Carboxyamidotriazole Protects Against Experimental Colitis By Inhibiting NF-?B And NLRP 3 Inflammasome

Background and aimsThe etiology and mechanism of two major chronic inflammatory bowel diseases(IBD),Crohn,s Disease(CD)and Ulcerative Colitis(UC)is still obscure.In the last few decades,basic and clinical studies have shownthat increased expression of cytokines like TNF-a were closely associated with autoimmune disease—inflammatory bowel disease.Animal models,cytological research,epidemiological studies and clinical trials suggested that TNF-a inhibitorsmay reduce the risk of IBD.It made TNF-ainhibitorsattracted considerable attention.All the TNF-ainhibitors including TNF-amonoclonal antibodies and water-soluble TNF-areceptorsthat were approved were biological productstill the present moment.Although the effectiveness and safety of TNF-ainhibitors have been confirmed yet,they did have some defects.For example,as a protein preparation,injection treatment is the common way to administer TNF-ainhibitors.Furthermore,long-term use of TNF-ainhibitors can cause the produce of antibody and other related problems,especially the cost of drugs were ultra high.A new promising drug still pressed for the treatment of IBD.Carboxyamidotriazole is a kind of non-cytotoxic anti-cancer drug,which is in its preclinical experiments period.Proliferation of tumor cells and antiogenesis can be inhibited by CAI;it can also induce apoptosis of several tumor cell lines in vivo and in vitro.In recent years,our lab found that CAI has potent effect on anti-inflammatory in a variety of animal models which were having acute and chronic inflammatory diseases,especially on decrease the expression levels of some pro-inflammatory cytokines,involving IL-1??TNF-??IL-6,which were played critical role in inflammatory bowel disease pathogenesis.It suggests us that CAI may potentially have effect on curing IBD and the mechanism of treatment effects may associated with activation of NF-?B pathway and NLRP3 inflammasome.Both pathways play crucial roles in the pathogenesis of IBD.Thus,related in vivo and in vitro models were built to help us further understanding the molecular mechanisms of IBD therapeutic efficacy of CAI.MethodsWe used the widely applied models of dextran sodium sulfate(DSS)-induced colitis and LPS-induced THP-I to confirm the novel pharmacological effect of carboxyamidotriazole both in vivo and in vitro.Once DSS-induced acute colitis mice model was build,mice should be monitored daily for body weight,shapes of stool and presence of blood in the stool.Evaluated the severity of disease with DAI(disease activity index)based on the data we recorded daily,scored the pathological changes of colonic tissue,made histological staining(H.E)and analogized the H.E stained colon sections.The activity of myeloperoxidase(MPO)in colonic tissue,the level of lipocalin-2 and the fluorescence intensity of FITC in serum were detected to evaluate the therapy of drugs.ELISA was used to measure the expression levels of IBD-related cytokines in colonic homogenates,such as IL-1?,IL-18,TNF-?,IL-6,IL-10,IL-17and TGF-p.Western-blot and immunohistochemical staining were employed to determine the expression levels of NF-?B pathway-associated proteins,including NF-?B p65?I?B? and phosphorylated I?B? and NLRP3 inflammasome components and related cytokines,including NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1? and IL-18.Furthermore,q-PCR can help test the mRNA levels of NLRP3 inflammasome complex(NLRP3.ASC and caspase-1).Human acute monocytic leukemia THP-1 cell line was chosen to be used in this study.Using phorbol 12-myristate 12-acetate(PMA)to differentiate THP-1 cells and treated with LPS in the absence or presence of CAI to evaluate the inflammatory response.ELISA was employed to test the secretion levels of some cytokines in cellar supernatant,including TNF-??IL-1? and IL-18 which were related to whether the model was successfully build and the anti-inflammatory effect in vitro.Western-blot was utilized in determing the protein expressions of NLRP3 components and NF-?B pathway factors,including NF-?B p65,I?B?,phosphorylated I?B?,NLRP3,ASC,pro-caspase-1 and caspase-1.The mechanism that CAI acted on IBD therapy can be confirmed by the results of these detections.Results1.A DSS concentration of 2%(w/v)in the drinking water for 7 days induces strong colitis,but low mortality rates.The phenotype of the DSS-induced mice was characterized by shorten intestinal tract,Intestinal wall thinning,increased intestinal permeability,severe bleeding,mucous hyperemia,edema,erosion and ulceration in gut,increased inflammatory cell infiltration in intestinal mucosa,crypt abscess and goblet cell depletion.2.Orally administrate CAI can significantly attenuate clinical symptoms such as