目的 探讨来氟米特对大鼠自身免疫性葡萄膜炎(EAU)的保护作用.方法 实验研究.建立大鼠光感受器间维生素A类结合蛋白(IRBP)诱导的自身免疫性葡萄膜炎动物模型,按随机数字表法完全随机设计分组为正常组、模型组、来氟米特给药组(3、6、12 mg/kg)、环孢霉素阳性对照组(10 mg/kg),每组6～8只.于造模第2天开始灌胃给药,每日1次,共13d.裂隙灯显微镜观察大鼠EAU的症状,造模后第14天制备大鼠眼球切片,以HE染色法进行组织病理学观察,参照Agarwal的标准对EAU各组大鼠进行临床症状和病理学分级进行评分,免疫组织化学染色(SP法),检测各组大鼠眼组织中IL-17的表达,应用ELISA检测血清中IL-17和干扰素(IFN)-γ含量,使用流式细胞仪检测各组大鼠脾脏CD4+ Th17细胞亚群纯度,使用逆转录-聚合酶链式反应(RT-PCR)检测各组大鼠眼组织IL-17 mRNA和IFN-γmRNA表达水平.计量资料的数据以均数±标准(差((x)±s)表示,组间比较采用t检验.等级资料间比较采用非参数检验,多重比较用Bonferroni方法进行校正.结果 在来氟米特给药组的大鼠中,14d时EAU发病的临床评分均低于模型组,并呈剂量依赖性.与模型组比较[3(3,4)],来氟米特低剂量组[1.5(1,2)]临床评分明显减低,差异有统计学意义(P=0.0006,P＜α',α'=0.05/15],病理学检查显示模型组大鼠眼部表现为重度炎症细胞浸润及视网膜全层破坏、受损,病理评分3(3,4);中剂量组病理评分2(1,3),明显低于模型组,差异有统计学意义(P=0.0014＜α',α'=0.05/15).免疫组织化学检查显示模型组虹膜、睫状体和视网膜部位可见IL-17蛋白表达,来氟米特各剂量组眼部IL-17蛋白表达明显减少.流式细胞检测发现,与正常组相比,模型组大鼠脾脏Th17细胞亚群比例显著升高(8.5％±1.3％vs.0.5％±0.2％;t=8.057,P=0.000＜α',α'=0.05/15),EAU大鼠口服不同剂量的来氟米特作用后,可显著抑制EAU大鼠脏Th17细胞亚群的分化增殖,且呈剂量依赖性,来氟米特低剂量组(4.1％±0.6％vs.8.5％±1.3％;t=6.372,P=0.01＜α',α'=0.05/15),中剂量组(2.8％±0.2％vs.8.5％±1.3％;t=4.49,P=0.002＜α',α'=0.05/15)和高剂量组(1.8％±0.2％ vs.8.5％±1.3％;t=5.743,P=0.000＜α',α'=0.05/15)与模型组比较差异有统计学意义.RT-PCR检测结果发现与模型组比较,来氟米特低剂量组(0.603±0.03 vs.0.787±0.104;t=0.183,P=0.002＜α',α' =0.05/15)、中剂量组(0.535±0.048 vs.0.787±0.104;t=0.252,P=0.000＜α',α'=0.05/15)和高剂量组(0.374±0.051 vs.0.787±0.104;t=0.412,P=0.000＜α',α'=0.05/15)眼组织中IL-17表达水平显著降低,比较差异有统计学意义;来氟米特中剂量组(0.375±0.018 vs.0.427±0.056;t=0.69,P=0.001＜α',α'=0.05/15)和高剂量组(0.367±0.018 vs.0.427±0.056;t=0.077,P=0.000＜α',α'=0.05/15)眼组织IFN-γ表达水平显著降低,比较差异有统计学意义.结论 来氟米特能有效抑制EAU大鼠的眼部葡萄膜炎的炎症反应,减轻虹膜血管充血渗出,减少眼底视网膜炎症细胞浸润损伤;其机制是通过抑制EAU眼组织中IL-17、IFN-γ等炎性因子、抑制大鼠血清中炎性因子的表达而对EAU起保护作用.
