall=toekETRFc7KEb9svb+9kBg==, figureFileBig=PGzEXG6wkjHtXyNWWcsOTA==, tableContent=null), ArticleFig(id=1194661969971090206, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=CN, label=图5, caption=抑制自噬后各组细胞中α-Syn蛋白表达水平比较(n=3)

α-Syn. α-突触核蛋白；PAE. 丹皮酚；与对照组比较，(1)P<0.01；与PAE组比较，(2)P<0.05，

, figureFileSmall=toekETRFc7KEb9svb+9kBg==, figureFileBig=PGzEXG6wkjHtXyNWWcsOTA==, tableContent=null), ArticleFig(id=1194661970038199071, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=EN, label=Fig.6, caption=Comparison of the expression levels of p-JNK and Bcl-2 in each group of cells (n=3), figureFileSmall=9zhUQma2usT07SdesUlEug==, figureFileBig=/OzWQVyDdaUD8rF+Xpxz0g==, tableContent=null), ArticleFig(id=1194661970201776928, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194649189859693389, language=CN, label=图6, caption=各组细胞中p-JNK、Bcl-2蛋白表达水平比较(n=3)

PAE. 丹皮酚；p-JNK. 磷酸化c-Jun氨基末端激酶；Bcl-2. B淋巴细胞瘤-2蛋白；与对照组比较，(1)P<0.05，(2)P<0.01；与模型组比较，(3)P<0.05

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丹皮酚对人神经母细胞瘤细胞帕金森病模型的保护作用及其初步机制
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孙胜男 1 , 和璐璐 2 , 秦劭晨 3 , 徐磊 1 , 王利然 4 , 于保锋 2 , 马存根 1 , 樊慧杰 1, * , 柴智 1, *
解放军医学杂志 | 基础研究 2025,50(1): 69-75
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解放军医学杂志 | 基础研究 2025, 50(1): 69-75
丹皮酚对人神经母细胞瘤细胞帕金森病模型的保护作用及其初步机制
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孙胜男1, 和璐璐2, 秦劭晨3, 徐磊1, 王利然4, 于保锋2, 马存根1, 樊慧杰1, * , 柴智1, *
作者信息
  • 1山西中医药大学多发性硬化益气活血重点研究室/神经生物学研究中心，山西晋中 030619
  • 2山西医科大学基础医学院生物化学与分子生物学教研室，山西太原 030001
  • 3山西中医药大学第一临床学院，山西太原 030024
  • 4山西中医药大学第二临床学院，山西太原 030012

    • 孙胜男，硕士研究生，主要从事神经系统疾病的中西医结合防治研究

通讯作者:

柴智，E-mail：
樊慧杰，E-mail：
Protective effects of paeonol on human neuroblastoma cells in Parkinson's disease model and its underlying mechanism
Sheng-Nan Sun1, Lu-Lu He2, Shao-Chen Qin3, Lei Xu1, Li-Ran Wang4, Bao-Feng Yu2, Cun-Gen Ma1, Hui-Jie Fan1, * , Zhi Chai1, *
Affiliations
  • 1The Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine/Neurobiology Research Center, Shanxi University of Chinese Medicine, Jinzhong, Shanxi 030619, China
  • 2Biochemistry and Molecular Biology Teaching and Research Office, School of Basic Medicine, Shanxi Medical University, Taiyuan, Shanxi 030001, China
  • 3The First Clinical College of Shanxi University of Chinese Medicine, Taiyuan, Shanxi 030024, China
  • 4The Second Clinical College of Shanxi University of Chinese Medicine, Taiyuan, Shanxi 030012, China
出版时间: 2025-01-28 doi: 10.11855/j.issn.0577-7402.0483.2024.0716
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摘要
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目的 探究丹皮酚(PAE)对过表达α-突触核蛋白(α-Syn)诱导的人神经母细胞瘤细胞(SH-SY5Y)损伤中自噬的影响及其相关作用机制。方法 以SH-SY5Y细胞为对照组，含A53T-α-Syn突变的SH-SY5Y细胞为模型组、PAE(150 μg/ml)组、3-甲基腺嘌呤(3-MA，1 mmol/L)组和PAE(150 μg/ml)+3-MA(1 mmol/L)组。采用CCK-8法检测各组细胞存活率，光学显微镜下观察细胞形态，Western blotting检测α-Syn和自噬通路相关蛋白Beclin-1、p62、微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、磷酸化c-Jun氨基末端激酶(p-JNK)、Bcl-2表达水平。结果 与对照组比较，模型组细胞存活率明显降低(P<0.01)，α-Syn蛋白表达水平明显增高(P<0.001)，自噬相关蛋白LC3-Ⅱ、Beclin-1蛋白表达水平明显降低(P<0.01或P<0.05)，自噬底物蛋白p62蛋白表达水平升高(P<0.05)，调节自噬通路相关蛋白p-JNK、Bcl-2蛋白表达水平降低(P<0.05或P<0.01)。与模型组比较，PAE组细胞存活率增高(P<0.01)，α-Syn、p62蛋白表达水平降低(P<0.01或P<0.05)，LC3-Ⅱ、Beclin-1、p-JNK、Bcl-2蛋白表达水平增高(P<0.05)。与PAE组比较，PAE+3-MA组细胞α-Syn蛋白表达水平增高(P<0.05)。结论 PAE可缓解A53T-α-Syn突变诱导的SH-SY5Y细胞损伤，并通过激活自噬途径清除过表达的α-Syn，其机制可能与上调JNK/Bcl-2介导的自噬途径有关。

