Study On The Mechanism Underlying That SphK1 Ameliorates APAP-induced Acute Liver Injury Via The Suppression Of Hepatocyte Pyroptosis

Aims:Sphingosine kinase 1?SphK1?plays crucial roles in liver injury.Acute liver injury induced by acetaminophen?APAP?overdose is one of the most important causes of acute liver failure all over the world.Accumulating evidence suggests that NOD-like receptor pyrin domain 3?NLRP3?inflammasome plays an important role in APAP-induced liver injury.Activation of NLRP3 inflammasome triggers pyroptosis.However,whether hepatocyte pyroptosis participates in the early stage of APAP-induced liver injury are not well understood.This study aims to investigate the role of hepatocellular SphK1 in APAP-induced hepatocyte pyroptosis and acute liver injury.Method:Primary hepatocytes were isolated from male C57BL/6J mice and were treated with 5 mM APAP to induced injury.The effect and molecular mechanism of primary mouse hepatocytes pyroptosis induced by excessive APAP were verified by living cell imaging system and transfection experiments.To induce liver injury,fasted male C57BL/6J mice were administered APAP intraperitoneally at a dose of 250 mg/kg.The expression levels of pyroptosis-related proteins and SphK1 in liver tissues were detected by immunofluorescence staining,western blot and real-time quantitative PCR.Hepatocyte-specific deficiency of SphK1(SphK1^Hep-/-)mice and primary mouse hepatocytes isolated from SphK1^Hep-/-mice were used to determine the effect and molecular mechanism of hepatocellular deletion SphK1 on acute drug-induced liver injury induced by APAP overdose.Finally,the protective effect of hepatocyte SphK1 on hepatocyte pyroptosis and acute drug-induced liver injury caused by excessive APAP was verified by transfection of primary mouse hepatocytes with adenovirus contained SphK1 overexpression vector.Results:Excessive APAP could induce primary mouse hepatocytes pyroptosis,which was dependent on the activation of Caspase 1.The expression of cl-Caspase 1,GSDMD-N and SphK1 were strongly induced in mice exposed to excess APAP.Surprisingly,SphK1^Hep-/-mice had significantly higher mortality and elevated early hepatotoxicity compared to WT mice after excessive APAP treatment.Conditional deletion of hepatocellular SphK1 increased hepatocyte pyroptosis and death.The enhanced hepatotoxicity in SphK1^Hep-/-mice was associated with increased activation of NLRP3 inflammasome and Caspase 1 maturation and membrane rupture.Moreover,SphK1 deficiency in hepatocytes upregulated thioredoxin interacting protein?TXNIP?and downregulated mi R-148a.Furthermore,miR-148a directly inhibited TXNIP expression.In contrast,APAP-induced NLRP3 activation and hepatocyte pyroptosis were reversed by miR-148a mimic or TXNIP siRNA.Finally,the protective role of SphK1in hepatocyte damage was further strengthened by the results of adenovirus-mediated SphK1 overexpression in APAP-induced primary mouse hepatocytes pyroptosis.Conclusion:These data highlight a novel mechanism that the early stage of APAP-induced liver injury is characterized by hepatocyte pyroptosis,and demonstrate for the first time a protective role of hepatocellular SphK1expression after APAP overdose via inhibition of NLRP3 inflammasome and pyroptosis.