补阳还五汤对自身免疫性脑脊髓炎模型小鼠神经保护作用的机制探讨

Neuroprotective Mechanism of Buyang Huanwu Tang on Experimental Autoimmune Encephalomyelitis Mice

目的:探讨补阳还五汤(BYHWT)在实验性自身免疫性脑脊髓炎(EAE)发展的不同阶段的神经保护作用和机制。方法:采用髓鞘少突胶质细胞糖蛋白35-55多肽(MOG35-55)诱导36只C57BL/6雌性小鼠建立实验性自身免疫性脑脊髓炎(EAE)模型,随机分为EAE 9,17,28 d组以及BYHWT 9,17,28 d组。BYHWT各组于免疫后第3天开始灌胃给药(50 g·kg^-1·d^-1),EAE组以相同方式等体积给予生理盐水,每天1次,连续至免疫后9,17,28 d。采用国际通用的五级临床症状评分观察BYHWT对EAE小鼠的干预作用;采集小鼠脊髓标本,进行苏木素-伊红(HE)染色和固蓝(LFB)染色观察BYHWT的神经保护作用;采用蛋白免疫印迹法(Western blot)检测脊髓神经营养因子(BDNF)和生长相关蛋白-43(GAP-43)等的表达。结果:补阳还五汤治疗后可明显抑制脊髓炎细胞浸润,减轻髓鞘脱失;与EAE组比较,BYHWT各给药组Nogo-A在脊髓表达显著下调(P <0. 01),与EAE 17,28 d组比较,BYHWT 17 d组和28 d组BDNF在脊髓中表达明显上调(P <0. 05,P <0. 01);与EAE 9,17 d组比较,BYHWT 9,17 d组GAP-43在脊髓中表达显著上调(P <0. 01)。结论:补阳还五汤可以通过上调NTFs类物质的表达,下调神经抑制因子的表达,改善局部神经生长微环境而发挥神经保护的作用。

Objective: To explore the neuroprotective effect and mechanism of Buyang Huanwu Tang ( BYHWT) on experimental autoimmune encephalomyelitis ( EAE) at different stages. Method: The 36 female C57BL /6 mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein peptides ( MOG35-55 ), then randomly divided into 9,17,28 d EAE control group. Each BYHWT group was orally given drugs on the 3rd day after immunization ( 50 g·kg ^- 1·d ^- 1 ),and EAE control group was given the same volume of normal saline in the same way once a day for 9,17 and 28 d after immunization. The effect of BYHWT on EAE mice was observed with internationally accepted clinical score. Brain and spinal cord specimens were collected at 9,17 and 28 d after immunization. The neuroprotective effect of BYHWT was observed by hematoxylin-eosin ( HE) staining and solid blue staining ( LFB). The expressions of BDNF and GAP-43 in spinal cord and brain were detected by Western blot. Result: After treatment,BYHWT can significantly inhibit myelitis cell infiltration and alleviate myelin loss. Compared with EAE group,the expression of Nogo-A in the spinal cord of each BYHWT group was significantly down-regulated ( P < 0.01),and the expression of BDNF in the spinal cord was significantly up-regulated ( P < 0.05,P < 0.01 ) in the BYHWT group 17 and 28 d group compared with EAE group 17 and 28 d group. Compared with EAE 9,17 d group,GAP-43 expression was significantly up-regulated in the spinal cord of BYHWT 9,17 d group ( P < 0.01). Conclusion: BYHWT can improve the local nerve growth microenvironment and promote the expression of NTFs,reduce the expressions of neuroinhibitory factors,and play a role in neuroprotection.