丹皮酚  /  帕金森病  /  α-突触核蛋白  /  自噬

Objective To investigate the protective effects of paeonol (PAE) on autophagy in human neuroblastoma cells (SH-SY5Y) induced by overexpression of α-synuclein (α-Syn), and to explore its related mechanism. Methods SH-SY5Y cells served as control group, while those induced with A53T-α-Syn mutation were used as model group. Additional groups included PAE (150 μg/ml) group, 3-MA (1 mmol/L) group, and PAE(150 μg/ml)+3-MA (1 mmol/L) group. Cell viability was assessed using CCK-8 method, cell morphology was observed under an optical microscope, and protein expressions of α-Syn, LC3-Ⅱ, p62, Beclin-1, phosphorylated c-Jun N-terminal kinase (p-JNK), and p-Bcl-2 were determined by Western blotting. Results Compared with control group, model control exhibited decreased cell survival (P<0.01), increased α-Syn expression (P<0.001), reduced expression of autophagy-related proteins LC3-Ⅱ and Beclin-1 (P<0.01, P<0.05), elevated autophagy substrate protein p62 (P<0.05), and decreased expression of autophagy pathway-related proteins p-JNK and Bcl-2 (P<0.05, P<0.01). Compared with model group, PAE group showed increased cell survival (P<0.01), decreased α‑Syn and p62 protein expression (P<0.01, P<0.05), and increased expression of LC3-Ⅱ, Beclin-1, p-JNK and Bcl-2 (P<0.05). Compared with PAE group, 3-MA+PAE group demonstrated increased α-Syn expression (P<0.05). Conclusions PAE could attenuate the injury of SH-SY5Y cells induced by A53T-α-Syn and eliminate over-expressed α‑Syn by activating autophagy pathway, which may be associated with the upregulation of JNK/Bcl-2 mediated autophagy pathway.

