CTLA4-Ig改善EAMG大鼠脑功能障碍的分子机制研究

Molecular Mechanism of CTLA4-Ig Improving Brain Dysfunction in EAMG Rats

目的:探讨CTLA4-Ig对实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)大鼠脑功能障碍的改善作用及可能涉及的分子机制。方法:采用乙酰胆碱受体(acetylcholine receptor,AChR)α97-116肽免疫35只Lewis大鼠制作EAMG大鼠模型,然后用地塞米松和CTLA4-Ig处理大鼠。ELISA法检测血清AChR IgG和AChR IgG2b水平及后肢肌AChR含量;Lennon临床评分评价各组大鼠临床症状;避暗实验观察大鼠空间学习记忆的变化;紫外分光法测海马丙二醛(malonaldehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)、Caspases-3、Caspases-9和Caspases-12活性;RT-PCR测海马Caspases-3、Caspases-9、Caspases-12及Trx-1 mRNA表达;Western-blot测海马Trx-1蛋白表达。结果:模型成功率为85.7%,与正常对照组比较,EAMG大鼠模型组Lennon临床评分显著增高;肌肉AChR含量明显减少;血清AChR IgG、AChR IgG2b浓度明显升高;避暗实验结果显示,EAMG组大鼠避暗潜伏期缩短,而3 min错误次数增加,海马MDA含量增加,SOD活性降低,且Trx-1 mRNA及蛋白表达下调;Caspases-3、Caspases-9和Caspases-12 mRNA表达及活性均增加。与模型组比较,地塞米松组和CTLA4-Ig组可显著改善EAMG大鼠临床症状,并降低血清AChR IgG、AChR IgG2b浓度,同时增加肌肉AChR含量;避暗潜伏期明显延长,错误次数减少;海马组织MDA含量降低,SOD活性增加,Trx-1 mRNA及蛋白表达上调;Caspases-3、Caspases-9和Caspases-12 mRNA及活性均降低。结论:EAMG大鼠可合并脑功能障碍,Trx-1表达下调介导的氧化应激及细胞凋亡可能是其CNS受损的主要原因之一;CTLA4-Ig通过上调Trx-1表达改善EAMG大鼠海马氧化/抗氧化系统功能紊乱并抑制细胞凋亡。

Objective:To study the effects and possible mechanisms of CTLA4-Ig on brain dysfunction in EAMG rats.Methods:An experimental autoimmune myasthenia gravis(EAMG)was initially established by immunization of Lewis rats with acetylcholine receptor(AChR)a97-116 peptide.Then the rats were treated with dexamethasone and CTLA4-Ig.Serum levels of AChR IgG and AChR IgG2b and the content of AChR in hindlimbs were then detected using ELISA.Lennon clinical score was used to evaluate the clinical symptoms of rats in each group.The changes of spatial learning and memory in rats were observed by dark avoidance test.The content of MDA,activities of SOD,Caspases-3,Caspases-9 and Caspases-12 in hippocampus were measured by ultraviolet spectrophotometry.Caspases-3,Caspases-9,Caspases-12 and Trx-1 mRNA expression in hippocampus were measured by RT-PCR and the protein expression of Trx-1 was detected by western blot.Results:CTLA4-Ig and dexamethasone were found to significantly improve clinical symptoms of EAMG rats,reduce serum levels of AChR IgG,AChR IgG2b in peripheral blood,and enhance AChR content in the muscle;CTLA4-Ig and dexamethasone could also prolonge the latency,and reduce the error counts per second.Meanwhile ruduce MDA content,Caspases-3,Caspases-9,Caspases-12 activity and increase SOD activity,and significantly induced Trx-1 mRNA and protein expression upregulation and Caspases-3,Caspases-9,Caspases-12 mRNA expression downregulation and activity of them decreased in hippocampus.Conclusion:EAMG rats can be complicated with brain dysfunction.Oxidative stress and apoptosis mediated by down-regulation of Trx-1 expression may be one of the main causes of CNS damage.CTLA4-Ig can improve the dysfunction of oxidative/antioxidant system and inhibit apoptosis in hippocampus of EAMG rats by up-regulating the mRNA expression of Trx-1.