目的 探討來氟米特對大鼠自身免疫性葡萄膜炎(EAU)的保護作用.方法 實驗研究.建立大鼠光感受器間維生素A類結閤蛋白(IRBP)誘導的自身免疫性葡萄膜炎動物模型,按隨機數字錶法完全隨機設計分組為正常組、模型組、來氟米特給藥組(3、6、12 mg/kg)、環孢黴素暘性對照組(10 mg/kg),每組6～8隻.于造模第2天開始灌胃給藥,每日1次,共13d.裂隙燈顯微鏡觀察大鼠EAU的癥狀,造模後第14天製備大鼠眼毬切片,以HE染色法進行組織病理學觀察,參照Agarwal的標準對EAU各組大鼠進行臨床癥狀和病理學分級進行評分,免疫組織化學染色(SP法),檢測各組大鼠眼組織中IL-17的錶達,應用ELISA檢測血清中IL-17和榦擾素(IFN)-γ含量,使用流式細胞儀檢測各組大鼠脾髒CD4+ Th17細胞亞群純度,使用逆轉錄-聚閤酶鏈式反應(RT-PCR)檢測各組大鼠眼組織IL-17 mRNA和IFN-γmRNA錶達水平.計量資料的數據以均數±標準(差((x)±s)錶示,組間比較採用t檢驗.等級資料間比較採用非參數檢驗,多重比較用Bonferroni方法進行校正.結果 在來氟米特給藥組的大鼠中,14d時EAU髮病的臨床評分均低于模型組,併呈劑量依賴性.與模型組比較[3(3,4)],來氟米特低劑量組[1.5(1,2)]臨床評分明顯減低,差異有統計學意義(P=0.0006,P＜α',α'=0.05/15],病理學檢查顯示模型組大鼠眼部錶現為重度炎癥細胞浸潤及視網膜全層破壞、受損,病理評分3(3,4);中劑量組病理評分2(1,3),明顯低于模型組,差異有統計學意義(P=0.0014＜α',α'=0.05/15).免疫組織化學檢查顯示模型組虹膜、睫狀體和視網膜部位可見IL-17蛋白錶達,來氟米特各劑量組眼部IL-17蛋白錶達明顯減少.流式細胞檢測髮現,與正常組相比,模型組大鼠脾髒Th17細胞亞群比例顯著升高(8.5％±1.3％vs.0.5％±0.2％;t=8.057,P=0.000＜α',α'=0.05/15),EAU大鼠口服不同劑量的來氟米特作用後,可顯著抑製EAU大鼠髒Th17細胞亞群的分化增殖,且呈劑量依賴性,來氟米特低劑量組(4.1％±0.6％vs.8.5％±1.3％;t=6.372,P=0.01＜α',α'=0.05/15),中劑量組(2.8％±0.2％vs.8.5％±1.3％;t=4.49,P=0.002＜α',α'=0.05/15)和高劑量組(1.8％±0.2％ vs.8.5％±1.3％;t=5.743,P=0.000＜α',α'=0.05/15)與模型組比較差異有統計學意義.RT-PCR檢測結果髮現與模型組比較,來氟米特低劑量組(0.603±0.03 vs.0.787±0.104;t=0.183,P=0.002＜α',α' =0.05/15)、中劑量組(0.535±0.048 vs.0.787±0.104;t=0.252,P=0.000＜α',α'=0.05/15)和高劑量組(0.374±0.051 vs.0.787±0.104;t=0.412,P=0.000＜α',α'=0.05/15)眼組織中IL-17錶達水平顯著降低,比較差異有統計學意義;來氟米特中劑量組(0.375±0.018 vs.0.427±0.056;t=0.69,P=0.001＜α',α'=0.05/15)和高劑量組(0.367±0.018 vs.0.427±0.056;t=0.077,P=0.000＜α',α'=0.05/15)眼組織IFN-γ錶達水平顯著降低,比較差異有統計學意義.結論 來氟米特能有效抑製EAU大鼠的眼部葡萄膜炎的炎癥反應,減輕虹膜血管充血滲齣,減少眼底視網膜炎癥細胞浸潤損傷;其機製是通過抑製EAU眼組織中IL-17、IFN-γ等炎性因子、抑製大鼠血清中炎性因子的錶達而對EAU起保護作用.
