杨桃根提取物DMDD对α-synuclein PFFs所致分化SH-SY5Y细胞损伤的保护作用及机制研究  

Study on the protective effects and mechanisms of DMDD isolated from Averrhoa carambola L.root on differentiated SH-SY5Y cell damage induced byα-Synuclein PFFs

作　　者：粟璟曦 宋琼 苏迎 周宇光 黄仁彬[3] 王丽惠 邹春林 SU Jingxi;SONG Qiong;SU Ying;ZHOU Yuguang;HUANG Renbin;WANG Lihui;ZOU Chunlin(Center for Translational Medicine,Key Laboratory of Longevity and Aging-related Diseases,Ministry of Edu-cation,Institute of Neuroscience and Guangxi Key Laboratory of Brain Science,School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China;Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry,Guangxi Key Laboratory of Regenerative Medicine,Guangxi Medical University,Nanning 530021,China;Pharmaceutical College,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学基础医学院转化医学研究中心长寿与老年相关疾病教育部重点实验室神经科学研究所广西脑科学研究重点实验室,南宁530021 [2]再生医学与医用生物资源开发应用省部共建协同创新中心广西再生医学重点实验室,南宁530021 [3]广西医科大学药学院,南宁530021

出　　处：《广西医科大学学报》2024年第10期1433-1441,共9页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.81971191);山东省自然科学基金资助项目(No.ZR2019ZD39)。

摘　　要：目的:探讨不同浓度杨桃根提取物DMDD对α-突触核蛋白预制原纤维(α-Syn PFFs)所致分化SH-SY5Y细胞损伤的保护作用及作用机制。方法:使用不同浓度DMDD作用于视黄酸分化的SH-SY5Y细胞,通过MTS测定细胞活力,确定DMDD作用于分化SH-SY5Y细胞的低、中、高浓度;使用不同浓度的DMDD处理5μg/mLα-Syn PFFs诱导的细胞损伤模型,采用Hoechst 33342染色和一氧化氮试剂盒分别检测细胞核损伤细胞数与细胞内一氧化氮产生量;采用蛋白质印迹法检测细胞内酪氨酸羟化酶(TH)、多聚腺苷酸二磷酸(PAR)及多聚腺苷酸二磷酸核糖酶-1(PARP-1)蛋白表达水平;采用免疫荧光技术检测细胞内129位丝氨酸磷酸化α-突触核蛋白(pS129-α-Syn)、磷酸化组蛋白H2AX(γH2AX)阳性细胞数及凋亡诱导因子(AIF)核易位细胞数。结果:Hoechst 33342染色结果显示,与α-Syn PFFs模型组比较,各浓度DMDD处理组出现细胞核损伤的细胞数明显减少(P<0.001);一氧化氮检测结果显示,与α-Syn PFFs模型组比较,各浓度DMDD处理组细胞一氧化氮生成量显著降低(P<0.01或P<0.001);蛋白质印迹法结果显示,与α-Syn PFFs模型组比较,高浓度DMDD处理组细胞TH蛋白表达水平明显升高(P<0.05),而中、高浓度DMDD处理组细胞PAR蛋白表达水平明显降低(P<0.05),高浓度DMDD处理组细胞PARP-1、Cleaved PARP-1蛋白表达水平明显降低(P<0.05);免疫荧光结果显示,与α-Syn PFFs模型组比较,中、高浓度DMDD处理组pS129-α-Syn阳性细胞数明显减少(P<0.05或P<0.01),各浓度DMDD处理组γH2AX阳性细胞数明显减少(P<0.01或P<0.001),AIF核易位细胞数也明显减少(P<0.01或P<0.001)。结论:DMDD可能通过抑制PARP-1/AIF通路介导的PARP-1依赖性死亡途径减轻α-Syn PFFs所致分化SH-SY5Y细胞损伤。Objective:To explore the protective effects and mechanisms of different concentrations of Averrhoa carambola L.root extract 2-Dodecyl-6-Methoxycyclohexa-2,5-Diene-1,4-Dione(DMDD)onα-Syn PFFsinduced differentiated SH-SY5Y cell damage.Methods:The SH-SY5Y cells differentiated by retinoic acid were treated with different concentrations of DMDD,followed by detecting the cell viability by MTS to determine the low,medium,and high concentrations of DMDD for use in these cells.The cells were treated with various concentrations of DMDD in an established model of cell damage induced by 5μg/mLα-Syn PFF.Hoechst 33342 staining and nitric oxide kit were used to detect the number of cells with nuclear damage and intracellular nitric oxide production,respectively.The protein expression levels of tyrosine hydroxylase(TH),polyadenylate diphosphate(PAR)and polyadenylate diphosphate ribozyme-1(PARP-1)were measured by western blotting.Immunofluorescence was used to detect the number of cells positive for phosphorylated serine 129α-synuclein(pS129-α-Syn),phosphorylated histone H2AX(γH2AX),and nuclear translocation of apoptosis-inducing factor(AIF).Results:Hoechst 33342 staining demonstrated that the number of cells with nuclear damage was significantly decreased in all DMDD treatment groups compared with theα-Syn PFFs model group(P<0.001).The nitric oxide assay results showed that nitric oxide production was significantly reduced in the DMDD treatment groups compared with theα-Syn PFFs model group(P<0.01 or P<0.001).Western blotting revealed that compared with theα-Syn PFFs model group,TH protein expression level was significantly increased in the high DMDD concentration treatment group(P<0.05),while PAR protein expression levels were significantly decreased in the medium and high DMDD concentration treatment groups(P<0.05),and the protein expression levels of PARP-1 and Cleaved PARP-1 were significantly decreased in the high DMDD concentration treatment group(P<0.05).Immunofluorescence results showed that compared with theα-

关 键 词：杨桃根 DMDD α-突触核蛋白预制原纤维 PARP-1依赖性细胞死亡 SH-SY5Y 

分 类 号：R285[医药卫生—中药学]