法舒地尔通过诱导EAE小鼠Occludin表达抑制其炎性细胞浸润

Fasudil inhibits the infiltration of inflammatory cells by upregulating occludin expression in experimental autoimmune encephalomyelitis mice

目的观察法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(EAE)小鼠模型的治疗效果并探讨其作用机制。方法采用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫建立国际标准的小鼠EAE模型。将免疫后的42只雌性C57BL/6小鼠随机分为EAE组、Fasudil早期治疗组和Fasudil晚期治疗组。Fasudil早期治疗组即免疫后第3天按体质量40mg/(kg.d)腹腔注射Fasudil,1次/d,至免疫后30d;Fasudil晚期治疗组即免疫后首只小鼠出现症状即开始腹腔注射Fasudil,注射方法和剂量同Fasudil早期治疗组;与晚期治疗组给药同一时间,给予EAE组等量生理盐水作为对照。免疫后隔天观察各组小鼠临床评分和体质量变化。于免疫30d后处死动物,分离小鼠脊髓腰膨大和脑冠状位中部1/3处做冷冻切片,行HE染色和免疫荧光染色。同时提取小鼠脊髓蛋白检测Occludin蛋白的表达。结果 Fasudil可改善EAE的临床症状,抑制脑血管内皮细胞Rho激酶(ROCK)活性。与EAE组相比,Fasudil早期治疗组和晚期治疗组CD31阳性内皮细胞上p-MYPT1表达均减弱(P<0.01,P<0.05),起病时间均延长(P<0.01,P<0.05),脊髓Occludin蛋白表达均增加(P<0.05)。结论 Fasudil对EAE具有治疗的潜能,其机制可能为通过抑制血管内皮细胞ROCK活性,参与诱导内皮细胞紧密连接蛋白Occludin的表达,从而抑制炎性细胞的中枢浸润,最终阻止疾病的发生和发展。

Objective To observe the therapeutic effects of Fasudil on experimental autoimmune encephalomyelitis （EAE） mice, and explore its possible mechanisms. Methods The EAE mouse was induced by myelin oligodendrocyte glycoprotein peptides 35-55 （MOG35-55） immunization. 42 immunized female C57BL/6 mice were then randomly divided into three groups： Fasudil early treatment [Fasudil was intraperitoneally injected with the dosage of 40mg/ （kg . d） on day 3 post-immunization （p. i. ）], Fasudil late treatment （Fasudil intraperitoneally injection was initiated when clinical symptom appeared） and EAE group （Saline was injected instead）. The injection of Fasudil was performed once a day till day 30 post immunization. Clinical signs were evaluated and body weight was recorded every other day after immunization. 30 days later, brain and spinal cord were dissected, and the pathological characteristics and Rho kinase activity in brain and spinal cord were detected by HE and immunofluorescence staining. The extraction of spinal cord was also collected for the detection of occludin expression. Results The treatment with Fasudil improved clinical symptoms. Pathological examination revealed that Fasudil inhibited Rho kinase activity in brain endothelial cells. Compared with the EAE group, the expression of p-MYPT1 in CD31 positive endothelial cells in the Fasudil early treatment group and late treatment group decreased （P〈0. 01, P〈0. 05）, the symptom onset was delayed （ both P〈0. 05）, and the activation of protein occludin in spinal cord increased （ both P〈0.05 ） in these 2 treatment groups. Conclusions Fasudil shows potential treatment effects for EAE. The possible mechanisms could be the inhibition on Rho kinase activity in vascular endothelial cells, and the upregulation of tight junction occludin protein expression, which limited the infiltration of inflammatory cells into the CNS, and ultimately prevented the development of EAE.