목적 탐토래불미특대대서자신면역성포도막염(EAU)적보호작용.방법 실험연구.건립대서광감수기간유생소A류결합단백(IRBP)유도적자신면역성포도막염동물모형,안수궤수자표법완전수궤설계분조위정상조、모형조、래불미특급약조(3、6、12 mg/kg)、배포매소양성대조조(10 mg/kg),매조6～8지.우조모제2천개시관위급약,매일1차,공13d.렬극등현미경관찰대서EAU적증상,조모후제14천제비대서안구절편,이HE염색법진행조직병이학관찰,삼조Agarwal적표준대EAU각조대서진행림상증상화병이학분급진행평분,면역조직화학염색(SP법),검측각조대서안조직중IL-17적표체,응용ELISA검측혈청중IL-17화간우소(IFN)-γ함량,사용류식세포의검측각조대서비장CD4+ Th17세포아군순도,사용역전록-취합매련식반응(RT-PCR)검측각조대서안조직IL-17 mRNA화IFN-γmRNA표체수평.계량자료적수거이균수±표준(차((x)±s)표시,조간비교채용t검험.등급자료간비교채용비삼수검험,다중비교용Bonferroni방법진행교정.결과 재래불미특급약조적대서중,14d시EAU발병적림상평분균저우모형조,병정제량의뢰성.여모형조비교[3(3,4)],래불미특저제량조[1.5(1,2)]림상평분명현감저,차이유통계학의의(P=0.0006,P＜α',α'=0.05/15],병이학검사현시모형조대서안부표현위중도염증세포침윤급시망막전층파배、수손,병리평분3(3,4);중제량조병리평분2(1,3),명현저우모형조,차이유통계학의의(P=0.0014＜α',α'=0.05/15).면역조직화학검사현시모형조홍막、첩상체화시망막부위가견IL-17단백표체,래불미특각제량조안부IL-17단백표체명현감소.류식세포검측발현,여정상조상비,모형조대서비장Th17세포아군비례현저승고(8.5％±1.3％vs.0.5％±0.2％;t=8.057,P=0.000＜α',α'=0.05/15),EAU대서구복불동제량적래불미특작용후,가현저억제EAU대서장Th17세포아군적분화증식,차정제량의뢰성,래불미특저제량조(4.1％±0.6％vs.8.5％±1.3％;t=6.372,P=0.01＜α',α'=0.05/15),중제량조(2.8％±0.2％vs.8.5％±1.3％;t=4.49,P=0.002＜α',α'=0.05/15)화고제량조(1.8％±0.2％ vs.8.5％±1.3％;t=5.743,P=0.000＜α',α'=0.05/15)여모형조비교차이유통계학의의.RT-PCR검측결과발현여모형조비교,래불미특저제량조(0.603±0.03 vs.0.787±0.104;t=0.183,P=0.002＜α',α' =0.05/15)、중제량조(0.535±0.048 vs.0.787±0.104;t=0.252,P=0.000＜α',α'=0.05/15)화고제량조(0.374±0.051 vs.0.787±0.104;t=0.412,P=0.000＜α',α'=0.05/15)안조직중IL-17표체수평현저강저,비교차이유통계학의의;래불미특중제량조(0.375±0.018 vs.0.427±0.056;t=0.69,P=0.001＜α',α'=0.05/15)화고제량조(0.367±0.018 vs.0.427±0.056;t=0.077,P=0.000＜α',α'=0.05/15)안조직IFN-γ표체수평현저강저,비교차이유통계학의의.결론 래불미특능유효억제EAU대서적안부포도막염적염증반응,감경홍막혈관충혈삼출,감소안저시망막염증세포침윤손상;기궤제시통과억제EAU안조직중IL-17、IFN-γ등염성인자、억제대서혈청중염성인자적표체이대EAU기보호작용.
