[Effects of leflunomide on experimental autoimmune uveitis in Lewis rats].

OBJECTIVES: 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.

TGF-ß1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-ß1/Smad pathway.

Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-ß1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-ß1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-ß1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-ß1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-ß1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-ß1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.

Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways.

Monocytes/macrophages are heterogeneous and versatile cells that could undergo their phenotypically/functionally dynamic switch in response to the microenvironment signals. Two major macrophage subpopulations with different functions which represent extreme of a continuum in a universe of activation states, including classically activated/inflammatory (M1) and alternatively activated/regenerative (M2) macrophages, have long been recognized. Emerging evidence through genetic or pharmacologic approaches has now been made in defining the actual fate in vivo and in vitro underlying M1 or M2-like polarized activation under physiological and pathological conditions. These cells are characterized by their expression of cell surface markers, secreted cytokines and chemokines, and transcription and epigenetic pathways. Here in this review, we shed new light on the contribution of several major signaling pathways and their modulators/targets involved in directing the macrophage plasticity and polarized function, assess the mechanisms of macrophage polarization by interacting endogenous cellular mechanisms and molecules associated with reciprocal skewing of macrophage polarization between the M1 and M2 states. The identification of mechanisms underlying functional polarization of macrophages into M1 or M2 cells might provide new insights into a basis for macrophage-centered diagnostic and therapeutic strategies for multiple diseases.

TRPM7 channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3K and ERK pathways.

TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl4-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increase of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.

Amelioration of experimental autoimmune uveitis by leflunomide in Lewis rats.

PURPOSE: To investigate the efficacy of leflunomide in experimental autoimmune uveitis (EAU) in rats. METHODS: Lewis rats were immunized with interphotoreceptor retinoid-binding peptide (IRBP) in order to generate EAU. Rats received three dose of leflunomide through intragastric administration (prevention or treatment protocols) after immunization at three separate doses (3 mg/kg/d; 6 mg/kg/d; 12 mg/kg/d). Cyclosporin A was administered as a positive) control. Rats were euthanized during peak disease activity (day 14 or 15). Treatment effectiveness was evaluated in vivo using clinical EAU scoring (d14) and histopathological evaluation of enucleated eyes after experimental termination. The expression levels of inflammatory cytokines in the serum were quantified by ELISA. Eyeball of rats were harvested and mRNA expression of interleukin 17 (IL17) and IFN-γ were quantified through RT-PCR. Intracellular expression of interleukin (IL)-17 in the activated CD4(+) T cells was assessed by flow cytometry. The effects of leflunomide inhibition on immune responses in rats were investigated in isolated lymphocytes. RESULTS: Histopathological and clinical data revealed severe intraocular inflammation in the immunized rat. Inflammation reached its peak on day 14 in this EAU model. Treatment with leflunomide significantly prevented and treated EAU-induced ocular inflammation and decreased clinical and pathological scores compared to vehicle-treated eyes. Gene expression of IL17 and IFN-γ was markedly reduced in leflunomide-treated eyes. Leflunomide significantly decreased the serum levels of IL17 and IFN-γ. The study of IL17+ T cells in peripheral blood and spleen by flow cytometry showed a decreased number of Th17 cell in rats of leflunomide prevented group. Lymphocytes from animals treated with leflunomide had decreased antigen-specific proliferation in vitro compared with lymphocytes from untreated animals. CONCLUSIONS: Oral administration of leflunomide effectively suppressed IRBP-induced uveitis in rats. These results suggest that leflunomide may be potentially clinical application in uveitis.