黄芪提取物抑制小鼠实验性自身免疫性脑脊髓炎的作用及机制

Inhibitory effect and mechanism of Radix Astragali extract on experimental autoimmune encephalomyelitis in mice

目的探讨黄芪提取物(RAE)对小鼠实验性自身免疫性脑脊髓炎(EAE)的作用及机制。方法采用髓鞘少突胶质糖蛋白(MOG35-55)诱导雌性C57BL/6小鼠建立EAE小鼠模型。将小鼠随机分为正常对照组、模型组及模型+RAE 125,250和500 mg·kg^-1组。模型+RAE组于造模前2 d ig给予RAE,每天1次,共23 d。给药的同时采用双盲法记录神经功能评分及体质量变化;HE和劳克坚劳蓝(LFB)髓鞘染色检测脊髓组织炎症浸润和脱髓鞘情况;流式细胞分析技术检测脾CD4,CD11b和CD11c阳性细胞百分比;Western印迹法检测脊髓组织NF-κB、磷酸化NF-κB(p-NF-κB)、NF-κB抑制蛋白(IκBα)、磷酸化NF-κB抑制蛋白(p-IκBα)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、Bax、Bcl2、活化胱天蛋白酶3及钙离子结合衔接分子1(Iba1)等蛋白表达。结果与正常对照组相比,模型组神经功能评分增高(P<0.01),体质量显著降低(P<0.05,P<0.01);在给药第3周,模型组相比,模型+RAE 125和500 mg·kg^-1组EAE小鼠的发病症状明显改善,体质量降低减轻(P<0.01)。与正常对照组相比,模型组小鼠脊髓组织炎症浸润、脱髓鞘现象明显(P<0.01),模型+RAE组脊髓组织炎症浸润及脱髓鞘明显改善(P<0.01);模型组脾CD4,CD11b和CD11c阳性细胞百分比显著增高(P<0.01),模型+RAE组其增高得到逆转(P<0.01)。与正常对照组相比,模型组脊髓NF-κB通路蛋白磷酸化及Iba1,Bax和活化胱天蛋白酶3等蛋白表达均增加,PI3K/Akt通路蛋白磷酸化及Bcl2/Bax蛋白表达比值降低(P<0.01);模型+RAE 125 mg·kg^-1组上述蛋白表达的变化明显被逆转(P<0.05,P<0.01)。结论RAE能有效缓解EAE小鼠的发病症状,减少中枢神经系统的炎症浸润和脱髓鞘,其作用机制可能与降低周边炎症细胞激活、抑制中枢神经系统神经炎症以及减少神经细胞的凋亡相关。

OBJECTIVE To investigate the effect and mechanism of Radix Astragali extract(RAE)on experimental autoimmune encephalomyelitis(EAE)in mice.METHODS EAE in female C57BL/6 mice was induced by myelin oligodendrocyte glycoprotein 35-55(MOG35-55)to establish an EAE mouse model.Mice were randomly divided into five groups:normal control,EAE model,model+RAE 125,250 and 500 mg·kg^-1 groups.RAE groups were given respective dosages of RAE two days prior to EAE induction and continuously once per day for 23 d.The neurological score and body mass were recorded in a double-blind manner.HE and Luxol fast blue staining were conducted to assess the inflammatory infil?tration and demyelination of the spinal cord.Flow cytometry was used to detect the percentage of CD4,CD11b and CD11c positive cells in the spleen.Western blotting was performed to analyze the protein expressions of NF-κB,phospho-NF-κB(p-NF-κB),NF-κB inhibitor alpha(IκBα),phospho-NF-κB inhibitor alpha(p-IκBα),phosphatidylinositol-3-kinase(PI3K),phospho-phosphatidylinositol-3-kinase(p-PI3K),protein kinase B(Akt),phospho-protein kinase B(p-Akt),Bcl2,Bax,cleaved-caspase 3 and ionized calcium binding adapter molecule 1(Iba1)in the spinal cord.RESULTS The neurological score of the model group mice was higher than that of the normal control group(P<0.01),but the body mass of the model group was significantly lower(P<0.05,P<0.01).Compared with model group,RAE 125 and 500 mg·kg-1 could effectively improve the symptoms and body mass loss of EAE mice in the third week of administration(P<0.01).Compared with the normal control group,the inflammatory infiltration and demyelination of the spinal cord in the model group mice were significantly increased(P<0.01).RAE could effectively attenuate the inflammatory infiltration and demyelination of the spinal cord in EAE mice(P<0.01).Compared with the normal control group,the percentages of CD4,CD11b and CD11c positive cells in the model group mice were significantly higher(P<0.01),which could be reversed by RAE(P<0.01).Compared with the normal control group,the phosphorylation of NF-κB pathway proteins,and Iba1,Bax and cleaved-caspase 3 proteins were significantly increased in the model group(P<0.01),which could be counteracted by RAE(125 mg·kg^-1).CONCLUSION RAE could effec?tively alleviate the severity of EAE mice,reduce the inflammatory infiltration and demyelination of the central nervous system.The action mechanism of RAE might be mediated by restraining the activation of peripheral inflammatory cells,suppressing neuroinflammation and inhibiting neuronal apoptosis.