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Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated SH-SY5Y cells
Journal of Biological Research-Thessaloniki ( IF 3.3 ) Pub Date : 2021-02-25 , DOI: 10.1186/s40709-021-00137-6
Junqiang Yan , Hongxia Ma , Xiaoyi Lai , Jiannan Wu , Anran Liu , Jiarui Huang , Wenjie Sun , Mengmeng Shen , Yude Zhang

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The oxidative stress is an important component of the pathogenesis of PD. Artemisinin (ART) has antioxidant and neuroprotective effects. The purpose of this study is to explore the neuroprotective effect of ART on 1-methyl-4-phenyliodine iodide (MPP +)-treated SH-SY5Y cells and underlying mechanism. We used MPP+-treated SH-SY5Y cells to study the neuroprotective effect of ART. Cell viability was measured by MTT assay after incubating the cells with MPP+ and/or ART for 24 h. DCFH-DA was used to detect the level of intracellular reactive oxygen species (ROS), and WST-8 was used to detect the level of superoxide dismutase (SOD). The level of intracellular reduced glutathione (GSH) was detected with 5,5΄-dithiobis-(2-nitrobenzoic acid), and the level of malondialdehyde (MDA) was assessed based on the reaction of MDA and thiobarbituric acid. A mitochondrial membrane potential detection kit (JC-1) was used to detect changes in the mitochondrial membrane potential (MMP), and an Annexin V-FITC cell apoptosis kit was used to detect cell apoptosis. The expression levels of caspase-3, cleaved caspase-3 and the autophagy-related proteins LC3, beclin-1, and p62 were detected by Western blotting. In addition, to verify the change in autophagy, we used immunofluorescence to detect the expression of LC3 and p62. No significant cytotoxicity was observed at ART concentrations up to 40 μM. ART could significantly increase the viability of SH-SY5Y cells treated with MPP+ and reduce oxidative stress damage and apoptosis. In addition, the Western blotting and immunofluorescence results showed that MPP+ treatment could increase the protein expression of beclin1 and LC3II/LC3I and decrease the protein expression of p62, indicating that MPP+ treatment could induce autophagy. Simultaneous treatment with ART and MPP+ could decrease the protein expression of beclin1 and LC3II/LC3I and increase the protein expression of p62, indicating that ART could decrease the level of autophagy induced by MPP+. Our results indicate that ART has a protective effect on MPP+-treated SH-SY5Y cells by the antioxidant, antiapoptotic activities and inhibition of autophagy. Our findings may provide new hope for the prevention and treatment of PD.

中文翻译:

青蒿素通过抑制MPP +处理的SH-SY5Y细胞的自噬作用来减轻氧化应激和细胞凋亡

帕金森氏病(PD)是仅次于阿尔茨海默氏病的第二大最常见的神经退行性疾病。氧化应激是PD发病机理的重要组成部分。青蒿素(ART)具有抗氧化和神经保护作用。本研究的目的是探讨ART对1-甲基-4-苯基碘碘化物(MPP +)处理的SH-SY5Y细胞的神经保护作用及其潜在机制。我们使用MPP +处理的SH-SY5Y细胞来研究ART的神经保护作用。将细胞与MPP +和/或ART孵育24小时后,通过MTT分析测量细胞活力。DCFH-DA用于检测细胞内活性氧(ROS)的水平,WST-8用于检测超氧化物歧化酶(SOD)的水平。用5,5′-二硫代双-(2-硝基苯甲酸)检测细胞内还原型谷胱甘肽(GSH)的水平,丙二醛(MDA)的含量根据MDA与硫代巴比妥酸的反应进行评估。线粒体膜电位检测试剂盒(JC-1)用于检测线粒体膜电位(MMP)的变化,膜联蛋白V-FITC细胞凋亡试剂盒用于检测细胞凋亡。通过Western印迹检测caspase-3,裂解的caspase-3和自噬相关蛋白LC3,beclin-1和p62的表达水平。此外,为了验证自噬的变化,我们使用了免疫荧光法检测了LC3和p62的表达。在最高40μM的ART浓度下未观察到明显的细胞毒性。ART可以显着提高MPP +处理的SH-SY5Y细胞的活力,并减少氧化应激损伤和细胞凋亡。此外,Western blotting和免疫荧光结果表明,MPP +处理可增加beclin1和LC3II / LC3I的蛋白表达,降低p62的蛋白表达,表明MPP +处理可诱导自噬。同时使用ART和MPP +处理可以降低beclin1和LC3II / LC3I的蛋白表达并增加p62的蛋白表达,表明ART可以降低MPP +诱导的自噬水平。我们的结果表明,ART通过抗氧化剂,抗凋亡活性和自噬抑制作用对MPP +处理的SH-SY5Y细胞具有保护作用。我们的发现可能为PD的预防和治疗提供新的希望。表明MPP +处理可诱导自噬。同时使用ART和MPP +处理可以降低beclin1和LC3II / LC3I的蛋白表达并增加p62的蛋白表达,表明ART可以降低MPP +诱导的自噬水平。我们的结果表明,ART通过抗氧化剂,抗凋亡活性和自噬抑制作用对MPP +处理的SH-SY5Y细胞具有保护作用。我们的发现可能为PD的预防和治疗提供新的希望。表明MPP +处理可诱导自噬。同时使用ART和MPP +处理可以降低beclin1和LC3II / LC3I的蛋白表达并增加p62的蛋白表达,表明ART可以降低MPP +诱导的自噬水平。我们的结果表明,ART通过抗氧化剂,抗凋亡活性和自噬抑制作用对MPP +处理的SH-SY5Y细胞具有保护作用。我们的发现可能为PD的预防和治疗提供新的希望。抗凋亡活性和自噬抑制。我们的发现可能为PD的预防和治疗提供新的希望。抗凋亡活性和自噬抑制。我们的发现可能为PD的预防和治疗提供新的希望。
更新日期:2021-02-26
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