壳寡糖对自由基的清除及对N9小胶质细胞的保护作用

Chitooligosaccharide:Free Radicals Scavenging Activity and Protective Effect against Lipopolysaccharide-induced Oxidative Injury in Microglial Cell Line N9

目的:研究壳寡糖(COS)的抗氧化作用及其对脂多糖(LPS)损伤N9小胶质细胞的保护作用。方法:首先在细胞外检测COS对DPPH自由基、H2O2和羟自由基的清除作用,然后采用LPS诱导建立体外培养N9小胶质细胞活化模型,将细胞分为正常对照组、LPS模型组、LPS+COS给药组(COS质量浓度分别为50、100、200、300、400μg/mL)。观察各组细胞的形态;检测各组细胞培养液中一氧化氮(NO)含量;用流式细胞仪检测细胞内活性氧(ROS)水平并观察其荧光图像;用DNA损伤实验观察各组细胞凋亡情况。结果:COS能有效清除DPPH自由基和羟自由基,对H2O2无清除能力;COS能减少活化的N9细胞数量并明显降低LPS活化的N9细胞培养液中NO水平,降低细胞内ROS水平,减少LPS诱导的细胞凋亡,保护N9小胶质细胞。结论:COS在N9小胶质细胞内外均具有较强的抗氧化能力,能保护N9小胶质细胞,减轻N9小胶质细胞的氧化损伤。

Objective：To study anti-oxidant activity of chitooligosaccharide（COS） and its protective effect against lipopolysaccharide（LPS）-induced oxidative injury in microglial cell line N9.Methods：The antioxidant activity of COS in vitro was evaluated by DPPH and hydroxyl free radical and hydrogen peroxide scavenging assays.Microglial cell line N9 was treated with LPS alone at the dose of 1 μg/mL or co-treated with LPS at the dose of 1 μg/mL and COS at doses of 50,100,200,300 μg/mL and 400 μg/mL,respectively.Cell morphology was examined under microscope with HE staining and NO level in cell culture supernatant was determined.Intracellular generation of reactive oxygen species（ROS） was detected using 2＇,5＇ dichlorofluorescin diacetate（DCFH-DA） and flow cytometry combined with fluorescence microscope.Cell apoptosis was detected by DNA ladder assay.Results：COS exhibited an effective scavenging activity against DPPH and hydroxyl free radicals but no scavenging activity against H2O2.Meanwhile,COS attenuated the activation of microglial cell line N9 induced by LPS.The level of NO in cell culture supernatant was significantly decreased due to the presence of COS,and ROS generation induced by LPS was inhibited by COS in a dose-dependent manner.COS also inhibited the apoptosis of microglial cell line N9 induced by LPS.Conclusion：The antioxidant activity of COS is beneficial for protecting microglial cell line N9 from oxidative injury induced by LPS.