骨化三醇对实验性自身免疫性脑脊髓炎的治疗作用及相关机制

目的探讨骨化三醇(Calcitriol)对实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)的治疗作用及相关机制。方法用含200μg髓鞘少突胶质细胞糖蛋白35-55肽段(Myelin oligodendrocyte glycoprotein 33-35,MOG33-35)、250μg结核菌素的50μl弗氏不完全佐剂(IFA)皮内免疫C57BL/6小鼠,并分别于免疫当天和第2天注射百日咳毒素,建立EAE实验性动物模型(EAE组);骨化三醇组从免疫当天起隔日腹腔注射骨化三醇100 ng进行治疗,观察两组小鼠临床评分的差异;于EAE发病高峰期处死小鼠,取脊髓及淋巴结,通过HE及LFB染色观察脊髓中炎细胞浸润及髓鞘脱失;采用流式细胞术检测两组淋巴细胞CD4+T细胞亚型的分布。结果与EAE组比较,骨化三醇组发病延缓且发病较轻,临床评分差异有统计学意义(P<0.05或P<0.01或P<0.001);骨化三醇组较EAE组小鼠脊髓白质炎性细胞浸润明显减少,脱髓鞘斑块明显减轻;骨化三醇组与EAE组相比,Th17细胞亚群明显受到抑制(P<0.05),而Th2和Treg细胞水平明显升高(P均<0.05)。结论骨化三醇可以延缓EAE发病,减轻临床症状及病理改变,并能够通过调节CD4+T细胞亚群平衡,即抑制Th17细胞,上调Th2和Treg细胞水平发挥对EAE的预防和治疗作用。

Curative Effect of Calcitriol on Experimental Autoimmune Encephalomyelitis and Relevant Mechanism

Objective To investigate the curative effect of calcitriol on experimental autoimmune encephalomyelitis(EAE) as well as the relevant mechanism.Methods C57BL / 6 mice were immunized with 50μof incomplete Freunds adjuvant(IFA) containing 200μg of myelin oligodendrocyte glycoprotein 33-35(MOG33-35) and 250μg of tuberculin,then injected i.v.twice with pertussis toxin on days 0 and 1 after immunization respectively to establish the animal model of EAE(EAE group).However,the mice in calcitriol group were injected i.p.with 100 ng of calcitriol every other day starting from day 0.The clinical scores of mice in the two groups were compared.Mice were killed in the peak phase of onset of EAE,of which spinal cords were collected and observed for inflammatory cell infiltration by HE staining and for demyelization by Luxol Fast Blue(LFB) staining,while lymph nodes were observed for distribution of CD4+ T lymphocyte subsets by flow cytometry.Results Compared with those in EAE group,the onset of EAE in mice in calcitriol group was delayed,while the disease was relieved,and the clinical score decreased significantly(P < 0.05,P < 0.01 or P < 0.001);the inflammatory cell infiltration and demyelization spots in spinal cord decreased significantly;the Th17 lymphocyte subsets were inhibited significantly(P < 0.05),while the Th2 and Treg lymphocyte levels increased significantly(P < 0.05).Conclusion Calcitriol delayed the onset of EAE,relieved clinical symptoms and pathological change,and showed preventive and curative effects on EAE by regulating the balance of CD4+ T lymphocyte subsets,i.e.inhibiting Th17 lymphocytes and up-regulating Th2 and Treg lymphocyte levels.