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

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。

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 < 0.05) when compared with model control group, which was abolished by H89 treatment (P < 0.05). Western blotting results revealed that the expression of CREB in the central nervous system decreased in model control group compared with normal control group (P < 0.05). Treatment with ICA promoted the expressions of CREB when compared with model control group (P < 0.05), which was reversed by H89 (P < 0.05). Conclusions Icariin ameliorates neurological symptoms of EAE potentially due to promotion of CREB expressions in the central nervous system through PKA pathway.