Study Of The Effects And Mechanism Of Augmenter Of Liver Regeneration In Acetaminophen-induced Acute Liver Injury

ObjectiveAcute liver injury(ALI)and even acute liver failure(ALF)are caused by acetaminophen(APAP)overdose.APAP overdose is not only one of the main causes of most ALF cases,but also a serious public health problem.Oxidative stress and mitochondrial dysfunction are the key factors of APAP hepatotoxicity.Augmenter of liver regeneration(ALR)has both antioxidative and anti-apoptotic effects and has been proven to be a protective factor in liver.However,no evidence showed the significance of ALR in APAP hepatotoxicity.Therefore,the aim of this study was to investigate the role of ALR in APAP hepatotoxicity in a mouse model of ALI.MethodsHealthy female BALB/c mice were purchased.Experiment was divided into 3 parts,to explore the survival rates,serum ALT and AST levels,liver necrosis,and ALR protein within 72 h of ALI model;overexpressing ALR by lentivirus and inhibiting autophagy by trimethyladenine(3-MA)to study their significance on autophagy related proteins(LC3 II,p62),autophagosomes and protective effect of liver in acetaminophen-induced liver injury(AILI);overexpressing ALR by lentivirus and inhibiting autophagy by 3-MA to study their significance on oxidative stress(ROS,MPO,MDA,SOD and GSH),mitochondrial dysfunction and apoptosis(AIF,Cyto C,cleaved-caspase 3 and TUNEL)in AILI.The ALI model was established using APAP,and the Control model was established by intraperitoneal injection of PBS solution.Survival,biochemistry,histology,and functional analysis were performed for each group of mice after modeling.Survival analysis was performed to describe the survival curves of ALI induced by different doses of APAP;serum ALT and AST levels were analyzed by automatic biochemical analyzer;histological analysis of liver was performed by HE staining;the expression of MPO,MDA,SOD and GSH in liver tissue was analyzed by spectrophotometer;the number of autophagosomes in liver tissue was analyzed by electron microscopy(EM);the protein expression of ALR,LC3 II,p62,AIF,Cyto C and cleavedcaspase 3 in liver tissue was detected by Western blot(WB);the distribution and expression of ROS in liver tissue were determined by immunofluorescence;the number of apoptotic cells in liver tissue was determined by TUNEL method.ResultsIn the first part,the higher the APAP dose,the lower the survival rate of the mice within 72 h;the serum ALT and AST,APAP groups were significantly higher than the Control group(P < 0.05),and reached the peak at APAP-24 h;intrahepatic hemorrhage and necrosis were observed in each APAP group;the higher the APAP dose,the more severe the intrahepatic hemorrhage and necrosis;the liver ALR,APAP-3 h,APAP-6 h and APAP-12 h groups were significantly higher than the Control group(P < 0.01);the APAP-24 h group began to decrease,and the APAP-48 h group was significantly lower than the Control group(P < 0.01).In the second part,serum ALT and AST,Normal Saline group and Lvnull group were not significantly different(P > 0.05);Normal Saline group and Lvnull group had no intrahepatic hemorrhage and necrosis;autophagosomes was significantly higher in the LvALR+APAP group than in the Lvnull+APAP group;ALR expression and LC3 II transformation and p62 degradation were significantly increased in the LvALR+APAP group compared with the Lvnull+APAP group(P < 0.01);autophagosomes was significantly higher in the LvALR+APAP group than in the Lvnull+APAP group;serum ALT and AST were significantly improved in the LvALR+APAP group compared with the Lvnull+APAP group(P < 0.01),while the LvALR+APAP+3-MA group was significantly worse(P < 0.01);intrahepatic hemorrhage and necrosis were improved in the LvALR+APAP group compared with the Lvnull+APAP group,while the LvALR+APAP+3-MA group was significantly worse.In the third part,liver MPO,MDA,SOD,GSH and ROS were significantly improved in the LvALR+APAP group compared with the Lvnull+APAP group(P < 0.05),while the LvALR+APAP+3-MA group was significantly worse(P < 0.05);liver AIF,Cyto C,and cleaved-caspase 3,compared with the Lvnull+APAP group,the LvALR+APAP group significantly improved(P < 0.05),whereas the LvALR+APAP+3-MA group was significantly worse(P < 0.05);compared with the Lvnull+APAP group,apoptotic cells in the LvALR+APAP group were significantly decreased,while the LvALR+APAP+3-MA group was significantly increased.ConclusionWithin 72 hours of APAP modeling,liver dysfunction and ALR expression increased at first and then decreased.Lentivirus overexpression of ALR after APAP modeling can enhance autophagy,improve liver function,and reduce liver oxidative stress,mitochondrial dysfunction and apoptosis.