| ObjectiveAcute pancreatitis(AP)is one of the most common clinical emergencies with high morbidity and mortality.This study aims to explore the role of acinar cell pyroptosis in AP and the molecular mechanism of its regulation,and provide a new research basis for the treatment of AP.MethodsC57BL/6J mice were selected by intraperitoneal injection of cerulein to construct AP model,and the control group was intraperitoneally injected with the same volume of normal saline.Collect pancreatic tissue and blood samples,and evaluate the severity of AP and degree of acinar cell damage by detecting pancreatic tissue HE staining,TUNEL staining,histological score,tissue water percentage,myeloperoxidase(MPO)level,and serum pancreatic amylase and lipase levels.266-6 cells were stimulated with octapeptide cholecystokinin(CCK8),and the level of lactate dehydrogenase(LDH)in the medium was detected to evaluate the degree of cell damage.The differentially expressed genes were screened by analyzing the sequencing results of the cerulein-induced AP model in the GEO database.The expression level of SPHK1,the activation level of endoplasmic reticulum stress-related proteins IRE1α,PERK,and ATF6,the expression level of TXNIP,the expression levels of inflammasome-related proteins NLRP1,NLRP3,NLRC4,and AIM2,as well as the activation levels of pyroptosis-related proteins Caspase-1 and GSDMD in pancreatic tissue of AP model mice and the 266-6 cells treated with CCK8 were detected by Western Blot experiment and quantitative PCR.,to evaluate the SPHK1,endoplasmic reticulum stress,TXNIP,inflammasome activation,pyroptosis signaling pathway changes in AP.Flow cytometry to detect the ratio of pyroptosis induced by CCK8 in 266-6 cellssi-SPHK1 knocked down the expression of SPHK1 in 266-6 cells,4μ8c,GSK2656157,and Melatonin inhibited the activation of IRE1α,PERK,and ATF6 in 266-6 cells,respectively.Ruscogenin and Z-YVAD-FMK inhibited TXNIP-mediated NLRP3 activation and activation of Caspase-1 in 266-6 cells,respectively.The effects of regulating the above signaling pathways on CCK8-induced 266-6 cell damage and pyroptosis were observed in vitro.SPHK1 knockout mice were used,and GSK2656157,Ruscogenin and Belnacasan were used in vivo to inhibit PERK activation,TXNIP-mediated NLRP3 activation and Caspase-1 activation in AP model mice respectively,and observe changes in AP severity and pyroptosis.ResultsWhen cerulein induced AP,the pancreatic tissue was obviously edematous;HE staining increased the spacing of acinar cells,disordered arrangement,inflammatory cell infiltration,and patchy pancreatic tissue hemorrhage and necrosis;histological score,MPO level increased significantly,serum pancreatic amylase level and serum pancreatic Lipase levels were significantly increased.The positive cells of tunnel staining in pancreatic tissue increased significantly.After CCK8 treatment of 266-6 cells,LDH levels in the medium were significantly increased.The target gene sphingosine kinase 1(SPHK1)was screened out by analyzing the sequencing results of the cerulein-induced AP model in the GEO database.In AP model mouse pancreatic tissue and CCK8-treated 266-6 cells,the expression of SPHK1 was upregulated,the activation levels of endoplasmic reticulum stress-related proteins IRE1α,PERK,and ATF6 were up-regulated,the expression level of TXNIP was up-regulated,the expression of inflammasome-related protein NLRP3 and the activation levels of pyroptosis related proteins Caspase-1 and GSDMD were increased.When 266-6 cells were stimulated with CCK8 by flow cytometry,pyroptosis was significantly increased.Knocking down the expression of SPHK1 in 266-6 cells,or applying GSK2656157,Ruscogenin,Z-YVAD-FMK to inhibit the activation of PERK,NLRP3,and Caspase-1 in266-6 cells,respectively,can significantly improve CCK8-induced 266-6 cell damage and pyroptosis.Knockout of mouse SPHK1 gene or application of GSK2656157,Ruscogenin,Belnacasan to inhibit PERK/NLRP3/Caspase-1 activation in vivo can significantly improve the severity of AP and pyroptosis in model mice.ConclusionOur study showed that up-regulation of SPHK1 expression during AP promotes PERK activation through endoplasmic reticulum stress,which in turn mediates NLRP3 inflammasome activation through up-regulation of TXNIP,thereby promoting acinar cell pyroptosis and participating in the pathogenesis of acute pancreatitis. |
