The Role And Its Mechanism Of Autophagy In Acetaminophen Hepatotoxicity Basing On NLRP3 Inflammasome

ObjectiveAcetaminophen(APAP),is a commonly used over-the-counter antipyretic and analgesic drug.APAP overdose can lead to severe liver injury,which is the most common cause of acute liver failure.It is found that mitochondria may be the main target of APAP hepatotoxicity.Mitochondrial dysfunction can not only induce oxidative stress leading to liver damage,but also can activate inflammation to further aggravate liver injury.As a highly conserved degradation system in eukaryotic cells,autophagy can wrap the misfolded proteins and damaged organelles,and transport them to lysosomes to degrade.It is found that autophagy may play an important role in the process of diseases.However,the study of autophagy and APAP hepatotoxicity is just beginning.Recent research just suggests that autophagy is likely to play an important role in APAP-induced acute liver injury as a cellular stress mechanism.Previous studies did not systematically detect the dynamic changes of autophagy in APAP-induced liver injury,nor did it explore the role and the possible mechanism of autophagy.Therefore,some key problems about autophagy and APAP hepatotoxicity have not been solved.In the present study,we aimed to observe the dynamic change of autophagy,especially the change of Pinkl-Parkin mediated mitophagy,as well as the effect of APAP on NLRP3 activation and the release of inflammation media by eatablishing models of APAP-induced acute liver injury and the intervention models of autophagy to investigate its possible role in APAP-induced hepatotoxicity as much as possible.Methods1.Established models of APAP-induced acute liver injury and intervation models of autophagy(1)Dose-response model:After three days of adaptive feeding,forty Male C57BL/6 mice were randomly divided into four groups:control group,APAP low dose group(180mg/kg.bw),APAP middle dose group(300mg/kg.bw)and APAP high dose group(500mg/kg.bw).After fasting 12h,mice were intraperitoneally(i.p.)injected with saline or APAP solution and then sacrificed at 24h.(2)Time-course model:Set up six groups:control group,APAP 3h group,APAP 6h group,APAP 12h group,APAP 24h group and APAP 48h group.And there were ten mice in each group.After fasting 12h,mice were intraperitoneally(i.p.)injected with 300mg/kg.bw APAP and then they were sacrificed at 3,6,12,24,48h respectively.(3)Autophagy intervention model:The first model is established by RAPA.Set up eight groups:control group,2mg/kg.bw RAPA group,4mg/kg.bw RAPA group,8mg/kg.bw RAPA group,300mg/kg.bw APAP group,2mg/kg.bw RAPA+300mg/kg.bw APAP group,4mg/kg.bw RAPA+300mg/kg.bw APAP group and 8mg/kg.bw RAPA+300mg/kg.bw APAP group.And there were ten mice in each group.After fasting 12h,mice in control group were intraperitoneally(i.p.)injected with saline.Mice in RAPA groups were intraperitoneally(i.p.)injected with RAPA solution.Mice in APAP group were intraperitoneally(i.p.)injected with APAP solution(300mg/kg.bw).Mice in RAPA intervention groups were intraperitoneally(i.p.)injected with RAPA solution,and then they were intraperitoneally(i.p.)injected with APAP solution(300mg/kg.bw)after 1h.Finally,all the mice were sacrificed at 24h.Serum and liver tissues were collected.The second model is established by CQ.Set up four groups:control group,60mg/kg.bw CQ group,300mg/kg.bw APAP group,60mg/kg.bw RAPA+300mg/kg.bw APAP group.And there were ten mice in each group.After fasting 12h,mice in control group were intraperitoneally(i.p.)injected with saline.Mice in CQ groups were intraperitoneally(i.p.)injected with CQ solution.Mice in APAP group were intraperitoneally(i.p.)injected with APAP solution(300mg/kg.bw).Mice in CQ intervention groups were intraperitoneally(i.p.)injected with CQ solution,and then they were intraperitoneally(i.p.)injected with APAP solution(300mg/kg.bw)after 1h.Finally,all the mice were sacrificed at 24h.Serum and liver tissues were collected.2.Biochemical analyses,histological examination and the detection of proteins and genes(1)The levels of serum ALT and AST were measured in all the mice.The liver of mice were dissected and weighed to calculate the liver index.The same sections of liver tissues in mice were stained with hematoxylin and eosin(HE)to evaluate histopathological changes.