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

doi:10.13481/j.1671-587X.20230602

摘要/Abstract

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（500 μmol·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24 h）、MPP^＋+mimics+FMF组（转染miR-145-3p mimics后0.25 g·L^－1 FMF作用24 h）和MPP^＋+ NC+FMF组（转染miR-145-3p NC后0.25 g·L^－1 FMF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500 μmol·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，自噬小体数量进一步增加。RT-qPCR法检测，与对照组比较，模型组细胞中miR-145-3p表达水平明显降低（P<0.01）；与模型组比较，MPP^＋+mimics组和MPP^＋+FMF组细胞中miR-145-3p表达水平均明显升高（P<0.01）；与MPP^＋+FMF组比较，MPP^＋+mimics+FMF组细胞中miR-145-3p表达水平明显升高（P<0.01）。Western blotting法检测，与对照组比较，模型组细胞中Beclin-1蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ比值均降低（P<0.05）；与模型组比较，MPP^＋+mimics组和MPP^＋+ FMF组细胞中Beclin-1蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ比值均明显升高（P<0.01）；与MPP^＋+FMF组比较，MPP^＋+mimics+FMF组细胞中Beclin-1蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ比值均明显升高（P<0.01）。结论 FMF能够促进MPP⁺诱导的SH-SY5Y细胞自噬作用，减轻线粒体功能障碍，缓解MPP⁺诱导的SH-SY5Y细胞损伤，其作用机制与上调miR-145-3p表达有关。

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

Abstract:

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 500 μmol·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； Annexin Ⅴ-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-Ⅰ in 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 500 μmol·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 group and MPP⁺+FMF group were significantly higher than that in model group（P<0.01）； the proliferation ability of the cells in MPP⁺+mimics+FMF group was significantly higher than that in MPP⁺+FMF group （P<0.01）. The Annexin V-FITC/PI staining results showed that the apoptotic rate of the cells in model group was significantly higher than that in control group （P<0.01）； the apoptotic rates of the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly lower than that in model group （P<0.01）； the apoptotic rate of the cells in MPP⁺+mimics+FMF group was significantly lower than that in MPP⁺+FMF group （P<0.01）. The transmission electron microscope results showed that the cells in control group showed the uniform and intact mitochondrial morphology， the cells in model group showed the enlarged and irregular mitochondria with vacuolar changes，and the cells in MPP⁺+mimics group and MPP⁺+FMF group showed the improved mitochondrial structure， compared with model group， the autophagosome numbers of the cells in MPP⁺+mimics group and MPP⁺+FMF group were increased； the cells in MPP⁺+mimics+FMF group showed more intact mitochondrial structure， compared with MPP⁺+FMF group， the autophagosome numbers in the cells was increased. The RT-qPCR results showed that the expression level of miR-145-3p in the cells in model group was significantly lower than that in control group （P<0.01）； the expression levels of miR-145-3p in the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than that in model group （P<0.01）； the expression level of miR-145-3p in the cells in MPP⁺+mimics+FMF group was significantly higher than that in MPP⁺+FMF group （P<0.01）.The Western blotting results showed that the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/LC3-Ⅰ in the cells in model group were lower than those in control group （P<0.05）； the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than those in model group （P<0.01）； the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in MPP⁺+mimics+FMF group were significantly higher than those in MPP⁺+FMF group （P<0.01）. Conclusion FMF may promote the autophagy， alleviate the mitochondrial dysfunction， and attenuate the injury of the SH-SY5Y cells induced by MPP⁺， and its mechamism is related to upregulating the miR-145-3p expression.

Key words: Fructus mume total flavone, Micro RNA-145-3p, Dopaminergic toxin 1-methyl-4-phenylpyridinium, Parkinson’s disease, Mitochondrial autophagy

中图分类号:

R285.5

文晓东,王春玲,蒋媛静,周欣梅,张艺,伍媛. 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[J]. 吉林大学学报(医学版), 2023, 49(6): 1415-1423.

Xiaodong WEN,Chunling WANG,Yuanjing JIANG,Xinmei ZHOU,Yi ZHANG,Yuan WU. Effect of fructus mume total flavone on injury of SH-SY5Y cells induced by MPP⁺ through regulating miR-145-3p expression and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(6): 1415-1423.

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