| Background: Acute pancreatitis is an inflammatory disorder of high incidence and high mortality.NLRP3 inflammasome plays an extremely important role in the progress of acute pancreatitis.Our previous study reveals that acetylation of NLRP3 is a key process in NLRP3 inflammasome activation.Objectives: Screening the acetylase inhibitors to find small molecule inhibitors for NLRP3 inflammasome,then investigate the mechanism of the effect on the NLRP3 inflammasome and provide a new therapeutic strategy for acute pancreatitis.Methods: Mouse peritoneal macrophages were pretreated with different small molecule inhibitors of acetyltransferase,then administrated with stimuli of NLRP3 inflammasome.We found that SI-2 hydrochloride(SI-2),a specific inhibitor of lysine acetyltransferase KAT13 B,could specifically block NLRP3 inflammasome activation by the ELISA method of detecting IL-1β.To explore the effect of SI-2:(1)Mouse peritoneal macrophages or activated THP-1 cells were pretreated with SI-2,then given diverse stimuli of different inflammasomes,supernatant and cell lysates were collected to detect the inflammasome activation.(2)AR42J cells were pretreated with SI-2,then administrated with sodium taurocholate;Mice were pretreated with SI-2,then induced localized peritonitis by MSU,induced endotoxemia by LPS or induced acute pancreatitis by cerulein+LPS.Sera,supernatant or cell lysates were collected for detecting inflammatory injury and NLRP3 inflammasome activation(such as IL-1β,LDH,P10,P17).To explore the mechanism of SI-2 inhibiting NLRP3 inflammasome activation.:(1)The effect of KAT13 B on NLRP3 inflammasome activation was detected by RNA interference;(2)The oligomerization of ASC protein was detected by protein cross-linking;(3)The formation of ASC speck was observed by immunofluorescence microscopy;(4)The acetylation of NLRP3,the interaction between NLRP3 and NEK7,NLRP3 and ASC were detected by immunoprecipitation;(5)Ligand docking and binding site analysis for the interaction between SI-2 and ASC and NLRP3.Result:(1)SI-2 specifically reduced the release of IL-1β and LDH in mouse primary peritoneal macrophages and human THP-1 cells after NLRP3 inflammasome activation,reduced the expression of P10 and P17 proteins,did not affect the release of IL-6 and TNF-α,nor did it affect the release of IL-1β after AIM2,NLRP1,NLRC4 inflammasomes activation.(2)SI-2 had protective effect against LPS-induced endotoxemia and MUSinduced localized peritonitis in mice,reduced IL-1β release and GSDMD cleavage induced by NLRP3 inflammasome activation.(3)SI-2 is protective in sodium taurocholate-induced AR42 J cell injury or cerulein+LPS induced acute pancreatitis,by reducing the release of amylase,IL-1β and the expression of P10 and P17.(4)Reducing the expression of KAT13 B had no significant effect on the activation of NLRP3 inflammasome.The protein cross-linking showed that SI-2 could inhibit the oligomerization of ASC.Consistently,Immunofluorescence microscopy observation was shown that SI-2 could reduce the formation of ASC speck.Moreover,Co-immunoprecipitation unveiled that SI-2obstructed the interaction between ASC and NLRP3.Through Ligand docking and binding site analysis,SI-2 had high binding energy to ASC and NLRP3.Conclusion: Our study reveals that SI-2 can specifically inhibit the activation of NLRP3 inflammasome in vitro and in vivo.SI-2 is protective in acute pancreatitis through inhibiting the activation of NLRP3 inflammasome.Mechanistically,SI-2 can block the oligomerization of ASC by interfering with the interaction between ASC and NLRP3.SI-2 has high binding energy with ASC and NLRP3,occupies the original binding site of ASC and NLRP3,destroys the interaction between ASC and NLRP3.Our findings provide a new therapeutic strategy for acute pancreatitis. |
