基于β-arrestin1沉默探讨益肾达络饮对EAE小鼠Rho/ROCK信号通路的影响

Effect of Yishen Daluo Prescription on Rho/ROCK Signaling Pathway in EAE Mice Based on Silencing of β-arrestin1

目的:基于β-抑制蛋白(β-arrestin1)基因沉默,研究益肾达络饮对实验性自身免疫性脑脊髓炎(EAE)小鼠Ras同源基因家族蛋白(Rho)/Rho相关卷曲螺旋蛋白激酶(ROCK)信号通路的影响。方法:60只C57BL/6雌性小鼠随机平均分为空白组、模型组、病毒组、益肾达络饮组(中药组)、病毒加益肾达络饮组(病毒加中药组)、醋酸泼尼松组(激素组)6组。除空白组外,每只小鼠均制备EAE模型,于免疫第4天病毒组、病毒加中药组每只小鼠尾静脉注射腺相关病毒(AAV)病毒溶液150μL(1×10^(11) vg·m L^(-1))。造模的第8天给药,空白组、模型组、病毒组给予生理盐水10 m L·kg^(-1),激素组给予醋酸泼尼松混悬液(3.9 g·kg^(-1)),其他组给予益肾达络饮(20 g·kg^(-1)),连续给药14 d并每日进行称重评分。免疫后每天记录各组小鼠的神经功能评分;苏木素-伊红(HE)染色测定小鼠脑组织、脊髓组织的炎症反应和病变位置;蛋白免疫印迹法(Western blot)测定EAE小鼠脊髓组织及脑组织中β-arrestin1、RhoA、ROCKⅠ的蛋白表达。结果:与模型组比较,病毒组、病毒加中药组的神经功能评分显著降低(P<0.01),中药组明显降低(P<0.05)。HE结果显示,模型组中枢神经系统(CNS)存在明显炎症反应,其他各组与模型组比较均有不同程度的减轻。Western blot结果显示,与空白组比较,模型组小鼠脊髓组织中β-arrestin1、RhoA及ROCKⅠ蛋白表达水平显著升高(P<0.01)。与模型组比较,病毒组、中药组、病毒加中药组、激素组小鼠脊髓组织中β-arrestin1、RhoA及ROCKⅠ蛋白表达水平显著降低(P<0.01)。与空白组比较,模型组小鼠脑组织中β-arrestin1、RhoA、ROCKⅠ蛋白表达水平均显著升高(P<0.01)。与模型组比较,病毒组、中药组、病毒加中药组、激素组小鼠脑组织中β-arrestin1蛋白表达水平均显著降低(P<0.01);病毒组、中药组小鼠脑组织中RhoA、ROCKⅠ蛋白表达水平均明显降低(P<0.05);病毒加中药组和激素组小鼠脑组织中RhoA、ROCKⅠ蛋白表达水平均显著降低(P<0.01)。结论:益肾达络饮可以改善EAE小鼠的临床症状,改善CNS的炎症反应,其机制可能为益肾达络饮抑制CNS中β-arrestin1的表达,从而降低Rho/ROCK信号通路中RhoA、ROCKⅠ蛋白的表达,并且益肾达络饮与抑制β-arrestin1的基因表达可能存在协同效果。

Objective:To investigate the effects of Yishen Daluo prescription(YSDL)on Ras homolog(Rho)/Rho-associated coiled-coil containing protein kinase(ROCK)signaling pathway in mice with experimental autoimmune encephalomyelitis(EAE)based on the silencing ofβ-arrestin1 gene.Method:Sixty C57BL/6 female mice were randomly divided into a blank group,a model group,a virus group,a YSDL group,a virus+YSDL group,and a prednisone acetate group(hormone group).The EAE model was induced in mice except for those in the normal group.Adeno-associated virus(AAV)solution(150μL,1×10^(11) vg·mL^(-1))was injected into the tail vein of each mouse in the virus group and the virus+YSDL group on the 4th day of immunization.Drugs were administered on the 8th day of modeling.Specifically,normal saline was given to the mice in the normal group,the model group,and the virus group at 10 mL∙kg^(-1),prednisone acetate suspension to those in the hormone group at 3.9 g∙kg^(-1),and YSDL to those in other groups at 20 g∙kg^(-1) for 14 consecutive days.The mice were weighed and scored every day.The neurological function scores of mice in each group were recorded every day after immunization.Hematoxylin-eosin(HE)staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice.The protein expression ofβ-arrestin1,Ras homolog gene family member A(RhoA),and Rho-associated coiled-coil forming protein kinaseⅠ(ROCKⅠ)in spinal cord and brain tissues of EAE mice was determined by Western blot.Result:Compared with the model group,the virus group and the virus+YSDL group showed decreased neurological function scores(P<0.01),and the YSDL group also showed decreased neurological function scores(P<0.05).HE results showed that there was obvious inflammatory reaction in the central nervous system(CNS)of the model group,which was alleviated to varying degrees in other groups compared with the model group.Western blot results showed that compared with the blank group,the model group showed increased protein expression levels ofβ-arrestin1,RhoA,and ROCKⅠin the spinal cord tissues(P<0.01).Compared with the model group,the virus group,the YSDL group,the virus+YSDL group,and the hormone group showed decreased protein expression levels ofβ-arrestin1,RhoA,and ROCKⅠin the spinal cord tissues(P<0.01).Compared with the blank group,the model group showed increased protein expression levels ofβ-arrestin1,RhoA,and ROCKⅠin the brain tissues(P<0.01).Compared with the model group,the virus group,the YSDL group,the virus+YSDL group,and the hormone group showed decreased protein expression level ofβ-arrestin1 in the brain tissues(P<0.01),and the virus group and the YSDL group showed decreased protein expression levels of RhoA,and ROCKⅠin the brain tissues(P<0.05).Additionally,the virus+YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠin the brain tissues(P<0.01).Conclusion:YSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS.The mechanism is presumably attributed to the fact that YSDL inhibits the expression ofβ-arrestin1 in CNS,thereby reducing the expression of Rho/ROCK signaling pathway.Furthermore,YSDL may have a synergistic effect with the inhibition ofβ-arrestin1 gene expression.