title: [Effects of leflunomide on experimental autoimmune uveitis in Lewis rats].
abstract: OBJECTIVE
  To investigate the protective efficacy of leflunomide 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, Th17 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', α' = 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.
authors.affiliation: Department of Ophthalmology, the 1st Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
authors.forename: Chengbo
authors.initials: C
authors.lastname: Fang
authors.forename: Dexi
authors.initials: D
authors.lastname: Zhou
authors.forename: Shuxiang
authors.initials: S
authors.lastname: Zhan
authors.forename: Yong
authors.initials: Y
authors.lastname: He
authors.forename: Zhen
authors.initials: Z
authors.lastname: Lin
authors.forename: Cheng
authors.initials: C
authors.lastname: Huang
authors.affiliation: Email: Lj@ahmu.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D001327:Autoimmune Diseases / Q000139:chemically induced / Q000517:prevention & control*; D016572:Cyclosporine / Q000627:therapeutic use; D015536:Down-Regulation; D005136:Eye Proteins; D007166:Immunosuppressive Agents / Q000627:therapeutic use*; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D020381:Interleukin-17 / Q000235:genetics / Q000378:metabolism*; D007555:Isoxazoles / Q000627:therapeutic use*; D000077339:Leflunomide; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D011917:Rats, Inbred Lew; D012173:Retinitis / Q000188:drug therapy / Q000473:pathology; D012177:Retinol-Binding Proteins; D058504:Th17 Cells; D014605:Uveitis / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D016572:Cyclosporine; D005136:Eye Proteins; D007166:Immunosuppressive Agents; D007371:Interferon-gamma; D020381:Interleukin-17; D007555:Isoxazoles; D000077339:Leflunomide; D012333:RNA, Messenger; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial