Study On The Protective Effect And Mechanism Of Licochalcone A On Acute Liver Failure

The liver is a vital organ that is responsible for detoxification and metabolic homeostasis in human and animals,but it is extremely vulnerable to multiple factors,including alcohol,drug toxicity,moldy foods,and hepatitis viruses,ultimately leads to acute hepatic failure（ALF）.ALF,also called acute liver injury,is a life-threatening and fatal clinical syndrome that is associated with a poor prognosis and high mortality.Meanwhile,inflammation and oxidative stress play an important role in the occurrence and development of various diseases.Recent studies have shown that nuclear erythroid 2-related factor 2（Nrf2）,as an important transcription factor,is an essential regulator of oxidative stress and inflammation and has been widely considered as a potential target for new therapeutics in liver diseases.Importantly,natural compounds,as important sources of Nrf2 activators,can alleviate the progress of many diseases via upregulating Nrf2 signaling pathway.Licochalcone A（Lico A）,a natural flavonoid compound that isolated from the root of licorice Glycyrrhiza inflate,possesses a variety of biological activities,such as anti-inflammatory and antioxidant effects.Accordingly,it is very important for exploring the protective effect and mechanism of Lico A on ALF through the upregulation of Nrf2 signaling pathway.In the current study,we firstly explored the protective effect and mechanism of licochalcone A（Lico A）against hepatotoxicity stimulated by a variety of xenobiotics,including t-BHP,AFB1,H₂O₂/LPS and APAP,in HepG2 cells.Then,we respectively chose two different mouse（C57BL/6）models of ALF induced by LPS/GalN and APAP to investigate the preventive effect and mechanism of Lico A on hepatic injury,thereby providing theoretical basis for the reasonable application of Lico A in clinic.Here,we demonstrated the mechanism from cellular and molecular levels to signal transduction using MTT assay,immunofluorescence,Western blotting,enzyme-linked immunosorbent（ELISA）,hematoxylin-eosin（H&E）staining,and flow cytometry（FCM）technology.Our findings indicated that Lico A effectively decreased t-BHP,AFB1,H₂O₂/LPS and APAP-stimulated hepatotoxicity and overproduction of reactive oxygen species（ROS）in HepG2 cells.Moreover,Lico A evidently inhibited cell apoptosis and Caspase-3 cleavage expression,increased various anti-oxidative enzymes expression,such as GCLC,GCLM,HO-1 and NQO1,which is largely dependent on upregulating Nrf2 nuclear translocation,reducing the Keap1 protein expression,and strengthening the antioxidant response element（ARE）promoter activity.However,Lico A suppressed APAP-induced hepatotoxicity and mediated many anti-oxidative enzymes expression,which were reversed in Nrf2^-/-HepG2cells.In vivo These results showed that Lico A dramatically protected against APAP-induced ALF by lessening the lethality,alleviating histopathological liver changes,and decreasing the alanine transaminase（ALT）and aspartate aminotransferase（AST）levels,malondialdehyde（MDA）formation,myeloperoxidase（MPO）level and superoxide dismutase（SOD）and glutathione（GSH）depletion.Furthermore,Lico A not only significantly modulated apoptosis-related protein by decreasing Caspase-3cleavage expression,but also efficiently alleviated mitochondrial dysfunction by reducing c-jun N-terminal kinase（JNK）phosphorylation and mitochondrial translocation,inhibiting Bax mitochondrial translocation,apoptosis-inducing factor（AIF）and cytochrome c release.Simultaneously,Lico A increased the expression of GCLC,GCLM,HO-1 and NQO1 protein,enhanced Nrf2 nuclear translocation,and reduced the Keap1 protein expression.However,Lico A-inhibited APAP-induced the lethality,histopathological changes,hepatic apoptosis and mitochondrial dysfunction in WT mice were evidently abrogated in Nrf2^-/-mice.These investigations firstly implicated that Lico A has potential protection against APAP-induced ALF which may be strongly involved in the Nrf2-mediated defense mechanisms.Furthermore,our results indicated that treatment with Lico A significantly improved LPS/GalN-induced ALF by lessening lethality,alleviating histopathological liver changes,decreasing the AST and ALT levels,reducing the secretion of inflammatory cytokines,and suppressing oxidative markers.In addition,Lico A efficiently alleviated LPS/GalN-induced inflammatory response by inhibiting TLR4-MAPK and-NF-κB,and the Txnip-NLRP3 signaling pathway.Moreover,Lico A obivously enhanced Nrf2/HO-1 siganling pathway and autophagy activation,as well as restored LPS/GalN-inhibited autophagy.Additional mechanistic investigations further evaluated that the dependence of the hepatoprotective role of Lico A on Nrf2signaling pathway,revealing that a lack of Nrf2 promoted Lico A-induced autophagy which contributed to the hepatoprotective effect of Lico A in Nrf2^-/-mice.Moreover,cotreatment with autophagy inhibitor（3-methyladenine,3-MA）alleviated the hepatoprotective effect of Lico A on LPS/GalN-induced ALF.Our study suggested that Lico A has protective potential against LPS/GalN-induced ALF,which may be strongly related to autophagy.To sum up,this study firstly demonstrated Lico A could effectively alleviate hepatotoxicity and the related mechanism in vitro and in vivo.In HepG2 cells,Lico A remarkably improved various xenobiotics-induced hepatotoxicity by inhibiting oxidative stress,mitochondrial dysfunction and apoptosis,which mainly targerting Nrf2 signaling pathway.In animal models,Lico A could ameliorate APAP-and LPS/GalN-induced ALF;the former played a role in inhibiting mitochondrial damage and cell apoptosis stimulated by oxidative stress,mainly through targeting Nrf2signaling pathway,the latter played a role in suppressing the inflammatory response,majorly through regulating autophagy activation.