Study On The Anti-Ulcerative Colitis Of Oxymatrine And The Involvement Of Pyroptosis Mechanism

Background: Ulcerative colitis is a chronic non-specific inflammatory disease of the intestinal tract with unknown mechanism,difficult to cure,and easy recurrence.In recent years,with the development of society,the prevalence of ulcerative colitis has been increasing year by year,which brings the inconvenience to the patients in work and daily life,and also a huge burden to the society.Although the extensive use of 5-aminosalicylic acid and immunosuppressive agents can relieve the patients’ suffereings,due to the unknown mechanism of ulcerative colitis,still there are the problems of large side effects and high recurrence rate.The uncontrolled amplified inflammatory response mediated by the NLRP3 inflammasome signaling pathway plays an important role in the occurrence and development of UC.Studies have shown that OMT can inhibit the NFκB signaling pathway,which is the upstream of the NLRP3 inflammasome signaling pathway,revealing that OMT may play a role in inhibiting pyroptosis.Therefore,present topic aims to investigate the improving effect and the underlying mechanism of oxymatrine on ulcerative colitis on the perspective of inhibiting the activation of NLRP3 inflammasome.Methods: The ulcerative colitis rat model was established by TNBS method;the in vitro pyroptosis model was established by LPS/ATP combination-induced macrophage pyroptosis;the rat colon histopathological change was evaluated by Hematoxylin and Eosin(HE)staining.Apperant and histological changes of TNBS-induced UC rats were investigated by using disease activity index(DAI)score,colonic mucosal injury index(CMDI)and Robarts histopathological score(RHI);The expression and activation levels of NLRP3,ASC,caspase-1,IL-1β and GSDMD in colon tissue of early and post-ulcerative colitis rats by oxymatrine were analyzed by Western blot method;LDH release and PI staining was used to detect the death of macrophages under the ATP stimulation in vitro.Results: Oxymatrine showed obvious improvement on TNBS-induced ulcerative colitis rat model.Compared with the normal group,the DAI index(6.0±0.6 vs 0.3±0.2,p<0.01),CMDI index(5.1±0.7 vs 0,p<0.01)and RHI score(30.2±0.8 vs 3.5±0.9,p<0.01)of the model group <0.01)showed a significant increase.DAI score(2.9±0.6 vs 6.0±0.6,p<0.01,),CMDI score(3.0±0.5 vs 5.1±0.7,p<0.05),RHI score(12.2±2.2 vs.30.2±0.8,p<0.01)were significantly improved with the OMT-treatment,which was consistent with the positive control drug 5-aminosalicylic acid.Six days after TNBS-treatment,the expresssion levels of ASC,IL-1β,and GSDMD in the model group were significantly up-regulated(p < 0.01).But with the 80 mg/kg oxymatrine treatment,the expression levels of NLRP3,caspase-1,and GSDMD were significantly decreased(p<0.01).By studying the expression levels of NLRP3 inflammasome-related proteins in rat colon tissue at different time points after TNBS injection,we found that mature proteins,active-caspase-1,cleaved-IL-1β and GSDMD-N relating to pyroptosis appeared during this proceeding.They peaked 24 hours after modeling and gradually decreased over the course of 24 hours to 6 days.Western blot analysis of these proteins at 8 hours and 24 hours were compared between TNBS-induced UC and 80 mg/kg oxymatrine pretreatment rats.It was found that compared to the UC-modeling,oxymatrine decreased the levels of caspase-1,active-caspase-1,IL-1β and cleaved-IL-1β.In RAW264.7 mouse macrophages,it was found that 3m M ATP could change cell morphology and cause cell death;in contrast,OMT inhibited ATP-induced changes in cell morphology and significantly inhibited ATP-induced peritoneal macrophages.Phage cells and RAW264.7 died(p<0.01);Western blot analysis showed that compared with the ATP-treated group,OMT decreased the protein expression levels of caspase-1,activated active-caspase-1and GSDMD-N.In rat primary peritoneal macrophages,LPS stimulation increased the expression of GSDMD,caspase-1 and IL-1β,and OMT could inhibit the up-regulation of GSDMD and caspase-1 caused by LPS;LPS and ATP co-stimulation,active-caspase-1 and cleaved-IL-1βexpression levels were up-regulated;in contrast,50-250 μM OMT dose-dependently inhibited active-caspase-1 and cleaved-IL-1β levels.Conclusion: Rectal administration of oxymatrine can improve TNBS-induced experimental ulcerative colitis in rats;TNBS-induced experimental rat ulcerative colitis is accompanied by NLRP3 inflammasome activation and cell pyroptosis;Oxymatrine Alkaline administration inhibits NLRP3 inflammasome-mediated pyroptosis in colonic tissue.Oxymatrine can inhibit the pyroptosis induced by LPS/ATP combined stimulation of primary peritoneal mucosal macrophages and RAW264.7 macrophages.In conclusion,pyroptosis is an important pro-inflammatory factor in the occurrence and development of ulcerative colitis.Both in vivo and in vitro experiments have shown that oxymatrine has a significant inhibitory effect on NLRP3 inflammasome-mediated pyroptosis.Oxymatrine can improve ulcerative colitis by inhibiting pyroptosis.