Part Of The Mechanism Of Anti-inflammatory Effects Of Carboxamide Triazole

Background and aimInflammatory bowel diseases(IBD),including ulcerative colitis(UC)and Crohn's disease(CD),is a chronic non-specific inflammatory bowel disease,whose etiology and pathogenesis are still not clear now.In recent years,the incidence of IBD has increased dramatically,and the drugs associated with clinic is facing severe challenges.At present,drugs for the clinical treatment of IBD include aminosalicylates,corticosteroids,antibiotics,probitotics,immunosuppressive agents,biological agents and surgical resection,which all is aim to alleviate the patient's condition,maintain remission,restore and maintain normal intestinal nutrition,and ensure the quality of patients' life to choose best timing of surgery.But all above treatmental methods have their limitations.Therefore,it is of importance to develop new therapeutic drugs for IBD,and explore mechanism of their action.Carboxyamidotriazole(CAI)is a non-cytotoxic anti-tumour drug in development,it can inhibit proliferation and suppress angiogenesis in vitro.CAI can inhibit the extracellular Ca2+ influx,and we discovered that CAI had considerable the anti-inflammmatory potency in a series of acute and chronic inflammatory model,also showed anti-inflammatory effects on autoimmune diseases model during years of our research.CAI could significantly decrease the level of proinflammatory cytokines such as TNF-?,IL-1? and IL-6 in inflammatory sites and serum of inflammatory animal model.Given that the mechanism of its anti-inflammatory is not clear,we did the following two parts studies to find out the mechanism of its anti-inflammatory.The first part is the study on TLR9 in anti-inflammatory mechanism of carboxyamidotriazole.Recently,many scholars believe that IBD is an autoimmune disease.TLRs receptors family act as an important role in the non-specific immune response.It is reported that the activation of TLR9 signal could lead to avtivation of NF-?B signal pathway.Hence,we investigated whether the anti-inflammatory effect of CAI on inflammatory models is associated with the expression of TLR9.It is the second part that effects of carboxyamidotriazole on the activation and degranulation of RBL-2H3 cells.Mast cells are not only the major cells that response to allergic diseases,but also play an important role in many chronic inflammatory diseases.However,there in no mast cells that could be in a serial subcultivation.But rat basophilice leukemia(RBL-2H3)is charactered with many features and functions of mast cells,so the cells are an important model to research mechanisms of associated drugs in vitro in mast cells.We observed the activation and degranulation of RBL-2H3 cells after administration of CAI,and the vitro model was stimulated by compound 48/80.Therefore,we could get the experimental basis for the research and development of CAI.Methods1.Mechanism of TLR9 in anti-inflammatory of carboxyamidotriazoleWe used the intestinal tissues of experimental colitis,rats model induced by TNBS in vivo,RT-qPCR methos tested the mRNA expression of TLR9,TLR7,IRF7 and IRF3,and Western Blot methods detected the protein expression of TLR9,MyD88,IL-1? and TRAF3.We choosed human acute myeloid leukemia cell line THP-1 to do experiments in vitro,which is commonly used in inflammatory cells,then induced THP-1 with PM A to form macrophage model.Give non-methylated oligonucleotides CpG-ODN 2216,a specific stimulation of TLR9,to activate the signaling pathway of TLR9 in macrophage model,while give CpG-ODN 2234 as negative control group.Collect the supernatant of cell culture,ELISA was employed to test the levels of pro-inflammatory cytokines TNF-?,IL-1? and IL-6 and the expression of anti-inflammatory cytokine IL-10.Western Blot methods detected the protein expression of TLR9,MyD88,IL-1? and TRAF3.To validate the hypothesis that the mechanism of CAI on inflammatory model by above detection in cell level.2.Effects of carboxyamidotriazole on the activation and degranulation of RBL-2H3 cellWe tested the activation of RBL-2H3 in release of histamine and ?-glucosidase hexose,also test the proliferation and apoptosis of RBL-2H3 cells after administration of CAI,and the vitro model was stimulated by compound 48/80.Results1.Mechanism of TLR9 in anti-inflammatory of carboxyamidotriazole1.1.The results of qPCR showed that the mRNA level of TLR9,TLR7,IRF7 and IRF3,which were significantly increased in the colonic tissues of TNBS-treated colitis rat,were reduced by CAI treatment.1.2.The results of Western Blot showed that the protein expression of TLR9 significantly increased,which were significantly increased in the colonic tissues of TNBS-treated colitis rat,namely,CAI inhibited TLR9 proteins expressed in the colonic tissues of the model.1.3.THP-1 cell lines were induced by the PMA in the concentration of 200ng/ml,a small amount cells could be differentiated into adherent macrophages with pseudopodia grow induced 24h.And they were basically induced into macrophages after 96h exposed to 200ng/ml PMA.1.4.The results showed that the dose of CAI for THP-1 cells did not significantly have toxic effects on proliferation.1.5.It was dramatically increased that the pro-inflammatory cytokines such as TNF-?,IL-1? and IL-6 and the anti-inflammatory cytokine IL-10,stimulated by CpG ODN 2216 group in macrophage model.The expression of the cytokines decreased significantly after administration of CAI.1.6.Western blot results showed that CAI could inhibit the expression of TLR9 protein,and the level of IL-1? protein also decreased.The down stream molecules of TLR9,MyD88 and TRAF3 decreased as well after administration of CAI compared with THP-1 macrophage stimulated by CpG ODN 2216 group in macrophage model.The expression of the cytokines decreased significantly after administration of CAI.2.Effects of carboxyamidotriazole on the activation and degranulation of RBL-2H3 cell2.1.The neutral red staining showed that the number of cells significantly reduced that were round in the model induced by compound 48/80 after administration of CAI,and most of cells were spindle shaped,the cells of degranulation decreased.Namely CAI had an obvious effect on degranulation of RBL-2H3 cells that was activated by compound 48/80,compared with control group.And the positive control drug,hydrocortisone sodium succinate,had also inhibited degranulation of RBL-2H3 cells.2.2.RBL-2H3 cells had significantly reduced the release of histamine after administration of CAI(20.40?mol/L)in the vitro model that induced by compound 48/80.And the positive control drug,hydrocortisone sodium succinate,had also inhibited release of histamine in RBL-2H3 cells.2.3.The group of CAI 40?mol/L and hydrocortisone sodium succinate both inhibited the release of ?-glucosidase hexose in RBL-2H3 cells that induced by compound 48/80.2.4.The results of CCK-8 method showed the dose of the treatment of CAI and hydrocortisone sodium succinate had no significant effect on viability of RBL-2H3 cells compared with normal control group.2.5.The results of Hoechst 33342 staining method showed the dose of the treatment of CAI and hydrocortisone sodium succinate had no significant effect on apoptosis of RBL-2H3 cells compared with normal control group.ConclusionsCAI could reduce the expression of TLR9 and associated factors in the intestinal tissues of TNBS-induced colitis rat model,and the same effects of THP-1 macrophage model stimulated by CpG ODN 2216.The expression of TLR9 and TLR9 related pathway molecules was inhibited both in level of mRNA and protein.Therefore,therapeutic effect of CAI on TNBS-induced colitis rat model and other inflammatory model may be mediated by the action of TLR9 signaling pathway.CAI can effectively inhibit the activation and degranulation of RBL-2H3 mast cells,and this effect is not through cytotoxicity.The anti-inflammatory effect of CAI may partially due to the down-regulation of mast cell activity.