乌梅总黄酮调控miR-145-3p表达对MPP+诱导SH-SY5Y细胞损伤的作用及其机制  

Effect of fructus mume total flavone on injury of SH-SY5Y cells induced by MPP+through regulating miR-145-3p expression and its mechanism

作　　者：文晓东[1] 王春玲 蒋媛静[1] 周欣梅[1] 张艺 伍媛 WEN Xiaodong;WANG Chunling;JIANG Yuanjing;ZHOU Xinmei;ZHANG Yi;WU Yuan(Department of Encephalopathy,Ruikang Hospital,Guangxi University of Traditional Chinese Medicine,Nanning 530011,China;Department of Pharmacology,School of Pharmacy,Guangxi University of Traditional Chinese Medicine,Nanning 530200,China)

机构地区：[1]广西中医药大学附属瑞康医院脑病一区,广西南宁530011 [2]广西中医药大学药学院药理教研室,广西南宁530200

出　　处：《吉林大学学报（医学版）》2023年第6期1415-1423,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金地区科学基金项目(82060888);广西壮族自治区中医药管理局科研项目(GZZC2020107);广西中医药大学校级课题面上项目(2020MS049)。

摘　　要：目的:探讨乌梅总黄酮(FMF)调控微小RNA (miR)-145-3p对多巴胺能毒素1-甲基-4-苯基吡啶(MPP+)诱导的SH-SY5Y细胞损伤的保护作用,并阐明其作用机制。方法:构建MPP+诱导帕金森病(PD) SH-SY5Y细胞模型,CCK-8法筛选MPP+和FMF最佳干预浓度和作用时间。将细胞分为对照组(未经处理)、模型组(500μmol·L^(-1)MPP+作用24 h)、MPP++mimics组(转染miR-145-3p mimics)、MPP++NC组(转染miR-145-3p NC)、MPP++FMF组(0.25 g·L^(-1)FMF作用24h)、 MPP++mimics+FMF组(转染miR-145-3pmimics后0.25g·L^(-1)FMF作用24h)和MPP++NC+FMF组(转染miR-145-3p NC后0.25 g·L-1FMF作用24 h)。CCK-8法检测各组细胞增殖能力,AnnexinⅤ-FITC/PI染色法检测各组细胞凋亡率,透射电镜观察各组细胞线粒体自噬超微结构表现,实时荧光定量PCR (RT-qPCR)法检测各组细胞中miR-145-3p表达水平,Western blotting法检测各组细胞中Beclin-1蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ比值。结果:不同浓度MPP+作用SH-SY5Y细胞后,细胞增殖能力明显降低(P<0.01),PD细胞模型构建成功。MPP+最佳作用浓度和时间为500μmol·L^(-1)和24 h,FMF最佳干预浓度和作用时间为0.25 g·L^(-1)和24 h。CCK-8法检测,与对照组比较,模型组细胞增殖能力明显降低(P<0.01);与模型组比较,MPP++mimics组和MPP++FMF组细胞增殖能力均明显升高(P<0.01);与MPP++FMF组比较,MPP++mimics+FMF组细胞增殖能力明显升高(P<0.01)。AnnexinⅤ-FITC/PI染色法检测,与对照组比较,模型组细胞凋亡率明显升高(P<0.01);与模型组比较,MPP++mimics组和MPP++FMF组细胞凋亡率均明显降低(P<0.01);与MPP++FMF组比较,MPP++mimics+FMF组细胞凋亡率明显降低(P<0.01)。透射电镜观察,对照组细胞线粒体形态均匀,结构完整;模型组细胞线粒体体积变大,结构不规则,出现空化现象;与模型组比较,MPP++mimics组和MPP++FMF组细胞线粒体结构改善,自噬小体数量增加;与MPP++FMF组比较,MPP++mimics+FMF组细胞线粒体结构较为完整,自噬小体�Objective:To discuss the protective effect of fructus mume total flavonoids(FMF)on the injury of the SH-SY5Y cells induced by 1-methyl-4-phenylpyridinium(MPP+)through regulating microRNA(miR)-145-3p expression,and to clarify the mechanism.Methods:The SH-SY5Y cell model of Parkinson’s disease(PD)was established by MPP+induction.The optimal intervention concentration and action time of MPP+and FMF were screened out by CCK-8 assay.The cells were divided into control group(untreated),model group(treated with 500μmol·L^(-1)MPP+for 24 h),MPP++mimics group(transfected with miR-145-3p mimics),MPP++NC group(transfected with miR-145-3p NC),MPP++FMF group treated with 0.25 g·L^(-1)FMF for 24 h),MPP++mimics+FMF group(transfected with miR-145-3p mimics and treated with 0.25 g·L^(-1)FMF for 24 h),and MPP++NC+FMF group(transfected with miR-145-3p NC,and(treated with 0.25 g·L^(-1)FMF for 24 h).CCK-8 assay was used to detect the proliferation abilities of the cells in various groups;AnnexinⅤ-FITC/PI staining was used to the detect the apoptotic rates of the cells in various groups;transmission electron microscope was used to observe the ultrastructure morphology of mitochondrial autophagy in the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-145-3p in the cells in various groups;Western blotting method was used to detect the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/LC3-Ⅰin the cells in various groups.Results:After treated with different concentrations of MPP+,the proliferation ability of the cells was significantly decreased(P<0.01),and the PD cell model was successfully constructed.The optimal concentrations and action time of MPP+and FMF intervention were 500μmol·L^(-1),24 h,and 0.25 g·L^(-1),24 h,respectively.The CCK-8 assay results showed that the proliferation ability of the cells in model group was significantly lower than that in control group(P<0.01);the proliferation abilities of the cells in MPP++mimics gro

关 键 词：乌梅总黄酮 微小RNA-145-3p 多巴胺能毒素1-甲基-4-苯基吡啶 帕金森病 线粒体自噬 

分 类 号：R285.5[医药卫生—中药学] R742[医药卫生—中医学]