黄芪甲苷通过PPARγ抑制Th1、Th17分化改善自身免疫性脑脊髓炎

Astragaloside Ⅳ inhibits Th1/Th17 differentiation through PPARγ to ameliorate autoimmune encephalomyelitis

目的探讨黄芪甲苷实验性自身免疫性脑脊髓炎(EAE)治疗作用及其机制。方法采用髓磷脂少突胶质细胞糖蛋白35-55(MOG35-55)免疫C57BL/6小鼠构建EAE模型,随机分为对照(PBS)组与黄芪甲苷(AS-IV)组(100 mg/kg)。免疫前3天连续腹腔注射黄芪甲苷,免疫后隔天注射,持续观察小鼠发病情况。取脊髓切片HE染色观察炎性细胞浸润,LFB染色评估脊髓髓鞘脱失,流式细胞数检测CD4^(+)T细胞向Th1与Th17细胞分化比例。提取正常小鼠脾脏中CD4^(+)T细胞,在细胞因子作用下诱导分化,加入不同剂量黄芪甲苷与PPARγ抑制剂T0070907探究其对CD4^(+)T细胞分化的影响。采用Western blot检测PPARγ、NF-κB与NLRP3表达。结果与对照组相比,AS-IV组EAE小鼠发病率降低,发病时间延迟,且骨髓炎性细胞浸润减少,髓鞘脱失抑制,脾脏与脊髓中Th1与Th17细胞表型的含量减少。体外实验中,黄芪甲苷剂量依赖性减少了CD4^(+)T细胞向Th1与Th17细胞表型分化,然而,T0070907逆转了黄芪甲苷的抑制作用。与此同时,无论是在体内还是体外实验中,黄芪甲苷有效增加了PPARγ表达,减少了p-NF-κB与NLRP3表达水平。结论黄芪甲苷通过激活PPARγ,介导NF-κB信号通路抑制,减少CD4^(+)T细胞向Th1与Th17细胞分化,减轻小鼠EAE症状。

Astragaloside Ⅳ has been widely used in treating immune system diseases,but there are few studies on the effect of astragaloside Ⅳ on experimental autoimmune encephalomyelitis(EAE).In this study,we aim to explore the therapeutic efficacy and mechanism of astragaloside Ⅳ in EAE.C57 BL/6 mice were immunized with myelin oligodendrocyte glycoprotein_(35-55)(MOG_(35-55)) for EAE model,and randomly divided into control(PBS) group and astragaloside Ⅳ(AS-Ⅳ) group(100 mg/kg).Astragaloside Ⅳ was continuously administrated for 3 days by intraperitoneal injection before MOG_(35-55) immunization and administrated every other day after the immunization.Then mice were continuously observed for the onset of symptoms.HE staining of spinal cord sections was performed to observe inflammatory cell infiltration and LFB staining was used to evaluate spinal cord demyelination;flow cytometry was used to detect the ratio of Th1 to Th17 cells.The CD4^(+) T cells from the spleen of normal mice were extracted and induced to differentiate in the presence of cytokines.Different doses of astragaloside Ⅳ and PPARγ inhibitor T0070907 were added to explore their effect on the differentiation of CD4^(+) T cells.Western blot was used to detect the expression of PPARγ,NF-κB and NLRP3.Data showed that as compared with PBS group,the incidence of EAE in the AS-Ⅳ group was reduced,the EAE onset was delayed,the infiltration of inflammatory cells was reduced,the demyelination was inhibited,and the phenotype of Thl and Th17 cells in the spleen and spinal cord were reduced.In vitro,astragaloside Ⅳ dose-dependently reduced the phenotypic differentiation of CD4^(+) T cells into Thl and Th17 cells.However,T0070907 reversed the inhibitory effect of astragaloside Ⅳ.At the same time,both in vivo and in vitro,astragaloside Ⅳ effectively increased the expression of PPARy and reduced the expression levels of p-NF-κB and NLRP3.Taken together,astragaloside Ⅳ activates PPARγ,mediates the inhibition of NF-κB signaling pathway,reduces the differentiation of CD4^(+) T cells into Thl and Th17 cells,and reduces EAE symptoms in mice.