药学学报, 2019, 54(11): 1976-1981
张雯, 宋俊科, 朱晓瑜, 杨海光, 周启蒙, 许启泰, 杜冠华. 异鼠李素激活Sirt1/PGC-1α信号通路抑制MPP+诱导的SH-SY5Y细胞损伤[J]. 药学学报, 2019, 54(11): 1976-1981.
ZHANG Wen, SONG Jun-ke, ZHU Xiao-yu, YANG Hai-guang, ZHOU Qi-meng, XU Qi-tai, DU Guan-hua. Isorhamnetin activates Sirt1/PGC-1α signaling pathway to inhibit MPP+-induced SH-SY5Y cell injury[J]. Acta Pharmaceutica Sinica, 2019, 54(11): 1976-1981.

张雯1, 宋俊科1, 朱晓瑜2, 杨海光1, 周启蒙1, 许启泰2, 杜冠华1
1. 中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 药物靶点研究与新药筛选北京市重点实验室, 北京 100050;
2. 海南绿槟榔科技发展有限公司, 海南 定安 571200
研究异鼠李素(isorhamnetin,ISO)对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridiniumion,MPP+)损伤SH-SY5Y细胞的保护作用及机制。建立MPP+损伤SH-SY5Y细胞模型,MTT和LDH法检测细胞活力,并测定细胞内超氧化物歧化酶(superoxidedismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的活性,考察细胞氧化应激水平。应用DCFH-DA和MitoSOX荧光探针检测细胞内活性氧自由基(ROS)和线粒体内超氧化物水平,应用JC-1检测线粒体膜电位变化。Western blot和免疫荧光测定细胞中沉默信息调节因子2相关酶1(silent mating type information regulation 2 homolog 1,Sirt1)和过氧化物酶体增殖物激活受体γ辅助激活因子1α(peroxisome proliferator-activatedreceptor γ coactivator-1α,PGC-1α)的蛋白表达,并且采用Western blot对凋亡相关蛋白Bax和Bcl-2的表达进行检测。500 μmol·L-1MPP+损伤导致SH-SY5Y细胞存活率显著降低为52.46%,LDH释放量显著增加至417.63%。5和15 μmol·L-1 ISO通过增加Sirt1和PGC-1α的表达,减轻MPP+诱导的SH-SY5Y细胞损伤,降低LDH释放,显著减少MPP+诱导的细胞总ROS增加,降低线粒体内超氧化物水平,抑制线粒体膜电位下降,其细胞存活率分别增加到61.61%和67.55%。此外,ISO能够降低MPP+损伤所致Bax升高,抑制Bcl-2的降低,抑制MPP+引起的SH-SY5Y细胞凋亡。而ISO所介导的细胞凋亡抑制,能够被Sirt1特异性抑制剂Sirtinol所逆转。ISO通过激活Sirt1/PGC-1α信号途径,减轻氧化应激损伤,抑制细胞凋亡,发挥神经细胞保护作用。
关键词:    异鼠李素      沉默信息调节因子2相关酶1      过氧化物酶体增殖物激活受体γ辅助激活因子1α      神经保护     
Isorhamnetin activates Sirt1/PGC-1α signaling pathway to inhibit MPP+-induced SH-SY5Y cell injury
ZHANG Wen1, SONG Jun-ke1, ZHU Xiao-yu2, YANG Hai-guang1, ZHOU Qi-meng1, XU Qi-tai2, DU Guan-hua1
1. State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of Drug Target and Screening Research, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
2. Hainan Green Areca Technology Development Co. LTD., Ding'an 571200, China
We studied the protective effect and mechanism of isorhamnetin (ISO) on 1-methyl-4-phenylpyridiniumion (MPP+)-induced SH-SY5Y cells injury. MPP+-induced SH-SY5Y cell injury model was established, and cell viability was measured by MTT and LDH methods. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in cells were determined to investigate the level of oxidative stress. DCFH-DA and MitoSOX fluorescence probes were used to detect the levels of intracellular reactive oxygen species (ROS) and mitochondria superoxide, respectively. JC-1 fluorescence probe was used to detect the changes of mitochondrial membrane potential. Western blot and immunofluorescence methods were used to determine the expressions of Sirt1 and PGC-1 proteins, as well as the expression levels of apoptosis-related proteins Bax and Bcl-2. MPP+ at the dose of 500 μmol·L-1 significantly reduced SH-SY5Y cells viability to 52.46% and increased LDH release to 417.63%. ISO at 5 and 15 μmol·L-1 significantly increased the expression of Sirt1 and PGC-1α, inhibited LDH release, reduced intracellular ROS and mitochondria superoxide, inhibited the decline of mitochondrial membrane potential and increased cell viability to 61.61% and 67.55%. In addition, ISO could downregulate the expression of Bax and upregulate the expression of Bcl-2 to reduce cell apoptosis. ISO-mediated inhibition of apoptosis could be reversed by Sirt1 specific inhibitor Sirtinol. Through activating Sirt1/PGC-1α signaling pathway, ISO could reduce oxidative stress injury and inhibit cell apoptosis to protect cells from MPP+ injury.
Key words:    isorhamnetin    silent mating type information regulation 2 homolog 1    peroxisome proliferator-activated receptor γ coactivator-1α    neuroprotection   
收稿日期: 2019-03-25
DOI: 10.16438/j.0513-4870.2019-0205
基金项目: 中国医学科学院医学与健康科技创新工程(2017-I2M-1-010);海南省重大科技计划资助项目(ZDKJ2016003).
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