(2)For mice in the dose-response and time-course model,total proteins,nuclear proteins and mitochondrial proteins were extracted to detect the change of autophagy,mitophagy,inflammation and p62-Keapl-Nrf2 pathway related proteins.Total RNA isolated from liver of mice was reverse-transcribed to cDNA to determine the mRNA levels of antioxidant genes.(3)For mice in RAPA intervention model,total proteins and mitochondrial proteins were extracted to determine the changes of autophagy,mitophagy and inflammation related proteins.(3)For mice in CQ intervention model,total proteins were extracted to determine the changes of inflammation related proteins.Results1.The changes of autophagy and mitophagy in APAP-induced liver injury(1)The dose-response relationship between APAP and liver injury:Compared with the control group,the liver index,levels of ALT and AST of mice in APAP groups were dose-dependently increased.Moreover,liver sections from APAP groups exhibited various degrees of hepatocyte necrosis accompanied with granular degeneration of hepatocytes and infiltration of inflammatory cells.(2)The time-course relationship between APAP and liver injury:Compared with the control group,the liver index,levels of ALT and AST of mice in APAP groups were time-dependently increased.The levels of ALT and AST reached the peak at 24h,and were decreased at 48h.The liver tissues of mice showed various degrees of hepatocyte necrosis over time.The range of hepatocyte necrosis was larger at 24h,and was obviously shrinked at 48h.(3)The effect of APAP on inflammation related proteins:Compared with the control group,administration of APAP markedly elevated the expression of NLRP3,caspase-1 and IL-1β.(4)The effect of APAP on autophagy related proteins:Compared with the control group,the expression of p62 in APAP groups was significantly increased.LC3-Ⅱ/LC3-I was also markedly increased.And both of them showed obvious relationship of dose-response and time-course.(5)The effect of APAP on mitophagy related proteins:Compared with the control group,OPTN,NDP52 and p62 in mitochondria were dose-dependently increased following the administration of APAP.Parkin and Pinkl in mitochondrial and cytoplasm also were dose-dependently increased.2.The changes of p62-keapl-Nrf2 pathway in APAP-induced liver injury(1)The changes of p62-Keapl-Nrf2 pathway related proteins:Compared with the control group,the expressions of p-TBK1,p62 and p-p62 were significantly increased by APAP.The expression of Nrf2 in nuclei and cytoplasm also were markedly increased.Especially for nuclear Nrf2,there was a statistically significant difference between APAP 6h group and control group.(2)The changes of antioxidant genes:In dose-response model,compared with the control group,the expression of HO-1,GCLC were decreased significantly and GSTA1 was significantly increased after APAP exposure.In time-course model,the expression of HO-1,GCLC and GSTA1 reached the maximum at 3h,6h and 24h respectively.3.The effects of intervention on autophagy,mitophagy and inflammation in APAP-induced liver injury(1)The effect of RAPA on APAP-induced liver injury:Compared with APAP group,the levels of ALT and AST were markedly decreased following the intervention of RAPA.Moreover,histopathological examination showed that the degree of liver damage was gradually decreased with the increase of RAPA doses.(2)The effect of RAPA on autophagy:Compared with APAP group,the expression of p62 was significantly decreased.LC3-II/LC3-I was markedly increased.(3)The effect of RAPA on mitophagy:Compared with control group,the expression of Parkin,Pinkl and Drpl in liver and mitochondria were significantly increased after APAP exposure.However,compared with APAP group,the expression of Parkin,Pinkl and Drpl in liver and mitochondria decreased with the intervention of RAPA.The expression of Opal was increased.(4)The effect of RAPA on inflammation:Compared with APAP group,the intervention of RAPA significantly reduced the expression of inflammation related proteins NLRP3,caspase-1,and IL-1β.(5)The effect of CQ on inflammation:Compared with APAP group,the intervention of CQ significantly increased the expression of inflammatory related proteins NLRP3,caspase-1,and IL-1β.Conclusions1.APAP overdose caused acute liver injury in mice,and activated mitophagy and NLRP3-mediated inflammation.2.p62-Keap1-Nrf2 pathway played a protective role in the early stage of APAP-induced liver injury.3.Autophagy activator-RAPA can further activate autophagy,remove damaged mitochondria,inhibit the activation of NLRP3,and has a protective effect on APAP-induced liver injury.