Abstract
BackgroundTo investigate the effects and potential mechanism of L161982 (a kind of EP4 antagonist) on the collagen-induced arthritis (CIA) mice model.MethodsThe CIA mice model were first established by immunizing with Chicken Type II Collagen on DBA/1 mice. The CIA groups were administered once a day for 2 weeks with either 5 mg/kg L161982 by intraperitoneal injections (IP), 200 U celecoxib by intragastrical injections, or 100 μl PBS (IP). At the end of the study, total arthritis score and histopathologic examination were assessed to determine CIA severity. The plasma and tissue expressions of IL-17 and monocyte chemoattractant protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) and Immunohistochemical staining (IHC) respectively; The number of CD4+CD25+Foxp3+ regulatory T cells (Treg) determined as a proportion of total CD4+ cells in the lymph nodes and spleen. We also tested the proliferation of isolated Tregs and the ratio of Th17 polarization of Naïve T cells under the treatment of L161982 by BrdU assay and flow cytometry respectively.ResultsCIA mice treated with L161982 showed reduced arthritis scores, joint swellings, cracked cartilage surface, and less hyperplasia in the connective tissue of the articular cavity. Plasma and tissue IL-17 and MCP-1 decreased, while the proportion of Treg cells is increased both in the spleen and lymph nodes of CIA mice. Otherwise, L161982 have no direct effect on Tregs proliferation; a decreased tendency of Th17 polarization in vitro were observed in L161982-treated naïve T cells.ConclusionAlthough less effective than Celecoxib, L161982 also resulted in a reduction of ankle joint inflammation in CIA mice. L161982 reduces the RA severity in CIA mice through inhibition of IL-17 and MCP-1, increasing Treg cells, and reducing inflammation. The mechanism of the reduction of IL-17 in plasma or tissue after administration of L161982 might be potentially derived from the suppression of CD4+ T cells differentiation into Th-17 cells.
Highlights
IntroductionTo investigate the effects and potential mechanism of L161982 (a kind of EP4 antagonist) on the collagen-induced arthritis (CIA) mice model
To investigate the effects and potential mechanism of L161982 on the collagen-induced arthritis (CIA) mice model
The following reagents were used in this study: EP4 receptor antagonist L161982 (N-[[4′-[[3Butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-4-yl]methyl] [1,1′-biphenyl]-2-yl]sulfonyl]-3-methyl-2-thiophenecarboxamide) (Tocris, UK); Chicken Type II Collagen (Sigma, USA); Celecoxib, prostaglandin E2 (PGE2) and BrdU Cell Proliferation enzyme-linked immunosorbent assay (ELISA) Kit (Abcam, USA); Dimethyl sulfoxide (DMSO, Sigma, USA); Freund’s Complete Adjuvant (Chondrex, USA); Interleukin-17 (IL-17), monocyte chemoattractant protein-1 (MCP-1), and ELISA kit; Mouse antibodies: FITC-anti-CD4, PE-anti-CD25, PeCY5-anti-Foxp3, PE-CD62L and PeCY5anti-IL-17, anti-IL-17, anti-MCP-1, anti-cleaved-caspas 3, soluble anti-CD3 and soluble anti-CD28; EasySep mouse CD4+CD62L+ naïve T cells isolation kit (Milenyi biotec, USA); Th17 cells inducement: mouse TGF-β1, IL-6, IL-23, anti-IFNγ and anti-IL-4 (Milenyi biotec, USA)
Summary
To investigate the effects and potential mechanism of L161982 (a kind of EP4 antagonist) on the collagen-induced arthritis (CIA) mice model. The activation of naïve helper T cells (Th0) can differentiate into a variety of phenotypes depending on cytokine environment: Th1, Th17, Th2, and regulatory T cells (Treg) [5]. In the pathogenesis of RA, the imbalanced secretion of cytokines results in increased inflammatory mediators, including the arachidonic acid metabolite prostaglandin E2 (PGE2) [10]. PGE2 has been identified as having an immunoregulatory role in the differentiation of Th1 and Th2 cells [11, 12]. The activation of the EP4 receptors [10] may promote both inflammatory or anti-inflammatory effects, with an inflammatory role in Th17 cell-dependent diseases [13]. EP4 knock-out mice have been found to be resistant to type-II collagen antibodyinduced arthritis [14]
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