| Objective:To investigate the effects of compound 3,4-methylenedioxy-β-nitrostyrene(MNS)on NLRP3 inflammasome,and to provide experimental basis for the clinical application of MNS and related anti-inflammatory drugs.Methods:1.MNS was applied to trophoblast cells and macrophages at different concentrations(0μM,0.5μM,1μM,5μM,10μM,20μM)for 2 h,and the effect of MNS on cell viability was detected by lactate dehydrogenase assay.2.The safe concentration of MNS was selected to act on different inflammatory body activation models in vitro,and IL-1β was detected by ELISA to explore its effect on each model in vitro.3.Western Blot was used to detect the expression of various proteins in the activation pathway of inflammasome.Cell immunofluorescence was used to detect the formation of ASC spots during the activation of inflammasome,and the effect of MNS on TNF-α secretion was detected by ELISA.4.Mice were induced by l-nitroarginine methyl ester(L-NAME)and treated with MNS after grouping.Changes of blood pressure and urinary protein were recorded in each group.5.Mice enteritis model was induced by sodium dextran sulfate(DSS).After grouping,mice were given MNS treatment.6.HE staining and immunohistochemistry were used to observe the pathological changes of intestinal tissue in mice.Flow cytometry was used to detect Th17 and Th1 infiltration of macrophages and helper T cells in intestinal tissue.The expression of inflammatory factors in intestinal tissues was detected by ELISA.Results:1.MNS concentration in the range of 0.5 ～ 20μM had no significant effect on the activity of trophoblast cells and macrophages,p > 0.05.2.MNS inhibited IL-1β secretion after activation of NLRP3 inflammasome induced by MSU,nigericin,ATP,Silica and in a concentration-dependent manner,with statistical significance(p < 0.05).3.MNS significantly inhibited the formation of ASC spots and caspase-1 and IL-1βsecretion during the activation of inflammasome,p < 0.05;However,there were no significant effects on the expression of NLRP3,Pro-IL-1β,Pro-caspase-1 and ASC proteins,and the phosphorylation of P65 and secretion of TNF-α in NF-κB pathway,p >0.05.4.In L-NAME eclampsia model,MNS did not significantly inhibit blood pressure and urinary protein in mice(p > 0.05);In DSS induced enteritis model,the weight loss of mice after MNS treatment was significantly reduced,and the symptoms of hematochezia were significantly reduced,p < 0.05.5.MNS alleviated pathological injury of intestinal tissue,decreased infiltration of macrophages and pro-inflammatory helper T cell differentiation,and decreased secretion of pro-inflammatory factors,p < 0.05.Conclusion:Compound MNS inhibits the formation of NLRP3-ASC complex,thereby inhibiting the activation of inflammasome and reducing IL-1β secretion.MNS can significantly relieve the intestinal inflammatory response in mice with enteritis and has strong anti-inflammatory effect in vivo. |
