The Pharmacological Effects And Mechanism Of CAI On Rat Colitis

Background and aimsInflammatory bowel diseases (IBD), which comprised of Crohn’s disease (CD) and ulcerative colitis (UC), are chronic and spontaneously relapsing inflammatory diseases. The worldwide incidence and mortality of IBD are increasing, while its etiology and mechanism have not been fully understood yet. At the present stage, there are four major therapeutic drugs for IBD, including amino acid, corticosteroids, immunosuppressive agents and various biological agents. However, prolonged use of these medications can cause significant side effects. Besides, many of them are effective in most case but unsustainable for their toxicities. Therefore, potent new agents are urgently needed. Carboxyamidotriazole (CAI) is a non-cytotoxic anti-tumour drug in development, it can inhibit proliferation and induce apoptosis of several tumour cell lines in vitro and in vivo models. Recently, we discovered that CAI also had considerable the anti-inflammatory potency in a variety of animal models of acute and chronic inflammatory diseases and autoimmune diseases. And the anti-inflammatory effect of CAI was associated with its ability to decrease the levels of pro inflammatory cytokines. Considering that excess proinflammatory cytokines owing to the activation of NF-κB and NLRP3 inflammasome play a key role in IBD, we investigated the therapeutic effects of CAI in 2,4, 6-trinitrobenzene sulfonic acid (TNBS)-induced rat colitis and the involvement of CAI action with NF-κB and NLRP3 inflammasome.MethodsThe TNBS-induced colitis rat model was used. The following criteria need to be recorded daily:body weight, stool change, and presence of hemafecia. The severity of colitis was assessed by clinical, macroscopic, microscopic and biochemical investigations, such as the activities of myeloperoxidase (MPO) and the level of lipocalin-2. ELISA was employed to test a series of cytokines in serum and colon homogenate, including:tumor necrosis factor (TNF)-a, interleukin (IL)-1β, IL-6, IL-10, IL-17A, IL-18, and transforming growth factor (TGF)-p. RT-qPCR was used to determine the mRNA levels of NLRP3, ASC and caspase-1. Western blot and immunohistochemistry were also utilized in determining the protein expressions of NLRP3 inflammasome components and NF-κB pathway factors, including NF-κB p65, IκBα, phosphorylated IκBα, NLRP3, ASC, pro-caspase-1, caspase-1, precursors and mature forms of IL-1β and IL-18.Results1. In Sprague Dawley rats, TNBS dissolved in ethanol (50:50 vol/vol) at a dose of 80 mg/kg body weight was sufficient to produce pathological changes similar to that seen in human CD. The phenotype of the model was characterized by intestinal stenoses, bowel wall thickening, mucous hyperemia, edema, erosion and ulceration in colon, as well as inflammatory cell (mainly neutrophils) infiltration, crypt abscess and goblet cell depletion.2. CAI (10,20 and 40mg/kg) significantly reduced weight loss and disease activity index (DAI) scores in colitis rats. CAI (20,40mg/kg) could ameliorate splenomegalia and colonic shortening, and reduce the macroscopic and histological scores. CAI (40mg/kg) could alleviate colonic weight increase.3. The rats treated with CAI (20,40mg/kg) showed alleviated enteremia, nodule and edema in colon and diminished mucous membrane lesions in macroscopic examination. Microscopy results revealed less severe telangiectasis, neutrophil infiltration, and goblet cell depletion and crypt disorganization compared with PEG 400 group.4. Compared with normal control group, TNBS-induced rats had an increased MPO activity in colonic tissues and a higher level of lipocalin-2 in serum. The FITC-dextran level in serum of PEG 400-treated colitis rats was much higher than that in the serum of the normal control rats, suggesting that the intestinal permeability increased. While CAI evidently decreased the MPO activity, the serum lipocalin-2 level, and the serum FITC-dextran level. The reduced FITC-dextran level indicated a significant resolution of epithelial integrity.5. Compared with normal control rats, TNBS-induced rats had significant high levels of TNF-α, IL-1β, IL-6, IL-10, IL-17A and IL-18 in their serum. The level of TGF-β in serum and colon homogenate also increased. CAI treatment can palliate these elevations.6. Western blot results showed that the expressions of pro-IL-1β, mature IL-1β p17, pro-IL-18 and mature IL-18 p18 were significantly increased in the colonic tissues from PEG 400-treated colitis rats when compared with those obtained from the normal rats. The administration of CAI inhibited the expressions of both the precursor and mature forms of IL-1β and IL-18.7. Immunohistochemical results showed that the NF-κB p65 subunit expression in the normal colon was in a low level and mainly located in the cytoplasm. However, the expression of that of PEG 400-treated colitis rats was much higher than that of the normal control and particularly located in the cell nucleus. The administration of CAI (20,40 mg/kg) significantly decreased the NF-κB p65 staining in the colonic tissues.8. Western blot results showed that in TNBS-induced colonic tissues the level of p-IκBα significantly increased, while the level of IκBα was obviously reduced. However, the degradation of IκBαand elevation of p-IκBαinduced by TNBS can be significantly inhibited by CAI.9. RT-qPCR results show that NLRP3 and caspase-1 mRNA levels, which were significantly increased in the colonic tissues of PEG 400-treated colitis rats, were reduced by CAI treatment. And no change in mRNA level of ASC between all groups was detected.10. Western blot and immunohistochemistry results show that NLRP3 and caspase-1 levels were up-regulated in PEG 400-treated colitis rats and they were suppressed by the treatment of CAI (20,40 mg/kg). However, ASC expression had no differences between all groups.ConclusionsCAI is proven to possess therapeutic action against TNBS-induced colitis in rats. Oral administration of CAI can alleviate the severity of experimental colitis, suppress the mucosal inflammation, and protect against the loss of intestinal integrity. The anti-inflammatory effect of CAI is associated with its ability to decrease the pro inflammatory cytokines, which was supported by the observation that CAI significantly reduced the levels of TNF-a, IL-1β, IL-6, IL-17A and IL-18 at the inflammatory sites and in serum of the experimental animals. In addition, CAI also had a downward effect on the expression level of TGF-β, which is a mediator of collagen fiber accumulation in colon, suggesting that CAI has anti-fibrosis effect. Moreover, CAI can inhibit the activation of the NF-κB signaling pathway and NLRP3 inflammasome, so as to reduce the inflammatory damage of the intestinal tract and improve the clinical symptoms. Hence, we highlight CAI, which is a promising oral available agent, a potential drug candidate for the treatment of human IBD.