paeonol  /  Parkinson's disease  /  α-synuclein  /  autophagy
孙胜男, 和璐璐, 秦劭晨, 徐磊, 王利然, 于保锋, 马存根, 樊慧杰, 柴智. 丹皮酚对人神经母细胞瘤细胞帕金森病模型的保护作用及其初步机制. 解放军医学杂志, 2025 , 50 (1) : 69 -75 . DOI: 10.11855/j.issn.0577-7402.0483.2024.0716
Sheng-Nan Sun, Lu-Lu He, Shao-Chen Qin, Lei Xu, Li-Ran Wang, Bao-Feng Yu, Cun-Gen Ma, Hui-Jie Fan, Zhi Chai. Protective effects of paeonol on human neuroblastoma cells in Parkinson's disease model and its underlying mechanism[J]. Medical Journal of Chinese People’s Liberation Army, 2025 , 50 (1) : 69 -75 . DOI: 10.11855/j.issn.0577-7402.0483.2024.0716
正文
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帕金森病(Parkinson's disease，PD)是一种常见的慢性中枢神经系统退行性病变，其典型运动症状包括运动迟缓、肌肉僵硬、静止性震颤、姿势和步态障碍等[1]。流行病学数据显示，PD已迎来潜在的“大流行”，预计到2040年患者数将超过1200万，对社会和家庭造成巨大负担[2-3]。PD的神经病理学特征是中脑黑质致密部多巴胺(dopamine，DA)能神经元缺失，以及一种细胞内包涵体——路易体(Lewy body，LB)的存在[4]。LB是PD的脑组织学标志，其主要成分是异常聚集的α-突触核蛋白(α-synuclein，α-Syn)。自噬在维持DA能神经元中α-Syn的可溶性方面发挥着重要作用，α-Syn的异常聚集与自噬通路降解障碍密切相关[5]。
丹皮酚(paeonol，PAE)是一种天然酚类化合物，主要分离自毛茛科芍药属的根皮或皮层[6]。研究显示，PAE对多种神经系统疾病具有潜在的保护作用[7-9]；提示PAE可能成为PD的潜在治疗药物。本研究采用α-Syn基因(SNCA)A53T基因突变而过表达α-Syn的SH-SY5Y细胞为PD细胞模型[10]，观察PAE对PD细胞模型自噬的影响，探讨PAE治疗帕金森病的作用机制，旨在为PD治疗药物的研发提供实验依据。
1 材料与方法
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1.1 仪器与试剂
倒置显微镜(德国Leica公司)；全波长酶标仪(美国Molecular Devices公司)；细胞培养箱(美国赛默飞世尔科技公司)；凝胶成像仪(美国Azure公司)；湿转膜仪、凝胶电泳仪(美国Bio-Rad公司)。神经母细胞瘤株SH-SY5Y细胞由山西中医药大学国家中医药管理局多发性硬化益气活血重点研究室提供；SNCA A53T突变而过表达α-Syn的SH-SY5Y细胞株(A53T-α-Syn细胞)购自苏州海星生物科技有限公司。
PAE(纯度≥98%；sp8040，北京索莱宝科技有限公司)；3-甲基腺嘌呤(3-methyladenine，3-MA；HY-19312，美国MCE公司)；MEM/F12基础培养基、青霉素-链霉素溶液(武汉普诺赛生命科技有限公司)；CCK-8试剂盒(MA02180，大连美仑生物技术有限公司)；RIPA裂解液、50× Cocktail蛋白酶抑制剂(武汉赛维尔生物科技有限公司)；BCA蛋白浓度测定试剂盒(AR0197，武汉博士德生物工程有限公司)；高灵敏度化学发光检测试剂盒(eECL，江苏康为世纪生物科技股份有限公司)；抗α-Syn抗体、兔抗sequestosome 1(SQSTM1/p62)抗体、抗微管相关蛋白轻链3(LC3)抗体(英国Abcam公司)；抗Beclin-1抗体、抗B淋巴细胞瘤-2(Bcl-2)蛋白抗体(美国CST公司)；抗微管蛋白β(β-tubulin)抗体、抗β-肌动蛋白(β-actin)抗体、羊抗兔IgG(南京Bioworld公司)。
1.2 方法
1.2.1 细胞培养
在MEM/F12基础培养基中添加10%的热灭活FBS和1%的青霉素/链霉素双抗配制为完全培养基，过滤除菌备用。将细胞接种至完全培养基培养，置于温度37 ℃、相对湿度95%、5% CO2的培养箱中培养，2~3 d更换一次培养液。当细胞生长密度为80%~90%时，按1:(3~5)进行传代，或进行铺板、冻存处理。
1.2.2 细胞分组和药物干预
