维生素D对实验性自身免疫性葡萄膜炎影响机制

Effect of vitamin D on experimental autoimmune uveitis

目的探讨维生素D活性形式1-25二羟维生素D_(3)[1,25-Dihydrovitamin D_(3),1,25(OH)2D_(3),VD]对实验性自身免疫性葡萄膜炎(Experimental autoimmune uveitis,EAU)的影响及其机制研究。方法选择6~8周龄,雌性Lewis大鼠40只,随机数字表法分为5组:Control组和EAU组,EAU+VD 0.5μg/kg组、EAU+VD 1.0μg/kg组、EAU+VD 2.0μg/kg组,每组8只。Control组在大鼠躯干上皮下、后肢足垫及两侧腹壁各点一次性注射200μL无菌磷酸盐缓冲液(phosphate buffer saline,PBS)。其余各组在相同位置一次性注射200μL含有光感受器间维生素A结合蛋白(Interphotoreceptor binding protein,IRBP)、结核菌素(tuberculin,TB)、完全弗氏佐剂(CFA)和无菌磷酸盐缓冲液的乳糜液免疫诱导EAU。EAU+VD 0.5μg/kg组、EAU+VD 1.0μg/kg组、EAU+VD 2.0μg/kg组均在建模前3天分别给予VD 0.5μg/kg、1.0μg/kg、2.0μg/kg,1次/d灌胃给药,持续19 d。免疫后10 d、12 d、14 d分别在裂隙灯显微镜、眼科90D前置镜观察大鼠眼部前节及视网膜炎症表现;免疫后16 d处死大鼠,HE染色观察眼部组织病理变化;q-PCR检测TNF-α、IFN-γ、IL-6、IL-17、IL-23、CD80,CXCL9、CXCL10、CXCL11、CXCR3、CCL2、CCR2表达;Western Blot检测蛋白激酶B(Protein kinase,AKT)及磷酸化蛋白激酶B(Phosphorylated protein kinase B,p-AKT)表达。结果与EAU组相比,EAU+VD 2.0μg/kg组眼前节及眼部组织病理均表现轻度炎性改变;与EAU组相比,EAU+VD 2.0μg/kg组TNF-α、IL-23、CXCL9、CXCL10及CXCR3 mRNA表达量降低(P<0.05);磷酸化AKT表达量明显降低(P<0.01)。结论VD 2.0μg/kg预防给药有可能通过干扰AKT磷酸化,抑制炎症相关因子表达,缓解实验性自身免疫性葡萄炎病情。

Objective To investigate the effect of 1-25 dihydroxyvitamin D_(3)(1,25(OH)2 D_(3),VD),the active metabolite of vitamin D,on experimental autoimmune uveitis and its mechanism.Methods A total of 40 female Lewis rats aged 6 to 8 weeks were selected and divided into 5 groups by random number table method:Control group,EAU group,EAU+VD 0.5μg/kg group,EAU+VD 1.0μg/kg group,EAU+VD 2.0μg/kg group,with 8 rats in each group.In the Control group,200μL of phosphate buffer saline(PBS)was injected under the trunk epithelium,the foot pad of the hind limb and each point of the abdominal wall.The other groups were immunized with a single injection of 200μL of emulsion containing interphotoreceptor binding protein(IRBP),tuberculin(TB),complete Fredrin adjuvant(CFA),and sterile phosphate buffers at the same site to induce EAU.The EAU+VD 0.5μg/kg group,EAU+VD 1.0μg/kg group and EAU+VD 2.0μg/kg group were all given vitamin D 0.5μg/kg,1.0μg/kg and 2.0μg/kg,respectively,once a day for 19 days,starting 3 days before modeling.The manifestations of ocular anterior segment and retinal inflammation were observed by slit-lamp microscope at 10,12,and 14 days after immunization,and the ophthalmic front lens at 90 days,respectively.The rats were killed 16 days after immunization,and the pathological changes of eye tissues were observed by HE staining.The expressions of TNF-α,IFN-γ,IL-6,IL-17,IL-23,CD80,CXCL9,CXCL10,CXCL11,CXCR3,CCL2 and CCR2 were detected by qRT-PCR.Western Blot detection of Protein kinase B(AKT)and Phosphorylated protein kinase B(p-AKT)expression.Results Compared with EAU group,EAU+VD 2.0μg/kg group showed slight inflammatory changes in anterior segment and eye tissue.Compared with EAU group,mRNA expressions of TNF-α,IL-23,CXCL9,CXCL10 and CXCR3 in EAU+VD 2.0μg/kg group were decreased(P<0.05).The expression of phosphorylated AKT was significantly decreased(P<0.01).Conclusion Prophylactic administration of vitamin D 2.0μg/kg may alleviate experimental autoimmune staphylitis by interfering with AKT phosphorylation and inhibiting the expression of inflammation-related factors.