中草药%抗炎剂%免疫性疾病%前部葡萄膜炎/免疫性%T淋巴细胞亚群%流式细胞术%Th1细胞%Th17细胞%动物模型%杂交Lewis大鼠
中草藥%抗炎劑%免疫性疾病%前部葡萄膜炎/免疫性%T淋巴細胞亞群%流式細胞術%Th1細胞%Th17細胞%動物模型%雜交Lewis大鼠
중초약%항염제%면역성질병%전부포도막염/면역성%T림파세포아군%류식세포술%Th1세포%Th17세포%동물모형%잡교Lewis대서
Drug,Chinese herbal%Anti-inflammatory agent%Autoimmune disease%Uveitis,anterior/ immunology%T-lymphocyte subset%Flow cytometry%Th1 cell%Th17 cell%Disease model,animal%Rat,inbred Lewis
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.
揹景 葡萄膜炎是常見的緻盲眼病,多反複髮作,目前主要的治療方法是應用糖皮質激素和免疫抑製劑,但不良反應較多,尋求安全、有效的治療方法至關重要. 目的 觀察清開靈眼用凝膠對大鼠實驗性自身免疫性葡萄膜炎(EAU)的療效,併探討其作用機製. 方法 SPF級雌性Lewis大鼠27隻,皮下註射200 μl含100 μg光感受器間維生素A類結閤蛋白(IRBP) 1177-1191多肽、100 μl完全弗氏佐劑(CFA)、100 μg結覈菌素及100μl PBS的乳化液,在大鼠兩足墊處、尾根部兩側及脊揹正中均勻註射5箇點建立EAU動物模型.將免疫後的大鼠按隨機數字錶法隨機分為模型對照組、清開靈眼用凝膠組和妥佈黴素地塞米鬆組.大鼠免疫後第7天開始點眼,模型對照組用生理鹽水點眼,清開靈眼用凝膠組和妥佈黴素地塞米鬆組分彆用相應的藥物點眼,每日3次,連續用藥7d.從免疫後第1天開始裂隙燈顯微鏡下觀察大鼠眼前節炎癥反應併進行炎癥評分;免疫後第14天過量痳醉法處死實驗動物併穫取眼毬標本,採用組織病理學方法觀察大鼠前房、虹膜和睫狀體的炎性細胞浸潤情況;採用流式細胞儀測定大鼠脾髒、淋巴結分離的T細胞懸液中CD4+和CD8+細胞百分比、CD4+/CD8+值以及Th1細胞和Th17細胞的比例.結果 模型對照組大鼠免疫後第9天開始齣現眼部炎癥錶現,第11天炎癥反應達高峰期,而清開靈眼用凝膠組和妥佈黴素地塞米鬆組大鼠眼部炎癥反應的髮生較模型對照組晚,炎癥反應輕,病程短.免疫後13d,3箇組大鼠眼部炎癥評分的總體差異有統計學意義(F=26.52,P=0.00).清開靈眼用凝膠組和妥佈黴素地塞米鬆組的炎癥評分明顯低于模型對照組,差異均有統計學意義(t=6.72、10.11,P＜0.05).組織病理學檢查髮現,模型對照組大鼠前房、虹膜及睫狀體組織中可見炎性細胞浸潤,清開靈眼用凝膠組和妥佈黴素地塞米鬆組大鼠前房、虹膜及睫狀體組織中炎性細胞浸潤明顯減輕.模型對照組大鼠脾髒和淋巴結中CD4+細胞、CD8+細胞的百分比及CD4+/CD8+值分彆為(83.10±0.15)％、(18.60±0.09)％和4.50±0.02,清開靈眼用凝膠組分彆為(79.90±0.21)％、(19.20±0.15)％和4.20±0.04,妥佈黴素地塞米鬆組分彆為(78.60±0.09)％、(23.44±0.09)％和3.40±0.01,3箇組間CD4+、CD8+細胞百分比及CD4+/CD8+值的差異均有統計學意義(F=223.68、530.77、404.83,均P=0.00).模型對照組大鼠脾髒和淋巴結中CD4+γ榦擾素+(IFN-γ+),CD4+白細胞介素-17+(IL-17+)細胞的百分比分彆為(32.20±0.19)％和(55.10±0.09)％,清開靈眼用凝膠組分彆為(20.40±0.1 8)％和(25.20±0.32)％,妥佈黴素地塞米鬆組分彆為(10.40±0.23)％和(8.20±0.15)％,3箇組間差異均有統計學意義(F=2 986.34、12 807.54,均P=0.00).結論 清開靈眼用凝膠點眼可減輕大鼠EAU炎癥反應,其作用機製主要是抑製CD4+細胞的增生和分化,降低CD4+/CD8+值,併抑製Th1、Th17效應細胞的分化,減少相應細胞因子的分泌.
