miR-138靶向OX40L对实验性自身免疫性脑脊髓炎幼鼠的作用机制研究

Mechanism of miR-138 targeting OX40L on experimental autoimmune encephalomyelitis immature mice

目的:探究miR-138/OX40L分子轴对实验性自身免疫性脑脊髓炎(EAE)鼠的影响。方法:建立EAE小鼠模型并设为EAE组,未作处理的小鼠为对照组;流式细胞术分选小胶质细胞并检测T细胞凋亡情况;免疫荧光染色检测活化小胶质细胞内TLR3、TLR4和FIZZ-1表达;RT-qPCR检测miR-138和OX40L mRNA表达水平;Western blot检测OX40L蛋白水平;MTT法检测T细胞增殖能力;双荧光素酶报告基因实验用于验证miR-138和OX40L的靶向关系。结果:与对照组相比,EAE组小鼠出现四肢瘫痪、大小便失禁、体重减轻等症状,同时神经功能评分降低。EAE组小鼠活化小胶质细胞比例显著高于对照组小鼠,且TLR3、TLR4和FIZZ-1表达水平增加。miR-138在EAE小鼠脑组织和分离的小胶质细胞中表达下调(P<0.05),在小胶质细胞中过表达miR-138导致与其共培养的T细胞增殖能力下降(P<0.05),凋亡率增加(P<0.01)。miR-138能靶向结合OX40L mRNA从而调节OX40L蛋白表达。在小胶质细胞中过表达OX40L能促进T细胞的活性(P<0.05)并降低其凋亡(P<0.001);但同时过表达miR-138和OX40L部分消除了OX40L对T细胞活性的促进作用。结论:EAE小鼠的小胶质细胞中miR-138通过调节OX40L的表达影响T细胞活性,可能对EAE的治疗产生一定的效果。

Objective:To explore the effect of miR-138/OX40L molecular axis on experimental autoimmune encephalomyelitis(EAE)mice.Methods:EAE mice model were established and set as EAE group,and untreated mice as control group.Flow cytometry was used to sorted microglia and detected T cell apoptosis.Immunofluorescence staining to detect expression of TLR3,TLR4,and FIZZ-1 in activated microglia.RT-qPCR was used to detect the expression levels of miR-138 and OX40L mRNA.Western blot was used to detect the protein level of OX40L.MTT method was used to detect T cell proliferation ability.Dual-luciferase reporter gene experiment was used to verify the targeting relationship between miR-138 and OX40L.Results:Compared with control group,mice in EAE group showed symptoms such as quadriplegia,incontinence and weight loss,while neurological function scores were reduced.Proportion of activated microglia in the EAE group was significantly higher than that in control group,and expression levels of TLR3,TLR4,and FIZZ-1 were increased.miR-138 was down-regulated in EAE mice brain tissue and isolated microglia(P<0.05),and overexpression of miR-138 in microglia led to a decrease in proliferation of T cells co-cultured with it(P<0.05),and the apoptosis rate was increased(P<0.01).miR-138 can target and bind to OX40L mRNA to regulate expression of OX40L protein.Overexpression of OX40L in microglia can promote activity(P<0.05)and reduce apoptosis of T cell(P<0.001),while overexpression of miR-138 and OX40L together partly eliminates the promotion effect of OX40L on T cell activity.Conclusion:miR-138 in the microglia of EAE mice affects T cell activity by regulating expression of OX40L,which may have a certain effect on the treatment of EAE.