目的 探讨法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(EAE)的血-脑屏障(BBB)保护机制.方法 采用MOG35-55肽段诱导C57BL/6小鼠建立EAE模型,于免疫后第3天起Fasudil组腹腔注射Fasudil〔40 mg/(kg·d)〕干预,EAE组注射等量生理盐水,持续至免疫后第20天.于免疫后第7 d、14 d、21 d采集小鼠脑和脊髓标本,行HE染色、脱髓鞘染色和免疫荧光染色;测定脑和脊髓伊文思蓝(EB)的渗透量;采用Western blot法检测脑内细胞紧密连接蛋白Occludin和ZO-1表达情况.结果 与EAE组相比,Fasudil组小鼠中枢神经系统(CNS)炎性细胞计数和髓鞘脱失面积均明显减少〔分别98.00±33.15 vs.417.70±78.89,t=3.736,P<0.05;(11.38±1.09)% vs.(38.21±7.94)%,t=3.473,P<0.05〕.在免疫后14 d、21d,Fasudil组脑和脊髓内EB渗透量均低于EAE组(脑:9.04±0.15 vs.9.93±0.25,t=5.776,P<0.05;9.09±0.089 vs.9.83±0.22,t=3.116,P<0.05;脊髓:17.28±0.38 vs.21.33±2.21,t=7.782,P<0.05;16.48±0.71 vs.21.77±0.17,t=7.256,P<0.01).与EAE组相比,Fasudil组小鼠脑内细胞紧密连接蛋白Occludin表达上调(7 d:0.068±0.045 vs.0.127±0.022,t=6.026,P<0.05),14 d:0.185±0.011 vs.0.233±0.014,t=2.609, P<0.05,21 d:0.248±0.021 vs.0.364±0.121,t=2.834, P<0.01)和ZO-1(7 d:2.013±0.073 vs.2.404±0.256,t=1.467,P>0.05;14 d:1.783±0.129 vs.2.003±0.184,t=2.409, P<0.05;21 d:1.332±0.052 vs.1.674±0.023,t=6.026,P<0.01).结论 Fasudil可能通过抑制小鼠脑内细胞紧密连接蛋白Occludin和ZO-1的下调而保护BBB的完整性.%Objective To explore the protective mechanisms of Fasudil on blood-brain-barrier (BBB) in the treatment of experimental autoimmune encephalomyelitis(EAE).Methods Chronic EAE model was induced by MOG35-55 in female C57BL/6 mice.Fasudil was injectedintraperitoneally in the intervention group at 40 mg/(kg·d) and normal saline was injected in the EAE group from day 3 to day 20 post-immunization (p.i.), The specimens were collected on day 7, 14, and 21 p.i.The brain and spinal cord were frozen for HE staining and myelin staining, immunofluorescence staining.The contents of Evans blue (EB) in the brain and spinal cord were detected after intravenous injection.The protein extracted from the brains was collected for the measuremens of Occludin and ZO-1 by Western blot.Results Fasudil significantly decreased inflammatory cell infiltration and demyelination in the CNS(number of inflammatory cell: 98.00±33.15 vs.417.7±78.89,t=3.736,P<0.05;demyelination area :(11.38±1.09)% vs.(38.21±7.94)%,t=3.473,P<0.05).Comparing with the EAE group,at day 14 d.and 21 d , the EB levels in brain and spinal cord of Fasudil-treated mice were significantly lower(brain:9.04±0.15 vs 9.93±0.25,t=5.776,P<0.05;9.09±0.089 vs 9.83±0.22,t=3.116,P<0.05;spinal cord:17.28±0.38 vs 21.33±2.21,t=7.782,P<0.05;16.48±0.71 vs 21.77±0.17,t=7.256,P<0.01),Fasudil also induced an up-regulation of Occludin (7 d:0.068±0.045 vs 0.127±0.022,t=6.026,P<0.05;14 d:0.185±0.011 vs 0.233±0.014,t=2.609,P<0.05;21 d:0.248±0.021 vs 0.364±0.121,t=2.834,P<0.01)and ZO-1 (7 d:2.013±0.073 vs 2.404±0.256,t=1.467,P>0.05;14 d:1.783±0.129 vs 2.003±0.184,t=2.409, P<0.05;21 d:1.332±0.052 vs 1.674±0.023, t=6.026,P<0.01) in the brain.Conclusions Fasudil may be related to inhibition of the down-regulation of Occludin and ZO-1 in the brain, revealing that Fasudil can maintain the integrity of BBB.
展开▼