采用SH-SY5Y细胞作为对照组；将A53T-α-Syn细胞随机分为模型组、PAE组、3-MA组与PAE+3-MA组。PAE溶于二甲基亚砜(DMSO)，3-MA溶于基础培养基。对照组和模型组细胞不加药物，常规培养。PAE组细胞根据实验需求用含不同浓度PAE药液的完全培养基培养。PAE+3-MA组细胞在含150 μg/ml PAE的完全培养基中加入浓度为1 mmol/L的3-MA，24 h后终止培养。
1.3 观察指标及检测方法
1.3.1 细胞形态和细胞活力检测
采用光学显微镜观察细胞形态，CCK-8试验检测细胞存活率。将各组细胞以每孔1×104个接种于96孔板中，置于细胞箱培养至细胞贴壁后，分别以0、25、50、75、100、125、150 μg/ml PAE处理；24 h后将CCK-8试剂与完全培养基按1:9的比例配制，吸去药物处理后的培养上清，依次沿侧壁加入10 μl配制好的CCK-8试剂，相应调零孔也加入试剂。将96孔板转移至细胞培养箱避光孵育1.5 h后，用酶标仪检测450 nm波长下的吸光度(OD)值，计算细胞存活率。细胞活力(%)=(实验组OD值－培养板空白OD值)/(对照组OD值－培养板空白OD值)×100%。
1.3.2 Western blotting检测α-Syn和自噬通路相关蛋白Beclin-1、p62、LC3Ⅱ、磷酸化c-Jun氨基末端激酶(p-JNK)、Bcl-2表达水平
取对数期细胞在6孔板中进行给药处理24 h；将细胞取出，弃去培养基，6孔板中加入蛋白裂解液，用细胞刮板将贴壁细胞刮落，冰上裂解30 min，4℃、12 000 r/min离心5 min，取上清；按照试剂盒说明书采用BCA法测定蛋白浓度，提取细胞总蛋白，100 ℃煮沸5 min进行蛋白变性，冷却后于-20 ℃保存备用。制备十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)，浓度为12%的分离胶和5%的浓缩胶。以每组蛋白量为20 μg上样，先用80 V电压至压平条带约20 min，再用120 V电压60 min电泳结束，后恒压转至0.45 μm孔径的聚偏二氟乙烯(polyvinylidene fluoride，PVDF)膜上。5%奶粉封闭，室温下摇床孵育1 h，TBST洗3次×10 min。分别加入TBST稀释的α-Syn(1:1000)、Beclin-1(1:1000)、p62(1:1000)、LC3-Ⅱ(1:1000)、p-JNK(1:1000)、Bcl-2(1:1000)、β-actin(1:2000)、β-tubulin(1:2000)一抗，4 ℃摇床上孵育过夜后，用TBST洗3次×10 min。加入二抗，室温避光摇床上孵育60 min，TBST洗3次×10 min。将eECL工作液按照A液:B液=1:1的比例配置好备用，避光条件下，均匀滴在PVDF膜上，擦去多余化学发光显影液。用凝胶成像分析仪检测条带，拍照保存条带图片。为便于实验显影时分辨条带位置，选用分子量差距更大的β-tubulin作为α-Syn的内参；其他蛋白的内参采用β-actin。用ImageJ软件分析每组条带灰度值，并做统计学分析。
1.4 统计学处理
采用SPSS 27.0软件进行统计分析。计量资料均符合正态分布，以$\bar{x}±s$表示，方差齐时，多组间比较采用单因素方差分析(one-way ANOVA)，进一步两两比较采用LSD法；方差不齐时，多组间比较采用非参数检验，进一步两两比较采用U检验。P<0.05为差异有统计学意义。
2 结果
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2.1 不同浓度PAE对A53T-α-Syn细胞存活率的影响
CCK-8法检测结果显示，与对照组(正常SH-SY5Y细胞)比较，模型组(A53T-α-Syn细胞)细胞存活率明显降低(P<0.01)。与模型组比较，PAE浓度为25、50、75 μg/ml时，A53T-α-Syn细胞存活率差异无统计学意义(P>0.05)；PAE浓度为100、125、150 μg/ml时，A53T-α-Syn细胞存活率明显增高(P<0.05或P<0.01)。其中PAE 150 μg/ml时细胞存活率最高，因此，采用150 μg/ml PAE进行后续实验(图1)。
2.2 各组细胞形态变化
对照组(正常SH-SY5Y细胞)胞体呈梭形、不规则三角形或多边形，突触完整，细胞生长密度均匀。模型组(A53T-α-Syn细胞)部分胞体变圆、皱缩、空泡变性，突触变短或消失，细胞密度不均匀，生长缓慢。PAE组(A53T-α-Syn细胞)皱缩、空泡变性现象较模型组减少，多数细胞胞体呈梭形、不规则三角形或多边形，且突触相对完整，细胞密度增高(图2)。
2.3 PAE对A53T-α-Syn细胞中α-Syn蛋白表达的影响
Western blotting检测结果显示，对照组α-Syn难以检测到；与对照组比较，模型组细胞中α-Syn蛋白表达水平明显增高(P<0.001)；与模型组比较，PAE组细胞中α-Syn表达水平明显降低(P<0.01，图3)。
