【摘要】 目的：研究褪黑素对过氧化氢（H₂O₂）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、H₂O₂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，H₂O₂组加入含有200μmol·L^-1H₂O₂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^-1H₂O₂的培养基，Luzindole组加入50μmol·L^-1Luzindole、 10μmol·L^-1褪黑素和含有200μmol·L^-1H₂O₂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，H₂O₂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H₂O₂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H₂O₂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。更多还原

【Abstract】 Objective:To investigate the protective effect of melatonin on the hydrogen peroxide（H₂O₂）-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,H₂O₂group,melatonin groups,and N-acetyl-2-benzyltryptamine（Luzindole）group. The medium containing 200 μmol·L^-1H₂O₂was added in H₂O₂group;different concentrations(1,5 and 10 μmol·L^-1) of melatonin and medium containing 200 μmol·L^-1H₂O₂were added in melatonin groups;50 μmol·L^-1Luzindole,10 μmol·L^-1melatonin and the medium containing 200 μmol·L^-1H₂O₂were 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 H₂O₂group was decreased（P<0. 01）,the ROS level and the apoptotic rate were increased（P<0. 01）. Compared with H₂O₂group,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-Ⅱ protein expression levels were significantly increased（P<0. 01）,and the fluorescence intensity of autophagic vesicles in 10 μmol·L^-1melatonin group was increased（P<0. 05）. Compared with 10 μmol·L^-1melatonin group, the survival rates of SH-SY5Y cells in1 μmol·L^-1melatonin 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 5 μmol·L^-1melatonin groups and Luzindole group were significantly increased（P<0. 05）,and the LC3-Ⅱ protein 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 1 μmol·L^-1melatonin group and Luzindole group were significantly decreased（P<0. 05）.Conclusion:Melatonin can inhibit the H₂O₂-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.更多还原