复方中药制剂对实验性自身免疫性脑脊髓炎小鼠神经保护作用的研究

Neuroprotective effect of compound Chinese medicine on experimental autoimmune encephalomyelitis

目的探讨自制复方中药制剂对实验性自身免疫性脑脊髓炎(EAE)小鼠的疗效及相关机制。方法选取SPF级雌性C57BL/6小鼠36只,采用皮下注射MOG_(35-55)抗原免疫诱导EAE模型。将36只小鼠随机分为EAE模型组、中药组和激素组,每组各12只。自造模第14天起,中药组按体重20 g/kg给予自制复方中药灌胃,激素组按体重3.9 mg/kg给予醋酸泼尼松灌胃治疗,EAE模型组按同样方法给予等量生理盐水,至造模第42天结束。采用Kono 5级评分法评估各组小鼠神经功能,采用HE染色法观察中枢神经系统(CNS)炎细胞浸润,采用LFB染色法观察CNS脱髓鞘情况,采用酶联免疫吸附法检测外周血γ干扰素(IFN-γ)、白细胞介素-4(IL-4)和IL-17A细胞因子水平,采用Western blot检测CNS组织血清/糖皮质激素诱导激酶-1(serum/glucocorticoid-inducible kinase-1,SGK-1)表达。结果中药组小鼠神经功能评分峰值低于EAE模型组(P<0.01),脑脊髓组织炎细胞浸润和脱髓鞘程度较EAE模型组减轻。与EAE模型组比较,中药组EAE小鼠EAE病程前期、高峰期以及缓解期外周血IFN-γ和IL-17A水平均低(均P<0.01),而IL-4水平均高(均P<0.01);与激素组比较,中药组小鼠不同EAE病程阶段IFN-γ和IL-4水平比较均无统计学差异(均P>0.05),而IL-17A水平于EAE高峰期和缓解期低于激素组(均P<0.05)。中药组小鼠脑脊髓组织SGK-1蛋白表达在病程高峰期及缓解期均低于EAE模型组和激素组(均P<0.01)。结论该复方中药制剂对EAE小鼠具有神经保护作用,可减轻EAE小鼠临床症状及CNS组织炎性浸润及脱髓鞘,其作用机制可能与通过SGK-1蛋白调节细胞因子水平有关。

Objective To investigate the efficacy and related effects of compound Chinese medicine on experimental autoimmune encephalomyelitis(EAE)mice.Methods Thirty six C57BL/6 mice were randomly divided into 3 groups,an EAE group,a Chinese medicine group and a steroid group,with 12 mice per group.From the 14th day of EAE,the Chinese medicine group and steroid group were given compound Chinese medicine(20 g/kg)and prednisone(3.9 mg/kg)respectively,and the EAE group was given normal saline.The disease condition of EAE mice was evaluated and compared by clinical scores.The inflammatory cell infiltration and demyelination of central nervous system(CNS)were observed by histological staining.The levels of interferon-gamma(IFN-γ),interleukin 4(IL-4)and IL-17A in peripheral blood were detected by ELISA.Western blot was used to detect the expression of serum/glucocorticoid-inducible kinase-1(SGK-1)in CNS.Results The compound Chinese medicine reduced the neurological clinical score of EAE mice(P<0.01),alleviated the inflammatory cell infiltration and demyelination of CNS;significantly reduced the levels of inflammatory cytokines IFN-γand IL-17A in peripheral blood(P<0.01);significantly increased the level of inhibitory cytokine IL-4(P<0.01).The expression of SGK-1 protein in CNS tissues of the compound Chinese medicine group was significantly decreased(P<0.01).Conclusions The compound Chinese medicine has a neuroprotective effect,can reduce the clinical scores of EAE mice and relieve the CNS tissue involvement,regulate the levels of cytokines in EAE mice,thus reduce the EAE inflammation.SGK-1 protein may participate in the regulation of EAE immune response of the compound Chinese medicine.