Hepatoprotective Effects And Molecular Pharmacological Mechanisms Of Isoliquiritigenin

Objective:The aim of this study was to investigate the hepatoprotective effect of isoliquiritigenin（ISL）and its molecular pharmacological mechanism,so as to provide a theoretical basis for the early application of ISL in clinic.Methods:（1）Protective effect of ISL on cholestatic liver injury induced by alpha-naphthalene isothiocyanate（ANIT）in mice.The mice were given ISL（10,20 and40 mg/kg）by single daily gavage for 7 consecutive days.On day 5,mice were gavaged with ANIT（75 mg/kg）to establish a cholestatic liver injury model.Alanine transaminase（ALT）,aspartate aminotransferase（AST）,total bile acid（TBA）,total bilirubin（T-BIL）and H&E staining were determined to investigate the effect of ISL on cholestatic liver injury induced by ANIT in mice.The expression levels of hepatic farnesoid X Receptor（FXR）and,small heterodimer partner（SHP）,the transporter sodium ion/taurocholate cotransporting polypeptide（Na+/taurocholate cotransporting polypeptide,Ntcp）,multidrug resistance-associated protein2（Mrp2）,multidrug resistance protein2（multidrug resistance protein,Mdr2）,bile salt export pump（Bsep）,cholesterol 7α-hydroxylase（Cyp7a1）,sterol 12α-hydroxylase（Cyp8b1）and hydroxysteroid sulfotransferase 2a1（Sult2a1）were examined to reveal the regulatory effects of ISL on liver bile acid related transporters,metabolic enzymes and synthetase at the molecular level.We evaluated the effect of ISL on miR-20b-5p and elucidated the regulatory mechanism of biliary liver injury mediated by miR-20b-5p/FXR through in vitro experiments.The liver protective effect and molecular pharmacological mechanism of ISL were further investigated by transfecting miR-20b-5p mimic,miR-20b-5p inhibitor,si-FXR and other experiments in vitro.（2）Protective effect of ISL on acute liver injury（ALI）induced by carbon tetrachloride（CCl4）in mice.The mice were given ISL（10,20 and 40 mg/kg）by single daily gavage for 7 consecutive days.On day 6,mice were injected with CCl4 to establish an ALI model.Alanine transaminase（ALT）,aspartate aminotransferase（AST）,total bile acid（TBA）,total bilirubin（T-BIL）and H&E staining were determined to investigate the hepatoprotective effect of ISL on ALI induced by CCl4 in mice.Serum tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）,interleukin-10（IL-10）were determined to evaluate the anti-inflammatory effect of ISL.The expression levels of hepatic farnesoid X Receptor（FXR）,small heterodimer partner（SHP）,bile salt export pump（Bsep）and Na+/taurocholate cotransporting polypeptide（Ntcp）were examined to reveal the bile acid regulation of ISL.Bcl2,and Bcl-2-associated X（Bax）which were involved in apoptosis were determined to investigate the role of ISL.NF-κB were determined to evaluate the anti-inflammatory effect of ISL.The liver protective effect and molecular pharmacological mechanism of ISL were further investigated by transfecting miR-20b-5p inhibitor and other experiments in vitro.Results:（1）The expression of FXR was modulated by miR-20b-5p in cholestatic liver injury model.Oral administration of ISL reduced the expression of Ntcp,promoted the expression of Bsep,Mrp2 and Mdr2,reduced bile acid transport into hepatocytes,and promoted the efflux of intrahepatic bile acid.By inhibiting Cyp7a1 and Cyp8b1,promoting Sult2a1 expression,bile acid synthesis is reduced,and bile acid metabolism is increased..In vitro experiments,ISL can activate FXR expression by down-regulating miR-20b-5p,subsequently up-regulating Shp,Bsep,Mrp2,Mdr2 and Sult2a1,down-regulating the expression of Ntcp,Cyp7a1 and Cyp8b1,and improving the ANIT-induced hepatocyte injury.Gene silencing experiments further verified the liver protective effect of ISL via miR-20b-5p/FXR.（2）The expression of FXR was modulated by miR-20b-5p in CCL₄-induced acute liver injury in mice.ISL has down-regulates ALT,AST,TBA and T-BIL to relieve liver injury.The expression of miR-20b-5p were reduced,FXR and its downstream protein Shp were increased by ISL,it maintains bile acid homeostasis,and alleviates liver injury.ISL down-regulates the levels of NF-κB and its downstream TNF-αand IL-6,inhibits the expression of liver inflammatory factors,exerts its anti-inflammatory effect,and alleviates the liver injury caused by CCL₄.ISL upregulated the expression of Bcl-2 and inhibited the expression of Bax and caspase-3,playing its role in anti-apoptotic effect.In vitro transfection experiment further showed that CCl₄ could significantly down-regulate FXR,Shp and Bsep,and up-regulate Ntcp.After the administration of ISL,FXR,Shp and Bsep were significantly up-regulated,while Ntcp was significantly down-regulated.While miR-20b-5p inhibitor also significantly reversed the expression of FXR,Shp,Bsep and Ntcp induced by CCl₄.Conclusions:ISL can promote the expression of FXR by down-regulating miR-20b-5p,thereby regulating the expression of downstream bile acid transporters,inhibiting bile acid synthesis,promoting bile acid efflux and metabolism,and reducing the level of bile acid in liver.Reduce inflammatory response,improve anti-apoptosis ability of mice,relieve cholestatic liver injury induced by ANIT and acute liver injury induced by CCL₄.