益肾达络饮对MRS2179干预下实验性自身免疫性脑脊髓炎小鼠的影响及机制

Effects and mechanism of Yishen Daluo Decoction on experimental autoimmune encephalomyelitis mice under the intervention of MRS2179

目的:从P2Y1受体探讨益肾达络饮治疗实验性自身免疫性脑脊髓炎(EAE)的机制。方法:60只C57BL/6J雌性小鼠随机分成6组:空白组、模型组、P2Y1受体拮抗剂(MRS2179)组、中药组、MRS2179+中药组、激素组。除空白组外均制作EAE模型,每日记录各组小鼠的体质量与神经功能评分。造模第4天,MRS2179组与MRS2179+中药组腹腔注射MRS2179,造模第8天开始给予相应药物连续干预14 d取材。HE染色观察各组小鼠脑与脊髓的病理改变;ELISA测定各组小鼠血清中白介素(IL)-6、IL-17、IL-23、γ干扰素(IFN-γ)的浓度;Western Blot检测各组小鼠脑及脊髓中P2Y1、细胞外信号调节激酶(ERK)、胶质纤维酸性蛋白(GFAP)、环磷腺苷效应元件结合蛋白(CREB)、神经胶质细胞源性神经营养因子(GDNF)、蛋白激酶A(PKA)、蛋白激酶C(PKC)蛋白的表达变化。结果:益肾达络饮可以减缓EAE小鼠体质量下降,降低神经功能评分,缓解脑与脊髓中的炎性浸润。与空白组比较,模型组血清中IL-6、IL-17、IL-23、IFN-γ浓度显著升高,脑与脊髓中P2Y1、ERK、GFAP、PKA表达显著升高,GDNF、CREB、PKC表达显著降低(P<0.05,P<0.01)。中药组明显逆转上述变化,MRS2179+中药组进一步逆转上述变化。结论:益肾达络饮的作用机制可能是通过影响中枢神经系统中的P2Y1受体活性,抑制神经炎症反应,抑制GFAP分泌,影响PKA/PKC平衡,促进GDNF的生成,从而抑制星形胶质细胞过度活化、缓解炎症、促进神经修复,缓解EAE的疾病状态。

Objective:To explore the mechanism of Yishen Daluo Decoction in treating of experimental autoimmune encephalomyelitis(EAE)from P2Y1 receptor.Methods:Sixty C57BL/6J female mice were randomly divided into 6 groups:Blank group,model group,P2Y1 receptor antagonist(MRS2179)group,traditional Chinese medicine(TCM)group,MRS2179+TCM group,hormone group.EAE models were made except blank group.Body weight and clinical scores were recorded dayly.MRS2179 and MRS2179+TCM groups were administered intraperitoneally with MRS2179 at 8th day after modeling,corresponding drugs were given and tissues were taken after 14 days.We observed the pathological changes of brain and spinal cord by HE staining,determined concentrations of IL-6,IL-17,IL-23 and IFN-γin serum by ELISA.The expression of P2Y1,extracellular signal-regulated kinase(ERK),glial fibrillary acidic protein(GFAP),cMAP response element binding protein(CREB),glial cell linederived neurotrophic factor(GDNF),protein kinase A(PKA)and protein kinase C(PKC)were detected by Western Blot.Results:Yishen Daluo Decoction could relieve the weight loss of EAE mice,decrease clinical scores and relieve inflammatory infiltration.Compared with blank group,the levels of IL-6,IL-17,IL-23,IFN-γin serum and P2Y1,ERK,GFAP,PKA in brain and spinal cord were significantly increased in model group,GDNF,CREB and PKC were significantly decreased(P<0.05,P<0.01).TCM group can effectively reverse the above changes,and has a synergistic effect with MRS2179.Conclusion:Mechanism of Yishen Daluo Decoction may be related to its effects on P2Y1 receptor activity,neuroinflammation,GFAP secretion,PKA/PKC balance and GDNF,therefore,it can protect the nervous system and relieve the state of EAE.