weight loss,bloody stools,diarrhea,and decrease disease activity index scores in experimental colitis.3.On the day of sacrifice,the mice treated with CAI(20mg/kg,40mg/kg)showed alleviate pathological changes,such as enteremia,edema,ulceration in colon.4.CAI can protect mice against inflammation by significantly decrease activity of MPO in colonic tissue,intestinal permeability and levels of lipocalin-2 in serum.The activity of MPO and the inflammatory marker lipocalin-2 were both detected by ELISA kits.Measure intestinal permeability,which correlates with fluorescence intensity of serum which associated with amount of FITC-dextran in mouse serum.5.Histological analysis of hematoxylin/eosin staining colon sections showed that the mice treated with CAI have improving DSS-induced pathology,including mucin and goblet cell depletion,epithelial erosion,crypt disorganization,neutrophil infiltration and ulceration.6.Compared with normal group,DSS-induced mice have markedly high levels of proinflammatory cytokines including IL-1?,IL-18,TNF-a,IL-6,IL-10,IL-17 and TGF-?.Administration of CAI can decrease the secretion of those cytokine in colonic tissue.And we assayed the cytokines by ELISA kits.7.Compared with normal group,Pro-IL-1?,pro-IL-18 and mature IL-1?,IL-18 were highly expressed in colonic tissues in solvent control mice.Treated with CAI can reduce the expression of both the precursor and mature forms of IL-1? and IL-18.And we use Western blot to measure the expression of these proteins.8.We use immunohistochemical staining to evaluate the expression of NF-?B p65 subunit.It shows that NF-?B p65 subunit keeps in a low level and mostly located in the cytoplasm in normal group,whereas the solvent control mice showed express relatively high level of NF-?B p65 and particularly located in the nucleus.Treated with CAI can significantly light the staining and decreased the expression of NF-?B p65.9.Compared to the vehicle-treated group,CAI can significantly decrease the level of p-I?B? and protect I?B? from being degraded.10.q-PCR was used to test the mRNA levels of NLRP3 components,unsurprisingly the levels of NLRP3 and caspase-1 were significantly high in vehicle-treated group,and be decreased by CAI treatment.There were no differences in expression of ASC among all the groups.11.In vehicle-treated group NLRP3 was up-regulated and caspase-1 was highly activated,both can be inhibited by the treatment with CAI;nevertheless ASC expression had no difference among all the seven groups.We use both western blot and immunohistochemical staining to assay the expression of NLRP3 components.12.CAI has no direct toxic effects on THP-1 cells because there has no evidence showed that CAI can inhibit the cell proliferation.In the study,we use both CCK-8 kit and SRB to detect the situation of the cell proliferation.13.Our in vitro study demonstrated that CAI reduced TNF-??IL-1? and IL-18 production whereas LPS can stimulate highly secretion of those cytokines.14.Western blot examined the levels of p-I?B? and I?B? protein in each group.Observation showed that in LPS stimulated THP-1 cells the level of p-I?B? was highly increased and the level of I?B? was obviously reduced.The degradation of p-I?B? and increased I?B? can be markedly inhibited by CAI in vitro.15.The activation of caspase-1 and the expression of NLRP3 protein can be inhibited by the treatment of CAI,however,the expression of ASC showed no difference among all the six groups in vitro.We detected all the proteins using western blot.ConclusionsOrally administered CAI can markedly attenuate the situation of weight loss,food consumption,clinical assessments,macroscopic scores and histopathological score.CAI can ameliorateintestinaltract erythematous and walls thinning,decrease theareaofdefect.The coumpound can also alleviateintestinal mucous membrane impairment,telangiectasis,follicular hyperplasia,edema and crypt destruction through an observation on the histological changes of the colon.The activity of MPO in tissue homogenate and expression level of lipocalin-2 in animal serum was significantly reduced after administration of CAI.CAI can significantly reduced the secretion of pro-inflammatory cytokines,specifically decreased levels of TNF-??IL-1??IL-6 and IL-18,down-regulates the activity of NF-?B signaling pathway in inflammatory sites of the experimental animals and supernatant of cells.Furthermore,experiments models both in vivo and in vitro illustrated its potential mechanism by inhibiting NF-?B signaling pathway and NLRP3 inflammasome activation.The study suggested that CAI might act as an effective candidate for colitis and its mechanism may related to inhibiting NF-?B signaling pathway and NLRP3 inflammasome activation,reducing the over expression of pro-inflammatory cytokines.