阿托伐他汀对自身免疫性脑脊髓炎小鼠髓鞘修复及RhoA/Rock1通路的影响

Effects of atorvastatin on myelin repair and the RhoA/Rock1 pathway in mice with autoimmune encephalomyelitis

目的探讨阿托伐他汀对自身免疫性脑脊髓炎(EAE)小鼠髓鞘修复及RhoA/Rock1通路的影响。方法采用MOG35-55免疫建立EAE小鼠模型,小鼠随机分为空白组、模型组、阿托伐他汀组、高脂饮食组、高脂饮食+阿托伐他汀组,每组6只,阿托伐他汀每只小鼠每天按0.5 mL混悬液灌服,连续28 d。各组小鼠神经功能评分,采用苏木精-伊红(HE)、固蓝(LFB)染色、透射电镜及免疫组化染色方法检测各组小鼠脊髓组织炎症反应、髓鞘脱失及髓鞘再生情况;酶联免疫吸附法(ELISA)检测小鼠血清中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、一氧化氮(NO)的表达;蛋白免疫印迹(Western blot)法检测脑组织RAS同源基因家族成员A(RhoA)、Rho相关蛋白激酶1(Rock1)蛋白表达;实时荧光定量PCR(qRT-PCR)检测脊髓组织硫酸软骨素蛋白多糖(NG2)、髓鞘碱性蛋白(MBP)及脑组织RhoA、Rock1 mRNA表达。结果与空白组比较,模型组小鼠见较多炎性细胞浸润、发生明显脱髓鞘改变、部分髓鞘崩解、断裂、脱失;模型小鼠血清中TNF-α、IL-6、NO的含量及脑组织RhoA、Rock1蛋白和mRNA表达均明显升高(P<0.01),脊髓组织NG2、MBP蛋白及mRNA表达明显降低(P<0.01)。与模型组比较,阿托伐他汀组小鼠见极少量炎性细胞浸润、脱髓鞘程度明显好转,明显降低小鼠血清中TNF-α、IL-6、NO的含量及脑组织RhoA、Rock1蛋白和mRNA表达(P<0.05),明显升高MBP、NG2蛋白和mRNA表达(P<0.05);高脂饮食+阿托伐他汀组明显降低小鼠神经功能评分、脑组织RhoA、Rock1蛋白表达,明显升高NG2 mRNA表达。结论阿托伐他汀能改善EAE小鼠炎性细胞浸润及脱髓鞘程度,降低高脂饮食EAE小鼠神经功能评分,其中作用机制可能与调节RhoA/Rock1通路改善脱髓鞘程度,从而发挥对EAE小鼠的治疗作用有关。

Objective To investigate the effect of atorvastatin on myelin repair and the RhoA/Rock1 pathway in mice with autoimmune encephalomyelitis(EAE).Methods MOG35-55 immunization was used to establish an EAE mouse model.The mice were randomly divided into the control group,model group,atorvastatin group,high fat diet group and high fat diet+atorvastatin group(n=6 per group).The atorvastatin was administered to each mouse daily by 0.5 mL suspension,for 28 consecutive days.Mice were scored for neurological function and clinical symptoms were observed.Hematoxylin-eosin(HE)staining,Luxol fast blue(LFB)staining,transmission electron microscopy and immunohistochemical staining method were used to detect inflammation and demyelination and remyelination of the spinal cord tissue of each group of mice.The expression of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and nitric oxide(NO)in serum was detected by enzyme linked immunosorbent assay(ELISA);protein immanoblotting assay(Western blot)method was used to detect the expression of Ras homologous gene family member A(RhoA)and Rho associated protein kinase 1(ROCK1)in brain tissue.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of chondroitin sulfate proteoglycan(NG2)and myelin basic protein(MBP)in spinal cord and RhoA and Rock1 mRNA expression in brain tissue.Results Compared with the control group,the model group showed more inflammatory cell infiltration,marked demyelination,partial myelination disintegration,breakage and demyelination;TNF-α,IL-6 and NO in serum and the expression of RhoA,Rock1 protein and mRNA in brain tissue were significantly increased,while the expression levels of NG2 and MBP protein and mRNA in spinal cord tissue were significantly decreased(P<0.01).Compared with the model group,the atorvastatin group showed significant improvement in inflammatory cell infiltration and demyelination,significantly decreased TNF-α,IL-6 and NO in serum,expression of RhoA and Rock1 protein and mRNA in brain tissue and increased expression of MBP,NG2 protein and mRNA in brain tissue(P<0.05).The high fat diet+atorvastatin group showed significantly decreased neurological function scores,brain tissue RhoA and Rock1 expression and significantly increased NG2 mRNA expression.Conclusions Atorvastatin improved inflammatory cell infiltration and demyelination in EAE mice and reduced neurological function scores in EAE mice on a high fat diet.The mechanism of action may be related to the regulation of the RhoA/Rock1 pathway to improve the degree of demyelination and thus exert a therapeutic effect on EAE mice.