摘要背景 葡萄膜炎是常见的致盲眼病,多反复发作,目前主要的治疗方法是应用糖皮质激素和免疫抑制剂,但不良反应较多,寻求安全、有效的治疗方法至关重要. 目的 观察清开灵眼用凝胶对大鼠实验性自身免疫性葡萄膜炎(EAU)的疗效,并探讨其作用机制. 方法 SPF级雌性Lewis大鼠27只,皮下注射200 μl含100 μg光感受器间维生素A类结合蛋白(IRBP) 1177-1191多肽、100 μl完全弗氏佐剂(CFA)、100 μg结核菌素及100μl PBS的乳化液,在大鼠两足垫处、尾根部两侧及脊背正中均匀注射5个点建立EAU动物模型.将免疫后的大鼠按随机数字表法随机分为模型对照组、清开灵眼用凝胶组和妥布霉素地塞米松组.大鼠免疫后第7天开始点眼,模型对照组用生理盐水点眼,清开灵眼用凝胶组和妥布霉素地塞米松组分别用相应的药物点眼,每日3次,连续用药7d.从免疫后第1天开始裂隙灯显微镜下观察大鼠眼前节炎症反应并进行炎症评分;免疫后第14天过量麻醉法处死实验动物并获取眼球标本,采用组织病理学方法观察大鼠前房、虹膜和睫状体的炎性细胞浸润情况;采用流式细胞仪测定大鼠脾脏、淋巴结分离的T细胞悬液中CD4+和CD8+细胞百分比、CD4+/CD8+值以及Th1细胞和Th17细胞的比例.结果 模型对照组大鼠免疫后第9天开始出现眼部炎症表现,第11天炎症反应达高峰期,而清开灵眼用凝胶组和妥布霉素地塞米松组大鼠眼部炎症反应的发生较模型对照组晚,炎症反应轻,病程短.免疫后13d,3个组大鼠眼部炎症评分的总体差异有统计学意义(F=26.52,P=0.00).清开灵眼用凝胶组和妥布霉素地塞米松组的炎症评分明显低于模型对照组,差异均有统计学意义(t=6.72、10.11,P＜0.05).组织病理学检查发现,模型对照组大鼠前房、虹膜及睫状体组织中可见炎性细胞浸润,清开灵眼用凝胶组和妥布霉素地塞米松组大鼠前房、虹膜及睫状体组织中炎性细胞浸润明显减轻.模型对照组大鼠脾脏和淋巴结中CD4+细胞、CD8+细胞的百分比及CD4+/CD8+值分别为(83.10±0.15)％、(18.60±0.09)％和4.50±0.02,清开灵眼用凝胶组分别为(79.90±0.21)％、(19.20±0.15)％和4.20±0.04,妥布霉素地塞米松组分别为(78.60±0.09)％、(23.44±0.09)％和3.40±0.01,3个组间CD4+、CD8+细胞百分比及CD4+/CD8+值的差异均有统计学意义(F=223.68、530.77、404.83,均P=0.00).模型对照组大鼠脾脏和淋巴结中CD4+γ干扰素+(IFN-γ+),CD4+白细胞介素-17+(IL-17+)细胞的百分比分别为(32.20±0.19)％和(55.10±0.09)％,清开灵眼用凝胶组分别为(20.40±0.1 8)％和(25.20±0.32)％,妥布霉素地塞米松组分别为(10.40±0.23)％和(8.20±0.15)％,3个组间差异均有统计学意义(F=2 986.34、12 807.54,均P=0.00).结论 清开灵眼用凝胶点眼可减轻大鼠EAU炎症反应,其作用机制主要是抑制CD4+细胞的增生和分化,降低CD4+/CD8+值,并抑制Th1、Th17效应细胞的分化,减少相应细胞因子的分泌.

abstractsBackground Uveitis is a common refractory eye disease with recurrent features.Glucocorticoids and immunosuppressor are often used to the management of uveitis,but their adversary responses can not be ignored.It is very important to seek a safe and effective treating approach.Objective This study was to observe the curative effect of qingkailing ophthalmic gel,a Chinese herbal on experimental autoimmune uveitis (EAU) and to explore the mechanism.Methods EAU was induced in 27 SPF female Lewis rats by injection of complete Freund adjuvant (CFA),interphotoreceptor retinoid binding protein (IRBP) 1177-1191,tuberculin and phosphate buffer solution.The rats were randomly assigned to the model control group,qingkailing ophthalmic gel group and tobradex eye drops group.Normal salt solution was topically administered in the model control group from post-injected day 7 through 13 for 3 times per day,and corresponding drugs were used in the same way in the qingkailing ophthalmic gel group and tobramycin dexamethasone eye drops group.Ocular anterior segment was examined by slit lamp microscope and the inflammatory response was scored.Fourteen days after injected,the rats were sacrificed and eye specimens were prepared for the histopathological examination.The flow cytometry was used to assay the percentages of CD4 + and CD8+T lymphocytes and the ratio of CD4+/CD8+as well as percentages of Th1 and Th17 cells in the lymphocytes suspension of immunomized rat spleen and lymphoglandula.The use and care of the rats complied with Statement of ARVO.Results Ocular inflammatory response appeared in the 9th day and peaked in the 1 1th day after immunomization in the model control group,but later onset and slighter inflammation were seen in the qingkailing ophthalmic gel group and tobramycin dexamethasone eye drops group,with a significant differences in inflammatory scores among the 3 groups (F=26.52,P=0.00).Infiltration of lots of inflammatory cells was visible in the anterior chamber,iris and ciliary body tissues in the model control group,however,the inflammatory cells were obviously decreased in the qingkailing ophthalmic gel group and tobradex eye drops group under the optical microscope.The percentages of CD4+,CD8+ and the ratios CD4+/CD8+ in the lymphocyte suspension were (79.90 ± 0.21) ％,(19.20±0.15) ％ and 4.20 ± 0.04 respectively in the qingkailing ophthalmic gel group,(78.60 ±0.09) ％,(23.44 ± 0.09) ％ and 3.40±0.01 in the tobramycin dexamethasone eye drops group,and they were significantly different with (83.10±0.15)％,(18.60±0.09)％ and 4.50±0.02 in the model control group,with significant differences among the 3 groups (F=223.68,530.77,404.83,all at P=0.00).The percentages of CD4+IFN-γ+ and CD4+IL-17+ in the lymphocyte suspension were (20.40 ± 0.18) ％ and (25.20 ± 0.32) ％ in the qingkailing ophthalmic gel group,(10.40±0.23)％ and (8.20±0.15)％ in the tobramycin dexamethasone eye drops group,which were significantly dropped than (32.20±0.19)％ and (55.10±0.09)％ in the model control group,showing statistically significant differences among the 3 groups (F =2 986.34,12 807.54,both at P =0.00).Conelusions Qingkailing ophthalmic gel can alleviate and treat inflammatory process of rat EAU mainly by inhibiting the differentiation of CD4+ cells,downregulating the ratio of CD4+/CD8+ cells,arresting the differentiation of Th1 and Th17 effector cells and suppressing the secretion of corresponding cytokines.