黄芪甲苷在多发性硬化症模型动物中的保护作用及机制研究

Protective Effects and Mechanism of AstragalosideⅣin Multiple Sclerosis Model Animals

目的:研究黄芪甲苷(ASI)对多发性硬化症(MS)模型实验性自身免疫脑脊髓炎(EAE)动物的保护作用及机制研究。方法:将C57BL/6小鼠随机分为4组:正常对照组、模型组、ASI组(20 mg·kg^(-1))和阳性对照组(甲泼尼龙琥珀酸钠,20 mg·kg^(-1)),每组10只。采用雌性C57BL小鼠建立EAE模型,观察ASI对EAE小鼠神经行为学评分及体质量变化的影响。用LC-MS/MS液质联用法检测小鼠大脑中ASI含量,实时定量PCR检测紧密连接分子闭锁连接蛋白-1(ZO-1)、闭合蛋白(Occludin)、密封蛋白-5(Claudin-5)、封闭蛋白-12(Claudin-12)mRNA表达,Western-blot检测紧密连接分子ZO-1、Occludin、Claudin-5蛋白表达,苏木素-伊红(HE)染色和固蓝(LFB)染色方法对小鼠脊髓组织病理分析。结果:与正常对照组比较,模型组小鼠神经行为学评分明显升高,体质量明显降低,紧密连接分子ZO-1、Occludin、Claudin-5蛋白表达明显下调,ASI单次入脑量明显增加(P<0.05或P<0.01),有脊髓组织炎症浸润和脱髓鞘发生,各紧密连接分子mRNA差异无统计学意义(P>0.05)。与模型组比较,ASI组能够抑制EAE小鼠神经行为学评分升高和体质量下降,减少脊髓炎症浸润和脱髓鞘发生,提高皮层紧密连接分子ZO-1、Occludin、Claudin-5 mRNA及蛋白表达(P<0.05或P<0.01),Claudin-12 mRNA差异无统计学意义(P>0.05)。结论:ASI能够抑制多发性硬化症模型EAE动物疾病进程,其作用可能是透过血脑屏障进入脑组织中ASI含量增加,诱导皮层紧密连接分子ZO-1、Occludin、Claudin-5基因表达上调,降低血脑屏障通透性,对中枢神经系统稳定起到保护作用,从而抑制疾病发展。

Objective:To study the protective effects and mechanism of astragalosideⅣ(ASI)on experimental autoimmune encephalomyelitis(EAE)in multiple sclerosis(MS)model animals.Methods:C57 BL/6 mice were randomly divided into 4 groups:normal control group,model group,ASI group(20 mg·kg^(-1))and positive control group(methylprednisolone sodium succinate,20 mg·kg^(-1))with 10 mice in each group.EAE model was established in female C57 BL mice,and the effects of ASI on neurobehavioral score and the weight change of EAE mice were observed.The content of ASI in mice brain was determined by LC-MS/MS.The mRNA expression levels of tight junction molecules ZO-1,Occludin,Claudin-5 and Claudin-12 were detected by real-time quantitative PCR.The levels of protein expression of tight junction molecules ZO-1,Occludin and Claudin-5 were detected by Western blot.Hematoxylin-eosin(HE)staining and Luxol fast blue(LFB)staining were used to analyze the histopathology of spinal cord in mice.Results:Compared with those in the normal control group,the neurobehavioral score of the model group increased significantly,the body weight decreased significantly,and protein expression of ZO-1,Occludin and Claudin-5 all decreased significantly,the single entry of ASI into the brain increased significantly(P<0.05 or P<0.01),the inflammation infiltration and demyelination of spinal cord tissue occurred,and there was no significant difference in mRNA of tight junction molecules(P>0.05).Compared with the model group,ASI could inhibit the increase of neurobehavioral score and weight loss in EAE mice,reduce the inflammatory infiltration and demyelination of spinal cord,increase the mRNA and protein expression of ZO-1,Occludin and Claudin-5 in cortex(P<0.05 or P<0.01),and there was no significant difference in mRNA of Claudin-12(P>0.05).Conclusion:ASI can inhibit the course of EAE in multiple sclerosis model mice.Its effects may be related to increasing the content of ASI entering brain tissue through blood-brain barrier,inducing the up-regulation of gene expression of ZO-1,Occludin and Claudin-5 in cortex,and reducing the permeability of blood-brain barrier and protecting the stability of central nervous system,so as to inhibit the development of the disease.