五子衍宗方免煎颗粒对实验性自身免疫性脑脊髓炎小鼠炎性反应的影响及机制

Effect and Mechanism of Wuzi Yanzong Prescription Granules on Inflammatory Response in Mice with Experimental Autoimmune Encephalomyelitis

目的探讨五子衍宗方免煎颗粒对实验性自身免疫性脑脊髓炎(EAE)小鼠炎性反应的影响及机制。方法将C57BL/6雌性小鼠随机分为正常组、模型组及五子衍宗方组(五子衍宗方免煎颗粒,16 g·kg^(-1)·d^(-1)),每组12只。采用皮下注射抗原乳剂[髓鞘少突胶质细胞糖蛋白多肽35-55(MOG35-55)+结核分枝杆菌]结合腹腔注射百日咳毒素制备EAE小鼠模型。从免疫后第3天开始灌胃给药,每天2次,持续干预25 d。每日进行神经功能评分;采用HE染色法检测脊髓炎性细胞浸润情况;LFB染色法检测脊髓髓鞘脱失情况;ELISA法检测小鼠脾细胞上清液中炎性因子干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-17、IL-12、IL-6及IL-1β水平;qRT-PCR法检测小鼠脾细胞TLR4、TNF-αmRNA表达情况;Western Blot法检测小鼠脾脏ROCKII、P-MYPT1及NF-κB蛋白表达情况。结果与正常组比较,模型组小鼠神经功能评分显著升高(P<0.05,P<0.01);脊髓炎性细胞浸润及髓鞘脱失显著增加(P<0.01);脾细胞IFN-γ、TNF-α、IL-17、IL-12、IL-6及IL-1β的表达水平显著上升(P<0.001),TLR4、TNF-αmRNA表达显著上调(P<0.01,P<0.001);脾脏组织ROCKII、P-MYPT1、NF-κB蛋白表达量显著升高(P<0.01,P<0.001)。与模型组比较,五子衍宗方组小鼠神经功能评分显著降低(P<0.05,P<0.01);脊髓炎性细胞浸润及髓鞘脱失显著减少(P<0.01);脾细胞IFN-γ、TNF-α、IL-17、IL-12、IL-6及IL-1β的表达水平显著降低(P<0.01,P<0.001),TLR4、TNF-αmRNA表达显著下调(P<0.01,P<0.001);脾脏组织ROCKII、P-MYPT1、NF-κB蛋白表达量均显著降低(P<0.01)。结论五子衍宗方免煎颗粒能够改善EAE小鼠的神经功能,减少炎症浸润及脱髓鞘等病理损害,具有神经保护作用,其作用机制可能与抑制Rho及NF-κB信号通路,降低相关炎性因子水平有关。

Objective To investigate the effect and mechanism of Wuzi Yanzong Prescription Granules on the inflammatory response of mice with experimental autoimmune encephalomyelitis(EAE).Methods C57BL/6 female mice were randomly divided into normal group,model group and Wuzi Yanzong Prescription group(Wuzi Yanzong Prescription Granules,16 g·kg^(-1)·d^(-1)),with 12 mice in each group.The EAE mouse model was prepared by subcutaneous injection of antigenic emulsion[myelin oligodendrocyte glycoprotein polypeptide 35-55(MOG35-55)+mycobacterium tuberculosis]combined with intraperitoneal injection of pertussis toxin.The rats were given intragastric administration twice a day for 25 consecutive days from the third day after immunization.Neurological function was scored daily;spinal cord inflammatory cell infiltration was detected by HE staining;spinal cord myelin loss was detected by LFB staining;the levels of inflammatory factors interferon(IFN-γ),tumor necrosis factor(TNF)-α,interleukin(IL)-17,IL-12,IL-6 and IL-1βwere detected in the supernatant of splenocytes by ELISA.The mRNA expressions of TLR4 and TNF-αin mouse splenocytes was measured by qRT-PCR;the protein expressions of ROCKII,P-MYPT1 and NF-κB in mouse spleen were measured by Western Blot.Results Compared with the normal group,the neurological function scores of mice in the model group were significantly increased(P<0.05,P<0.01);inflammatory cell infiltration in the spinal cord was significantly increased(P<0.001)and myelin loss was significantly increased(P<0.01);the expression levels of IFN-γ,TNF-α,IL-17,IL-12,IL-6 and IL-1βin splenocytes were significantly increased(P<0.001),mRNA expression levels of TLR4 and TNF-αwere significantly increased(P<0.01,P<0.001);protein expressions of ROCKII,P-MYPT1 and NF-κB in splenic tissue were significantly increased(P<0.01,P<0.001).Compared with the model group,the neurological function scores of mice in the Wuzi Yanzong Prescription group were significantly reduced(P<0.05,P<0.01);inflammatory cell infiltration in the spinal cord and myelin loss were significantly reduced(P<0.01);the expression levels of IFN-γ,TNF-α,IL-17,IL-12,IL-6 and IL-1βin splenocytes were significantly reduced(P<0.01,P<0.001),mRNA expression levels of TLR4 and TNF-αwere significantly down-regulated(P<0.01,P<0.001);protein expressions of ROCKII,P-MYPT1 and NF-κB in splenic tissue were significantly reduced(P<0.01).Conclusion Wuzi Yanzong Prescription Granules can improve the neurological function of EAE mice,reduce pathological damage such as inflammatory infiltration and demyelination,and have neuroprotective effects.The mechanism of action may be related to the inhibition of Rho and NF-κB signaling pathways and reduction of the level of related inflammatory factors.