2.4 PAE对A53T-α-Syn细胞自噬相关蛋白Beclin-1、p62、LC3-Ⅱ表达的影响
Western blotting检测结果显示，与对照组比较，模型组细胞中自噬相关蛋白Beclin-1、LC3-Ⅱ表达水平明显降低(P<0.01，P<0.05)，p62蛋白表达水平明显增高(P<0.05)。与模型组比较，PAE组细胞中Beclin-1、LC3-Ⅱ蛋白表达水平明显增高(P<0.05)，p62蛋白表达水平明显降低(P<0.05，图4)。
2.5 抑制自噬后PAE对A53T-α-Syn细胞中α-Syn蛋白表达的影响
Western blotting检测结果显示，与模型组比较，PAE组细胞中α-Syn蛋白表达水平明显降低(P<0.05)；与PAE组比较，PAE+3-MA组细胞中α-Syn表达水平明显增高(P<0.05，图5)。
2.6 PAE对A53T-α-Syn细胞中p-JNK、Bcl-2蛋白表达的影响
Western blotting检测结果显示，与对照组比较，模型组细胞中自噬上游通路关键蛋白p-JNK蛋白双条带总和、该通路下游蛋白Bcl-2表达水平均明显降低(P<0.05，P<0.01)；与模型组比较，PAE组细胞中p-JNK蛋白双条带总和、Bcl-2表达水平均明显增高(P<0.05，图6)。
3 讨论
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PD的病因目前仍然不甚清楚，业界认为PD是遗传因素和环境因素复杂的、相互作用的结果，这些因素影响着许多基本的细胞过程[11-12]。α-Syn是PD病理学标志物LB的主要成分；α-Syn表达水平较低时其细胞毒性不明显，而表达水平较高时可诱导氧化应激、蛋白酶体和溶酶体功能障碍、多巴胺缺失及细胞死亡，因此，α-Syn被认为是PD的潜在治疗靶标。α-Syn的编码基因SNCA A53T突变最早见于罕见的常染色体显性遗传早发型PD，基因序列分析显示该基因第209位碱基由G变为A(G209A)，导致其编码氨基酸序列第53位的丙氨酸被苏氨酸替换(Ala 53 Thr，A53T)；该突变能够加速α-Syn的聚集，在细胞内形成寡聚体。有研究显示，A53T突变型的α-Syn比野生型更具有细胞毒性[13-15]。本研究在光学显微镜下观察过表达α-Syn诱导建立的PD细胞模型中，发现PAE可促进A53T细胞生长，CCK-8法检测结果显示PAE可提高细胞存活率，Western blotting检测结果显示PAE可降低α-Syn蛋白表达水平；提示PAE可降低过表达α-Syn所致的神经毒性作用，保护DA能神经元。
在心脑血管疾病中，PAE可通过减轻氧化应激，抑制活性氧的过度产生并下调p38 MAPK磷酸化、NF-κB核转位、caspase-3激活和Bcl-2产生途径，减少低密度脂蛋白氧化诱导的细胞凋亡[7]。Shi等[8]报道，PAE对MPTP诱导的PD模型小鼠有治疗效果，可减轻氧化应激和神经炎症引起的损伤，并对多巴胺能神经元产生神经营养作用。此外，有研究显示，PAE在6-OHDA小鼠PD模型中可缓解氧化应激，抑制细胞凋亡和星形胶质细胞增生，增加纹状体酪氨酸羟化酶(tyrosine hydroxylase，TH)的表达，发挥神经保护作用[9]。
自噬-溶酶体途径(autophagy-lysosome pathway，ALP)可降解真核细胞中的细碎片和异常蛋白[16]，按其机制可划分为3类，分别是巨自噬(即通常意义上说到的自噬)、小自噬及分子伴侣介导的自噬[17]。
α-Syn的聚集和清除更多依赖于自噬途径，当α-Syn过表达时会损害巨自噬，其中A53T型突变可诱导α-Syn延缓自噬，且在酵母中对自噬的抑制作用增加[18-20]。自噬始于最初膜结构的形成，即吞噬细胞。当吞噬细胞伸长时，它非选择性地隔离货物，并关闭成一个双膜囊泡称为自噬体。LC3-Ⅱ定位于前自噬体和自噬体膜，促使自噬体膜聚集、延伸并导致吞噬细胞伸长，因此被广泛作为自噬活性评价指标。p62蛋白对蛋白聚集体的清除也很重要，其作为衔接蛋白与LC3-Ⅱ相互作用，以靶向聚集体，进行巨自噬特异性降解[21-22]。在A53T细胞中，LC3-Ⅱ蛋白表达水平降低，p62蛋白表达水平增高，提示自噬活性受到抑制，导致蛋白质和受损细胞器清除障碍，使细胞损伤及凋亡增加。PAE干预后，LC3-Ⅱ蛋白表达水平明显增高，并促进p62及α-Syn降解，提示PAE可有效拮抗A53T突变过表达α-Syn对大自噬通路的抑制作用，靶向α-Syn的降解通路被激活，发挥保护细胞的作用。