Objectives To investigate the protective efficacv of]eflunomide on the Lewis rats with experimental autoimmune uveitis (EAU).Methods Complete randomized controlled trials research.Lewis rats were immunized with interphotoreceptor retinoid-binding peptide (IRBP) in order to build the model of EAU.Rats were randomized assigned into four groups, that is control group as A, model group without leflunomide as B, model group with leflunomide administrations as C, and model group with cyclosporine A as D.Rats in group C received intragastric administration of three doses of leflunomide at 3mg/kg/d;6mg/kg/ d;12mg/kg/d.Rats in group D received 10 mg/kg cyclosporin A were considered as a positive control.Each group has 6 to 8 rats.At the second day of immunization with IRBP, rats were intragastric administrated one time every day till day 13.Rats were investigated for EAU symptoms under slit lamp.Enucleated eyes were collected for sections with HE staining as histopathological evidences at the peak point of disease activity day 14.Treatment effectiveness was evaluated referred by Agarwal standard for clinical EAU and histopathological scoring.The expression of IL-17 in ocular sections was detected by immunohistochemistry (SP method).The expression levels of IL-17 and IFN-γ in the serum were quantified by ELISA.Intracellular expression of IL-17 in the activated CD4+T cells was assessed by flow cytometry.Ocular of rats were harvested and mRNA expression of IL-17 and IFN-γ were quantified through RT-PCR.Continuous variables were reported as mean ±SD.The comparison among groups was done by using analysis of students't test.Nonparametric test was used in Hierarchical data comparison and multiple comparison method was Bonferroni.Results The model of EAU disease was built successfully in Lewis rats.With giving IRBP for 14 days, the clinic EAU scores were lower in model rats than those without leflunomide.Moreover, the effects of leflunomide on the clinic EAU scores was dose-dependent.Comparing to vehicle-treated eyes, treatment with leflunomide significantly prevented the onset of EAU-induced ocular inflammation [1.5 (1,2)vs.3 (3,4), P=0.0006, P＜α', α'=0.05/15].The pathological examination showed model rats eye characterized by severe inflammatory cells infiltration and all layers of retina damaged.The pathologic grade was significant higher in model group than in medium dose leflunomide.[3(3, 4) vs.2(1,3), P=0.0014, P＜α', α'=0.05/15].IL-17 was positively expressed in iris, ciliary and retina in model group.While, it was markedly reduced in leflunomide-treated eyes.Flow cytometry detection found that compared with normal group, Th 17 cells rates in rats' spleen of model group also increased significantly (8.5％± 1.3％ vs.0.5％±0.2％;t=8.057, P=0.000, P＜α', α'=0.05/15).Compared with model group, Th17 cells in spleen of rats in leflunomide groups showed a decreased number by flow cytometry.And it showed dosage dependent.It was significant different between different doses leflunomide treated group compared with control group.The results showed as below, in low dose group (4.1％±0.6％ vs.8.5％±1.3％;t=6.372, P=0.01, P＜α', α'=0.05/15), in medium dose group (2.8％±0.2％ vs.8.5％± 1.3％;t=4.49, P=0.002, P＜α', C=0.05/15) and in high dose group (1.8％±0.2％ vs.8.5％± 1.3％;t=5.743, P=0.000, P＜α', α'=0.05/15).Gene expression of IL-17 and IFN-γ were markedly reduced in leflunomide-treated eyes.Leflunomide significantly decreased the serum levels of IL-17 and IFN-γ.Compared with model group, in leflunomide-treated low dosage group (0.603 ±0.03 vs.0.787 ±0.104;t=0.183, P=0.002, P＜α', α'=0.05/15), medium dosage group (0.535±0.048 vs.0.787±0.104;t=0.252, P=0.000, P＜α', α'=0.05/15) and high dosage group (0.374±0.051 vs.0.787±0.104;t=0.412, P=0.000, P＜α', α'=0.05/ 15), IL-17 mRNA showed lower expression.Moreover, IFN-γ mRNA in the tissue of EAU eyes were suppressed by medium dosage leflunomide group (0.375±0.018 vs.0.427±0.056;t=0.69, P=0.001, P＜α', α'=0.05/15) and high leflunomide dosage group respectively (0.367±0.018 vs.0.427±0.056;t=0.077, P=0.000,P＜α', α'=0.05/15).The difference was statistically significant.All the results suggested that IL-17, which was secreted by Th17 cell, a subtype of T lymphocytes, might play an important role in the pathogenesis of uveitis.Conclusions Oral administration of leflunomide effectively suppressed IRBP-induced uveitis in rats, not only reduced exudation in iris but also alleviated the infiltration damage of inflammation cells in fundus.It might be ascribed to the effect that leflunomide could treat inflammation by down-regulating the expressions of IL-17 and IFN-γ.Therefore, it suggested that leflunomide had protective effects against EAU in Lewis rats.
