Study On The Mechanism Of PARK7 In Acute Liver Injury Caused By APAP

Background:Acetaminophen(APAP)is a widely used drug for the treatment of cold and fever,arthralgia,neuralgia,migraine,cancer pain,and postoperative pain.At the same time,as an over-the-counter drug,APAP is easy to cause intentional or unintentional overdose and poisoning.Especially during the outbreak of COVID-19,many residents who lack knowledge of rational drug use go to pharmacies to buy APAP and APAP-containing drugs by themselves,resulting in many cases of drug abuse and poisoning injury,which has become a serious public health problem.The literature shows that APAP is the drug that causes the most drug-induced liver injury,and cases of toxic liver injury caused by such drugs are also commonly reported in the world.Patients with severe APAP poisoning even require liver transplantation to save their lives.China has listed APAP as the second most important factor causing liver failure.Although some progress has been made in the research of APAP poisoning mechanism and treatment measures at home and abroad,due to the complexity of APAP poisoning mechanism and clinical manifestations after poisoning,the specific molecular mechanism of APAP poisoning is still unclear.Therefore,the prevention and treatment of APAP poisoning remains one of the major public health challenges that needs to be addressed.Recent advances in the literature and our study found that APAP induced acute liver injury first caused oxidative stress and mitochondrial damage.Transcription factor NRF2 as a core molecule in the regulation of oxidative stress.Its release and expression are related to DJ1,and another form of DJ1 is parkinson disease protein 7(PARK7).As a recessive gene,PARK7 was first identified on the third locus of chromosome 1in patients with Parkinson’s disease,and its biological function has been extensively studied in brain astrocytes and neurons.At the subcellular level,PARK7 was mainly located in the cytoplasm,but also to a lesser extent in the mitochondria and nucleus.The PARK7 protein has a variety of functions,such as oxidative stress regulation,mitochondrial regulation and transcriptional regulation.PARK7 not only exists in glial cells and neurons,but is also widely distributed in other human tissues.At the same time,it is also involved in the occurrence and development of a variety of diseases.Literature studies have shown that in the CCL4-induced liver fibrosis model and liver ischemiareperfusion injury model,knockout of PARK7 gene can reduce liver fibrosis and liver ischemia-reperfusion injury in mice,which provides very important clues for the study of the mechanism of APAP toxic liver injury.However,whether PARK7 can be used as a therapeutic target for liver injury in APAP is still unclear.Therefore,this study started with the important public health problem of APAP poisoning,in-depth study on the pathogenesis of APAP-induced acute liver injury,systematically explore the role of PARK7 in APAP-induced acute liver injury,and to supply new insights for its early diagnosis and valid treatment..Objective:1.To clarify the change pattern of PARK7 in APAP-induced acute liver injury.2.To investigate the role of PARK7 in the regulation of oxidative stress and autophagy in APAP-induced acute liver injury.3.To discuss the preliminary regulatory mechanism of PARK7 on related molecules in APAP-induced acute liver injury.Methods:1.L02 cells,AML12 cells and male C57 mice were used to construct APAP-induced acute liver injury models.CCK-8 kit for cell viability.Annexin V-FITC/PI double staining for apoptosis.Mito Sox staining for mitochondrial reactive oxygen species(mtROS).JC-1staining for mitochondrial membrane potential.The detection of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)was used to evaluate the degree of hepatocyte injury.HE staining was used to examine the histological changes of liver tissue,and ROS staining of frozen sections of liver tissue was utilized to assess ROS changes.2.Expression of PARK7 in acute liver injury induced by APAP.L02 cells were grouped with 0,5,10,and 20 m M APAP,and AML12 cells were grouped with 0,10,20,and 40 m M APAP.C57 mice were divided into 0,300,400 and 500 mg/kg APAP groups.PARK7 expression was detected by RT-PCR,Western blot,immunofluorescence and immunohistochemistry.3.The PARK7 silencing/knockdown model was established.Lentivirus was used to silence PARK7 in L02 cells,small interfering RNA was used to silence PARK7 in AML12 cells,and adeno-associated virus was used to knock down PARK7 in C57 mice.The related indicators of hepatocyte injury induced by APAP after PARK7 downregulation were detected.4.L02 and AML12 cells were divided into four groups: normal control group,APAP exposure group,PARK7 silence control group,and PARK7 silence +APAP group.The experimental mice were divided into 4 groups: normal control group,APAP exposure group,PARK7 knockdown control group,and PARK7 knockdown +APAP,with 10 mice in each group.The observation indexes and measurement methods were as follows:Electron microscopic observation of mitochondrial and autophagosomal ultrastructure,autophagy double-labeled lentivirus to detect autophagic flow changes.Western blot was used to detect KEAP1,NRF2,NQO1,HO-1,SOD2,CAT,LC3,P62,PGC-1α,NRF1 and TFAM protein expression.RT-PCR was used to detect the mRNA levels of NQO1,HO-1,SOD2,CAT,PGC-1α,NRF1 and TFAM.Seahorse test was used to detect cellular respiratory function and glycolysis level.Results:1.With the increase of APAP concentration,the damage degree of L02 cells and AML12 cells increased,mtROS increased and mitochondrial membrane potential decreased.ALT and AST indexes in serum of C57 mice increased,liver tissue damage increased,ROS increased gradually.2.Immunofluorescence and Western blot results showed that 20 m M APAP induced PARK7 into the nucleus of L02 cells.Immunofluorescence and Western blot results showed that PARK7 was transported to the nucleus in the liver tissue of C57 mice.These results indicated that the mRNA and protein expression of PARK7 molecule increased with the increase of APAP concentration/dose.3.L02 cells were treated with sh1 lentivirus,and AML12 cells were treated with si3 small interfering RNA.Silencing PARK7 significantly alleviated APAP-induced cell viability damage in L02 and AML12 cells.After PARK7 molecular knockdown,the levels of ALT and AST in serum of APAP-exposed mice were decreased,and the degree of liver injury was alleviated.AAV infection was shown to be successful in both cell and animal experiments.4.The level of mtROS and the expression of NRF2,NQO1,HO-1,SOD2 and CAT were decreased after silencing/knockdown of PARK7 in the APAP-induced acute liver injury model.The expression levels of PGC-1α,NRF1 and TFAM were increased,the level of cell glycolysis was increased,and the level of cell energy metabolism was shifted to ECAR.The expression of autophagy protein LC3 was increased,the expression of P62 was decreased,mitochondrial swelling was alleviated,autophagosome formation was increased,and autophagic flow was increased.Conclusion:1.We found that PARK7 expression was increased in APAP-induced liver injury and played a role in promoting injury through nuclear translocation.2.It is clear that PARK7 plays an important role in the mechanism of APAP liver injury,and knockdown or inhibition of PARK7 has a beneficial role in protecting against acute liver injury caused by APAP.3.We demonstrated that the regulation of APAP-induced liver injury by PARK7 can affect antioxidant function,but mainly by up-regulating autophagy and promoting mitochondrial repair pathway.