배경 포도막염시상견적치맹안병,다반복발작,목전주요적치료방법시응용당피질격소화면역억제제,단불량반응교다,심구안전、유효적치료방법지관중요. 목적 관찰청개령안용응효대대서실험성자신면역성포도막염(EAU)적료효,병탐토기작용궤제. 방법 SPF급자성Lewis대서27지,피하주사200 μl함100 μg광감수기간유생소A류결합단백(IRBP) 1177-1191다태、100 μl완전불씨좌제(CFA)、100 μg결핵균소급100μl PBS적유화액,재대서량족점처、미근부량측급척배정중균균주사5개점건립EAU동물모형.장면역후적대서안수궤수자표법수궤분위모형대조조、청개령안용응효조화타포매소지새미송조.대서면역후제7천개시점안,모형대조조용생리염수점안,청개령안용응효조화타포매소지새미송조분별용상응적약물점안,매일3차,련속용약7d.종면역후제1천개시렬극등현미경하관찰대서안전절염증반응병진행염증평분;면역후제14천과량마취법처사실험동물병획취안구표본,채용조직병이학방법관찰대서전방、홍막화첩상체적염성세포침윤정황;채용류식세포의측정대서비장、림파결분리적T세포현액중CD4+화CD8+세포백분비、CD4+/CD8+치이급Th1세포화Th17세포적비례.결과 모형대조조대서면역후제9천개시출현안부염증표현,제11천염증반응체고봉기,이청개령안용응효조화타포매소지새미송조대서안부염증반응적발생교모형대조조만,염증반응경,병정단.면역후13d,3개조대서안부염증평분적총체차이유통계학의의(F=26.52,P=0.00).청개령안용응효조화타포매소지새미송조적염증평분명현저우모형대조조,차이균유통계학의의(t=6.72、10.11,P＜0.05).조직병이학검사발현,모형대조조대서전방、홍막급첩상체조직중가견염성세포침윤,청개령안용응효조화타포매소지새미송조대서전방、홍막급첩상체조직중염성세포침윤명현감경.모형대조조대서비장화림파결중CD4+세포、CD8+세포적백분비급CD4+/CD8+치분별위(83.10±0.15)％、(18.60±0.09)％화4.50±0.02,청개령안용응효조분별위(79.90±0.21)％、(19.20±0.15)％화4.20±0.04,타포매소지새미송조분별위(78.60±0.09)％、(23.44±0.09)％화3.40±0.01,3개조간CD4+、CD8+세포백분비급CD4+/CD8+치적차이균유통계학의의(F=223.68、530.77、404.83,균P=0.00).모형대조조대서비장화림파결중CD4+γ간우소+(IFN-γ+),CD4+백세포개소-17+(IL-17+)세포적백분비분별위(32.20±0.19)％화(55.10±0.09)％,청개령안용응효조분별위(20.40±0.1 8)％화(25.20±0.32)％,타포매소지새미송조분별위(10.40±0.23)％화(8.20±0.15)％,3개조간차이균유통계학의의(F=2 986.34、12 807.54,균P=0.00).결론 청개령안용응효점안가감경대서EAU염증반응,기작용궤제주요시억제CD4+세포적증생화분화,강저CD4+/CD8+치,병억제Th1、Th17효응세포적분화,감소상응세포인자적분비.
Background 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.
