PKA干预淫羊藿苷治疗实验性自身免疫性脑脊髓炎对中枢神经CREB表达的影响*

Therapeutic effect of Icariin on experimental autoimmune encephalomyelitis in mice*

摘要:

目的 研究淫羊藿苷（ICA）治疗实验性自身免疫性脑脊髓炎（EAE）对环磷腺苷效应元件结合蛋白（CREB）的影响，并探讨ICA治疗的作用机制。方法 复制C57BL/6小鼠EAE模型，并将其分为3组，每组 6只。模型对照组：予生理盐水3?ml/d灌胃；ICA组：予以ICA 300?mg/（kg·d）灌胃；ICA+H89组：予以ICA 300?mg/（kg·d）灌胃并予以蛋白激酶A（PKA）特异性阻断剂H89 5?mg/（kg·d）腹腔注射；另取6只未经模型复制的C57BL/6小鼠同等条件下饲养作为正常对照组，予生理盐水3?ml/d灌胃。EAE小鼠在发病达高峰时开始给药，1次/d，连续给药5?d。每日进行神经损害评分，至给药结束后次日。给药结束后次日进行神经损害评分后处死小鼠，立即采集脊髓颈膨大部分进行Western blotting检测，检测CREB的表达。结果 ICA组小鼠神经损害表现明显改善，与治疗前比较差异有统计学意义（P?<0.05），而ICA+H89组小鼠及模型对照组小鼠神经损害评分均无改善，治疗前后比较差异无统计学意义（P?>0.05）。模型对照组脊髓组织中CREB的表达较正常对照组降低（P?<0.05）。ICA组治疗后CREB的表达与模型对照组比较，差异有统计学意义（P?<0.05），ICA组CREB的表达升高。但同时给予ICA与H89治疗的小鼠并不能提高脊髓组织中CREB的表达，与模型组比较差异无统计学意义（P?>0.05）。结论 ICA可能是通过PKA途径提高中枢神经系统中CREB的表达，从而发挥对EAE的治疗作用。

Abstract:

Objective To study the expression of cAMP-response element binding protein (CREB) in the treatment of experimental autoimmune encephalomyelitis (EAE) by Icariin (ICA), and explore the possible therapeutic mechanisms of ICA. Methods EAE was established successfully on C57BL/6 EAE mice. Mice were divided randomly into three groups (n?=?6): model control group (saline 3?ml/d gavage), ICA group (ICA 300?mg/(kg?d) gavage), and ICA plus H89 group [ICA: 300?mg/(kg?d) gavage, H89: 5?mg/(kg?d), intraperitoneal injection]. H89 is a specific blocker of protein kinase A (PKA). Six normal mice were set up as normal control group and received saline 3 ml/d gavage. Treatment was started when EAE mice was presented with severe clinical symptoms. Meanwhile, the neurological signs were evaluated on daily basis. Mice were treated once a day for five days and were sacrificed at the sixth day after treatment. The cervical enlargement of the spinal cords was separated and used to investigate the expressions of CREB by western blotting. Results The mean clinical scores were significantly reduced by the treatment of ICA (P?