黄芪多糖抑制小鼠EAE 及调控BV-2 神经小胶质细胞活化的实验研究

目的:研究黄芪多糖(APS)对实验性自身免疫性脑脊髓膜炎(EAE)的治疗作用以及对参与EAE 发病机制的神经小胶质细胞活化的调控作用及其可能的作用机制。方法:动物实验:MOG35-55 诱导C57BL/6 小鼠建立EAE 模型,予APS 给药干预,通过5 级临床症状评分观察APS 对小鼠EAE 的治疗作用。细胞实验:MTT 法检测脂多糖(LPS)对于BV-2 神经小胶质细胞的增殖抑制作用,筛选合适的LPS 刺激浓度活化神经小胶质细胞,构建BV-2 神经小胶质细胞活化的模型;倒置显微镜观察BV-2 神经小胶质细胞形态学的改变;ELISA 法检测BV-2 神经小胶质细胞IFN-酌、TNF-α的分泌水平变化;观察不同浓度的APS 对BV-2 神经小胶质细胞活化的调控作用;APS 干预后,Western blot、Real-time PCR 方法分别检测BV-2 神经小胶质细胞PD-L1 蛋白和mRNA 表达水平的变化。结果:APS 能够有效治疗小鼠EAE 的临床症状,成功建立了体外BV-2 神经小胶质细胞活化模型,一定浓度的APS 能够抑制BV-2 神经小胶质细胞的活化,提高活化的BV-2 细胞的生存活性,降低IFN-酌、TNF-α的分泌水平,促进活化的BV-2 神经小胶质细胞PD-L1 基因及蛋白表达上调。结论:APS 对小鼠EAE 具有明显的治疗作用,其发挥作用的机制可能是APS 能够有效抑制神经小胶质细胞的活化,降低炎性细胞因子IFN-酌、TNF-α的分泌,对神经小胶质细胞有抗炎保护作用,PD-1/ PD-L1 通路可能是APS 发挥抗炎作用的重要途径。

Experimental study on effects of astragalus polysaccharide on treating EAE in mice and regulating activation of BV-2 microglial cell line

Objective:To investigate the therapeutic effects of astragalus polysaccharide (APS) on experimental autoimmune encephalomyelitis (EAE) and to explore the regulating effects on microglia activation that is associated with the pathogenesis of EAE and its possible mechanisms.Methods:Animal experiments:EAE model was induced by MOG35-55 in C57BL/6 mice.APS was given by gavage.EAE was scored according to a 0-5 scale to observe the therapeutic effects of APS.Cell experiments:The effects of lipopolysac-charide (LPS) on cell viability of BV-2 microglial cell line were investigated by MTT assay and then the appropriate concentration of LPS to activate the BV-2 microglial cell line was selected.The microglia activation model was established.The changes in BV-2 microglial cell line morphology were observed with an inverted microscope.The cytokines of TNF-αand IFN-γin the cell culture supernatant of BV-2 microglial cell line were detected by ELISA.The activated BV-2 microglial cells were treated with APS in different concentrations.The regulatory roles of the APS on the BV-2 microglial cell activation were observed.Western blot and Real-time PCR method were used to measure the protein and mRNA level of the PD-L1 on the cell surface of BV-2 microglial cells treated with APS.Results: APS could effectively ameliorate the symptoms in EAE mice and could suppress neuroinflammation of EAE significantly.The microglia activation model in vitro induced by LPS was successful.APS in certain concentration could inhibit the activation of microglia,increase the viabilily of the active microglia.Meanwhile,it could downregulated the level of the cytokines including IFN-γand TNF-αand upregulated the expression of protein and mRNA of PD-L1 on activated microglia.Conclusion:APS can effectively inhibit the autoimmune reaction of EAE and effectively suppress the microglia activation induced by LPS,reduce the pro- duction of IFN-γand TNF-α.APS plays a crucial role in reducing the inflammation induced by microglia activation.The potential mechanisms might be related to the upregualtion of the PD-1/ PD=L1 pathway.