3-MA是一种选择性自噬和磷酸肌醇3-激酶(PI3K)抑制剂[23]。本研究采用1 mmol/L 3-MA与PAE共同处理细胞24 h，结果显示，加入抑制剂3-MA后细胞α-Syn表达明显高于PAE组，显示自噬通路受到抑制时PAE清除过表达α-Syn的效果受到影响；提示PAE可参与调控自噬-溶酶体通路，促进α-Syn降解。本研究进一步对PAE增强自噬的具体机制进行了探讨。自噬的上游调控成分较多，JNK信号通路主要通过Bcl-2家族蛋白的磷酸化及其与Beclin-1的解离来调控自噬。p-JNK诱导下游蛋白Bcl-2的磷酸化，Bcl-2磷酸化使Beclin-1从Bcl-2蛋白复合物上解离，Beclin-1结合Vps34蛋白形成Vps34/Beclin-1激酶复合物；Vps34/Beclin-1复合物可促进初始自噬体的形成[24]。本研究结果显示，PAE处理后细胞JNK磷酸化增加，Bcl-2表达增高，下游的Beclin-1表达也增高，再次提示PAE可有效增强细胞自噬功能，其具体机制可能是通过激活JNK/Bcl-2通路增强细胞自噬功能，进而促进α-Syn的清除。
此外，自噬的发生还受AMPK/mTOR等信号通路的调节。AMPK通过磷酸化mTORC1、ULK1和Vps34复合物中的自噬相关蛋白直接促进自噬，或通过调节下游自噬相关基因的表达间接促进自噬。高mTORC1活性通过磷酸化依赖性抑制ULK1和Vps34复合物来抑制自噬，并阻止溶酶体和自噬基因的全局表达，从而调节自噬[25-26]。在PD中，PAE是否参与AMPK/mTOR信号通路的调控和激活细胞自噬，尚待进一步研究。此外，PAE激活自噬通路能否在PD动物模型发挥保护作用，尚未见相关报道，因此，探究PAE在PD动物水平的作用应作为未来的工作重点。错误折叠后的α-Syn在具有α-Syn沉积的神经退行性疾病(如PD、路易体痴呆和多系统萎缩症)中发挥中心病理作用，这些疾病被称为突触核蛋白病[27]。因此，研究PAE靶向α-Syn的自噬清除，不仅可为PD提供潜在的治疗方向，还可能为其他突触核蛋白病的治疗提供参考。
综上所述，PAE可缓解A53T-α-Syn突变诱导的SH-SY5Y细胞损伤，并通过激活自噬途径清除过表达的α-Syn，其机制可能与上调JNK/Bcl-2介导的自噬途径有关。
基金
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  • 国家中医药管理局青年岐黄学者培养项目[国中医药人教函(2022)256号]
  • 山西省重点国别科技合作项目(202204041101002)
  • 山西省青年拔尖人才支持计划项目(晋组办字〔2019〕35号文)
  • 山西省省筹资金资助回国留学人员科研项目(2021-142)
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doi: 10.11855/j.issn.0577-7402.0483.2024.0716
  • 接收时间：2023-04-03
  • 首发时间：2025-11-10
  • 出版时间：2025-01-28
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  • 收稿日期：2023-04-03
  • 录用日期：2023-05-25
基金
National Young Qihuang Scholars Training Program(2022-256)
国家中医药管理局青年岐黄学者培养项目[国中医药人教函(2022)256号]
Key National Science and Technology Cooperation Project of Shanxi Province(202204041101002)
山西省重点国别科技合作项目(202204041101002)
Outstanding Youth Talents Program of Shanxi Province(Jin2019-35)
山西省青年拔尖人才支持计划项目(晋组办字〔2019〕35号文)
Research Project Supported by Shanxi Scholarship Council(2021-142)
山西省省筹资金资助回国留学人员科研项目(2021-142)
作者信息
    1山西中医药大学多发性硬化益气活血重点研究室/神经生物学研究中心，山西晋中 030619
    2山西医科大学基础医学院生物化学与分子生物学教研室，山西太原 030001
    3山西中医药大学第一临床学院，山西太原 030024
    4山西中医药大学第二临床学院，山西太原 030012

通讯作者:

柴智，E-mail：
樊慧杰，E-mail：
参考文献
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https://castjournals.cast.org.cn/joweb/jfjyxzz/CN/10.11855/j.issn.0577-7402.0483.2024.0716
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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