Study On Anti-inflammatory Activity And Mechanism Of Brevilin A Based On NLRP3 Inflammasome

Brevilin A（BA）is a kind of sesquiterpene lactone isolated from Centipeda minima with anti-tumor,anti-bacterial,anti-neuroinflammatory,antioxidant and other pharmacological activities.NLRP3 inflammasome is currently the most widely studied inflammasome,which abnormal activation can lead to a variety of inflammatory diseases.Targeting NLRP3inflammasome is essential to prevent host damage and excessive inflammation.Studies have shown that BA can inhibit the activation of NF-κB and oxidative stress in a dose-dependent manner,thereby alleviating the inflammatory response in nerve cells.However,whether BA can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasomes has not been reported.Purpose:In this study,we constructed in vitro inflammation model of NLRP3,NLRC4,AIM2,non-classical NLRP3 inflammasome and LPS-induced septic shock model,MSU-induced peritonitis model in vivo to explore the influence of BA on NLRP3 inflammasome activation and its mechanism.The aim is expected to provide candidate drug and scientific basis for the prevention and control of NLRP3-driven diseases.Methods:1.LPS-primed BMDMs or PMA-primed THP-1 cells were treated with BA and then stimulated with nigericin to activate NLRP3 inflammasome.Caspase-Glo^?1 Inflammasome Assay kit,ELISA kits and LDH kit were used to detect the caspase-1 activity,the secretion of IL-1βand TNF-αand LDH release;Western blot（WB）was used to detect the expression of IL-1βand caspase-1 in the cell supernatant,NLRP3,pro-caspase-1,pro-IL-1βand ASC in the cell lysate.At the same time,NLRP3 inflammasome was activated by other agonists including ATP,Si O₂,poly（I:C）,non-canonical NLRP3 inflammasome was activated by c LPS,AIM2inflammasome was activated by poly（d A:d T）,and NLRC4 inflammasome was activated by Salmonella to further study the effect of BA on the activation of inflammasomes.2.WB was used to detect the influence of BA on the expression of NF-κB signaling pathway-related precursor proteins and the effects on ATP,nigericin,Si O₂,poly（I:C）,poly（d A:d T）,Salmonella and c LPS-mediated ASC oligomerization.At the same time,the generation of ROS,calcium influx and potassium efflux were detected to explore the molecular mechanism of BA regulating NLRP3 inflammasome.3.ELISA was used to detect the influence of BA intervention on related inflammatory factors in LPS septic shock and MSU peritonitis model,and flow cytometry was used to measure the recruitment of inflammatory cells in LPS model to evaluate the anti-inflammatory effect of BA in vivo.Results:1.BA dose-dependently inhibits nigericin-stimulated caspase-1 activation,IL-1βsecretion and pyroptosis in both BMDMs and THP-1 cells.Moreover,it inhibits the activation of NLRP3 inflammasome activated by a variety of agonists,and can inhibit the activation of non-canonical NLRP3 inflammasome and NLRC4 inflammasome,but has no inhibitory effect on the activation of AIM2 inflammasome in BMDMs.2.Mechanism studies have shown that BA inhibits NF-κB signaling pathway,while in this experimental system,the inhibitory effect of BA on the NLRP3 inflammasome is not related to the expression of NF-κB-dependent precursor proteins.BA inhibits NLRP3-dependet ASC oligomerization and ROS production,but does not affect calcium influx and potassium efflux.3.In vivo experiments show that BA can inhibit the secretion of the inflammatory factor IL-1β,reduce the recruitment of macrophages and neutrophils to the peritoneum,and significantly inhibits the activation of NLRP3 inflammasome.In contrast,the production of TNF-αand IL-6 are not affected by BA administration.Conclusion:BA inhibits the activation of NLRP3 inflammasomes by acting on the upstream of ASC oligomerization and inhibiting the production of ROS.And it significantly alleviates the inflammatory response in vivo,suggesting that BA is a novel and potent inhibitor of NLRP3inflammasome and can be used as a potential drug candidate to treat NLRP3 inflammasome-related diseases.