Study On Mechanism Of Bmil Action In Inhibition Of Acute Liver Injury

When acute liver injury(ALI)occurs,a large number of liver cells died in patterns of apoptosis,necrosis and others,accompanying inflammatory reaction and abnormal hepatic function,which are the main reasons for acute liver failure.Previous studies have demonstrated that the activation of p53 pathway plays an important role in the pathogenesis of acute liver injury,and Bmi1 deletion leads to the activation of p53 pathway,cell apoptosis and necrosis in a variety of mechanisms.However,in the course of acute liver injury,whether Bmil deletion contributes to liver damage by activating p53 pathway in hepatic cells and how the activation of p53 pathway aggravates liver injury are not clear.To answer these questions,acute liver injury model was first successfully generated by intraperitoneal injection of carbon tetrachloride(CCl4)in 5-week-old mice,and the mice injected vegetable oil used as control(Ctrl).The mice were harvested 48 hours post injection.Hepatic phenotypes were analyzed using techniques of histopathology,serology and cell molecular biology,so on.Bmi1 expression levels in liver were compared between CCl4-treated and Ctrl mice.Furthermore,the hepatic phenotypes were compared between CCl4-treated and Ctrl mice with or without Bmi1 gene mutation.Finally,p53 and Bmi1 double gene mutant(p53-/-Bmi1-/-)mice were generated and treated with CCl4,and their hepatic phenotypes were compared with WT and Bmi1-/-mice treated with CCl4.We found that Bmil expression in liver of Ctrl mice was extremely low,while its expression significantly up-regulated in liver of CCl4-treated WT mice.Meanwhile,Bmil were not detectable in livers of vehicle-or CCl4-treated Bmi1-/-mice.Compared with vehicle-treated WT mice,vehicle-treated Bmi1-/-mice displayed normal hepatic tissue structure and function,including the indexes of liver cell proliferation and apoptosis,necrosis,inflammatory reaction,oxidative stress,DNA damage and responsive actions,the expression levels of key molecules involved in p53 signaling pathway and hepatic function.Compared with vehicle-treated WT mice,CCl4-treated WT mice displayed 1)obvious hepatic cell necrosis in point or patchy distribution,accompanied with inflammatory cell infiltration,and liver cells generally swollen up;2)reduced hepatic function,accompanied with significantly increased serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels;3)increased cell proliferation and apoptosis,describing by the elevated both of the percentage of PCNA positive and TUNEL-positive hepatic cells;4)increased necroptosis,including increased percentages of PI positive,or receptor-interacting serine/threonine-protein kinase 3(RIPK3)positive hepatic cells,as well as significantly increased RIPK3 and the mixed lineage kinase domain-likeprotein(MLKL)expression levels;5)increased inflammatory reaction with up-regulated expression of inflammatory cytokine like TNF-alpha,IL-1 beta and IL-17,and increased,percentage of CD3 e-positive lymphocytes and F4/80-positive macrophages;6)increased oxidative stress like increased level of reactive oxygen species(ROS);7)accumulated DNA damage,including elevated percentages of phosphorylate H2A histone,member X(yH2A.X)and p53 binding protein 1(53BP1)positive hepatic cells;8)significantly up-regulated expression levels of key molecules of p53 signaling pathway,for example,increased protein levels of p53 and p21.Compared with CCl4-treated WT mice,CCl4-treated Bmi1-/-mice were detected to have more serious liver injury,which included 1)increased large diffused necrosis,bridging necrosis and total necrotic area in hepatic tissue;2)more hepatic functional loss interpreted by multiple parameters,containing hepatic cell proliferation,apoptosis,necroptosis,inflammatory reaction,oxidative stress,and DNA damage;3)more significantly increased expression levels of key molecules involved in p53 signaling pathway.Compared with CCl4-treated Bmi1-/-mice,liver damage was partially rescued and even restored to the levels of CCl4-treated WT mice in CCl4-treated p53-/-Bmi1-/-mice.Hepatic cell proliferation,apoptosis,necroptosis,inflammatory reaction,oxidative stress and DNA damage detected in CCl4-treated p53-/-Bmi1-/-mice have been restored to those levels of CCl4-treated WT mice,in addition to their ROS levels,which was still higher than CCl4-treated WT mice but significantly lower than CCl4-treated Bmi1-/-mice.This study show that Bmil plays a criticalrole in protecting liver from CCl4 induced acute liver injury through inhibiting oxidative stress and DNA damage,promoting hepatic cells proliferation,restricting cell apoptosis,necroptosis and inflammation reaction,and inactivating p53 signaling pathway,,which may regulate necroptosis induced by acute liver injury through transcriptional activation RIPK3.Therefore,these findings indicate the molecular mechanisms of Bmi1 protection in aggravating acute liver injury induced by CCl4,which further provides new potential targets and experimental basis for clinical treatment of acute liver injury.