白芍总苷对实验性自身免疫性葡萄膜炎大鼠肝脏中自然杀伤T细胞表达的影响

摘要

目的:探讨白芍总苷(TGP)对实验性自身免疫性葡萄膜炎(EAU)大鼠肝脏中自然杀伤T(NKT)细胞基因表达的影响.方法:实验研究.45只Lewis大鼠采用随机数字表法分为正常对照组、EAU模型组和TGP干预组,每组15只.EAU模型组和TGP干预组注射含光感受器间维生素A结合蛋白(IRBP 1177-1191)的乳糜液以诱导出葡萄膜炎,正常对照组注射等体积的不含IRBP多肽的乳糜液.于免疫后每天对TGP干预组大鼠进行TGP灌胃给药,其余2组给予0.9％氯化钠溶液灌胃,并每天采用裂隙灯显微镜观察各组大鼠眼前节炎症反应,记录发病程度,并进行炎症评分.免疫后第12天分别提取3组大鼠的眼球制成病理切片,观察炎症反应.收集3组大鼠肝脏CD4+T细胞,流式细胞仪检测NKT细胞的表达水平,观察变化趋势;采用RT-PCR和ELISA分别检测白细胞介素-4(IL-4)、干扰素-γ(IFN-γ)mRNA和蛋白表达情况.采用单因素方差分析进行数据处理.结果:免疫后第12天,TGP干预组炎症评分和病理分级明显低于EAU模型组,但是高于正常对照组;与正常对照组相比,EAU模型组和TGP干预组NKT表达水平明显较高,且TGP干预组高于EAU模型组(P<0.05);TGP干预组大鼠肝脏中IFN-γ的mRNA表达显著低于EAU模型组(P<0.001);TGP干预组大鼠肝脏中IL-4 mRNA低于正常对照组(P<0.001),但明显高于EAU模型组(P<0.001);TGP干预组大鼠肝脏IFN-γ蛋白浓度高于正常对照组(P<0.001),低于EAU模型组(P=0.01);TGP干预组大鼠肝脏中IL-4蛋白表达高于EAU模型组(P<0.001).结论:TGP可通过调节EAU大鼠IL-4、IFN-γ基因的表达水平,增加EAU大鼠中NKT细胞的表达数量,达到治疗葡萄膜炎的目的.%Objective: To investigate the effects of total glucosides of paeony (TGP) on natural killer T (NKT) cell gene expression in liver of experimental autoimmune uveitis (EAU) rats. Methods: Experimental study. Forty-five Lewis rats were randomly divided into three groups: normal controls, EAU-stimulated rats (model group), and EAU-stimulated rats treated with TGP (TGP intervention group). Control animals received an emulsion of phosphate-buffered saline (PBS) and CFA, which was injected subcutaneously. The other two groups were injected an equal volume of chylomicron containing interphotoreceptor retinoid-binding protein polypeptide (IRBP). Inflammation was observed by daily slit-lamp microscopy of the anterior segments after immunization. The incidence of inflammation and the inflammation scores were recorded. At 12 days after immunization, we extracted the eyes for pathologic examination, and CD4+ T cells were collected from the livers. The expression levels of NKT cells were detected by flow cytometry. Reverse transcription polymerase change reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the gene and protein expression levels of interleukin (IL)-4 and interferon (IFN)-γ. Data were analyzed by one-way ANOVA. Results: Twelve days after immunization, the inflammatory score and pathological grade of the TGP group were significantly lower than in the EAU group, but higher than in the normal group. Flow cytometry showed that compared with normal control group, the number of liver NKT cells in the EAU group and in the TGP intervention group were significantly increased. Further, the increased in the number of NKT expression in the TGP intervention group was higher than in the EAU group (P<0.05) during the inflammatory phase. RT-PCR showed that on day 12 the expression of IFN-γ mRNA in the liver of the TGP intervention group was significantly higher than in the control group (P<0.001), but lower than in the EAU group (P<0.001). At the same time, the expression of liver IL-4 mRNA in the TGP group was lower than that of the normal group (P<0.001), but significantly higher than the EAU group (P<0.001). ELISA showed that the liver IFN-γ levels in the TGP group were significantly lower in the EAU group after 12 days (P=0.01). The concentration of liver IL-4 protein was higher in the TGP group than in the EAU group (P<0.001). Conclusions: TGP can treat uveitis in EAU rats by regulating the expression of liver IL-4 and IFN-γ genes and increasing the number of NKT cells.