褪黑素对过氧化氢诱导人神经母细胞瘤SH-SY5Y细胞氧化应激的改善作用及其机制

Ameliorative effect of melatonin on oxidative stress of human neuroblastoma SHSY5Y cells induced by hydrogen peroxide and its mechanism

目的:研究褪黑素对过氧化氢(H_(2)O_(2))诱导的人神经母细胞瘤细胞(SH-SY5Y细胞)氧化应激损伤的保护作用,并探讨其作用机制。方法:体外培养SH-SY5Y细胞,将SH-SY5Y细胞分为对照组、H_(2)O_(2)组、不同浓度褪黑素组和2-苯基-N-乙酰色胺(Luzindole)组。对照组为正常培养SHSY5Y细胞,H_(2)O_(2)组加入含有200μmol·L^(-1)H_(2)O_(2)的培养基,不同浓度褪黑素组加入不同浓度(1、5和10μmol·L^(-1))褪黑素和含有200μmol·L^(-1)H_(2)O_(2)的培养基,Luzindole组加入50μmol·L^(-1)Luzindole、10μmol·L^(-1)褪黑素和含有200μmol·L^(-1)H_(2)O_(2)的培养基。采用CCK-8法检测各组SHSY5Y细胞存活率,流式细胞术检测各组细胞凋亡率,DCFH-DA荧光探针检测各组细胞中活性氧(ROS)水平,Western blotting法检测各组细胞中微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)蛋白表达水平,荧光显微镜观察各组自噬泡荧光强度。结果:与对照组比较,H_(2)O_(2)组SH-SY5Y细胞存活率降低(P<0.01),ROS水平和细胞凋亡率升高(P<0.01)。与H_(2)O_(2)组比较,不同浓度褪黑素组SH-SY5Y细胞存活率升高(P<0.05),ROS水平和细胞凋亡率降低(P<0.01),LC3-Ⅱ蛋白表达水平明显升高(P<0.01),10μmol·L^(-1)褪黑素组自噬泡荧光强度升高(P<0.05)。与10μmol·L^(-1)褪黑素组比较,1μmol·L^(-1)褪黑素组和Luzindole组SH-SY5Y细胞存活率明显降低(P<0.05);1和5μmol·L^(-1)褪黑素组及Luzindole组SH-SY5Y细胞中ROS水平和细胞凋亡率升高(P<0.05),SH-SY5Y细胞中LC3-Ⅱ表达水平明显降低(P<0.05或P<0.01);1μmol·L^(-1)褪黑素组和Luzindole组自噬泡荧光强度降低(P<0.05)。结论:褪黑素可以抑制H_(2)O_(2)诱导的SH-SY5Y细胞氧化应激损伤,具有神经保护作用,其机制可能与降低ROS水平和提高细胞自噬有关。

Objective:To investigate the protective effect of melatonin on the hydrogen peroxide(H_(2)O_(2))-induced oxidative stress injury of human neuroblastoma SH-SY5Y cells,and to explore its mechanism.Methods:The SH-SY5Y cells were cultured in vitro and divided into control group,H_(2)O_(2)group,melatonin groups,and N-acetyl-2-benzyltryptamine(Luzindole)group.The medium containing 200μmol·L^(-1)H_(2)O_(2)was added in H_(2)O_(2)group;different concentrations(1,5 and 10μmol·L^(-1))of melatonin and medium containing 200μmol·L^(-1)H_(2)O_(2)were added in melatonin groups;50μmol·L^(-1)Luzindole,10μmol·L^(-1)melatonin and the medium containing 200μmol·L^(-1)H_(2)O_(2)were added in Luzindole group.The survival rates of human neuroblastoma SH-SY5Y cells were measured by CCK-8 method,the apoptotic rates of SHSY5Y cells in various groups were detected by flow cytometry,and DCFH-DA fluorescence probe was used to detect the levels of reactive oxygen species(ROS)in the cells in various groups.The expression levels of microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ)protein in the cells in various groups were detected by Western blotting method,and the fluorescence intensities of autophagic vesicles in various groups were observed by fluorescence microscope.Results:Compared with control group,the survival rate of SH-SY5Y cells in H_(2)O_(2)group was decreased(P<0.01),the ROS level and the apoptotic rate were increased(P<0.01).Compared with H_(2)O_(2)group,the survival rates of SH-SY5Y cells in different concentrations of melatonin groups were increased(P<0.05),the ROS levels and the apoptotic rates were significantly decreased(P<0.01),the LC3-Ⅱprotein expression levels were significantly increased(P<0.01),and the fluorescence intensity of autophagic vesicles in 10μmol·L^(-1)melatonin group was increased(P<0.05).Compared with 10μmol·L^(-1)melatonin group,the survival rates of SH-SY5Y cells in1μmol·L^(-1)melatonin group and Luzindole group were significantly decreased(P<0.05);the ROS levels and the apoptotic rates in the SH-SY5Y cells in 1 and 5μmol·L^(-1)melatonin groups and Luzindole group were significantly increased(P<0.05),and the LC3-Ⅱprotein expression levels in the SH-SY5Y cells were decreased(P<0.05 or(P<0.01),and the fluorescence intensities of autophagic vessicles in the SHSY5Y cells in 1μmol·L^(-1)melatonin group and Luzindole group were significantly decreased(P<0.05).Conclusion:Melatonin can inhibit the H_(2)O_(2)-induced oxidative stress injury of SH-SY5Y cells and has a neuroprotective effect,and its mechanism may be related to reducing the ROS levels and enhancing the autophagy of cells.