The Protective Effect And Molecular Mechanism Of Glycyrrhetinic Acid On Acute Liver Injury

PART ONETHE PROTECTIVE EFFECT AND MOLECULARMECHANISM OF GLYCYRRHETINIC ACID ONACETAMINOPHEN-INDUCED LIVER INJURYObjective: To investigate the protective effect of glycyrrhetinic acid(GA) on acetaminophen-induced liver injury, and further explored themolecular mechanism.Methods: Balb/c mice were pretreated with GA (100mg/kg)30minbefore APAP administration. The serum ALT/AST, hepatic tissue histologyand glutathione (GSH) levels were analyzed. In addition, the hepaticapoptosis were determined by TUNEL analysis. The level ofactived-caspase3, high mobility group box1(HMGB1) and F4/80weredetermined by immunohistochemistry or immunofluorescence. The level oftumor necrosis factor (TNF)-α and interleukin (IL)-1β production weredetermined by enzyme-linked immunosorbent assay (ELISA). The protein expression of cytochrome enzyme P4502E1, Toll-like receptor (TLR)4and p38mitogen-activated protein kinases (MAPKs), p-IκB andinterleukin-1receptor-associated kinase-M (IRAK-M) were determined bywestern blot. The CYP2E1mRNA levels were determined by QRT-PCR.Hepatic neutrophil recruitments were determined by flow cytometryResults: Our results showed that pretreatment with GA protected miceagainst APAP-induced liver injury as indicated by decreased ALT and ASTactivities, alleviated hepatic pathological damage and hepatic apoptosis. Inaddition, GA regulated APAP metabolism by down-regulation of CYP2E1protein and mRNA levels, and elevating hepatic GSH levels. Moreover, GAsuppressed APAP-induced HMGB1secretion, p38MAPK and IκBsignaling activation and TNF-α and IL-1β production. However, theexpression of TLR4was not affected. Increasingly, the level of IRAK-Mwas elevated. Our results also showed that APAP-induced hepaticneutrophil recruitments and macrophage infiltration was inhibited by GA.Conclusion: Taken together, GA is a potential agent forAPAP-induced liver injury. On one hand, GA modulated APAP metabolismby down-regulating CYP2E1expression and elevated the hepatic GSHlevels. On the other hand, GA decreased HMGB1secretion, hepaticneutrophil recruitments and macrophage infiltration, further inhibitedTLR4-mediated signaling pathway and pro-inflammatory cytokinesproduction, which is critical for avoiding the second hit suffered form. Nevertheless, there is no effect on TLR4expression. The underlyingmolecular mechanism may be associated with up-regulation of IRAK-M. PART TWOTHE PROTECTIVE EFFECT AND MOLECULARMECHANISM OF GLYCYRRHETINIC ACID ONLIPOPOLYSACCHARIDE-INDUCED FULMINANTHEPATIC FAILUREObjective: To investigate the protective effect of glycyrrhetinic acid(GA) on lipopolysaccharide (LPS)-induced fulminant hepatic failure (FHF),and further explored the molecular mechanism.Methods: Balb/c mice were pretreated with GA (10,30,100mg/kg)30min before LPS/D-GalN administration. The mortality, hepatic tissuehistology, serum ALT/AST activities were analyzed. The expression ofserum and hepatic tumor necrosis factor-α (TNF-α) levels were determinedby enzyme-linked immunosorbent assay (ELISA). The levels of hepaticTNF-α mRNA were determined QRT-PCR. The protein expression oftoll-like receptor4(TLR4), interleukin-1receptor-associated kinases(IRAKs), p38mitogen-activated protein kinases (MAPKs), IκB andIRAK-M were determined by western blot. The interaction between IRAK1and tumor necrosis factor receptor-associated factor (TRAF) weredetermined by immunoprecipitation. In addition, mouse macrophages cellline RAW264.7were pretreated with GA (10-5M) before LPS. The levelsof TNF-α production were determined by ELISA. The expression of TLR4/MD2was determined by flow cytometry. The AP-1and NF-κBtranscriptional activities were determined by luciferase reporter geneexpression assay. IRAK-M siRNA were used for macrophage IRAK-Mdepletion.Results: Our results showed that pretreatment with GA protected miceagainst LPS/D-GalN-induced FHF, including dose-dependent decrease ofmortality and ALT/AST activities, as well as attenuated hepaticpathological damage. Meanwhile, GA reduced LPS/D-GalN-induced serumand hepatic TNF-α levels and inhibited p38MAPK and IκB activation. Italso showed that GA did not influence the expression of TLR4, butinhibited IRAK-1activity with upregulation of IRAK-M expression.Moreover, GA-pretreated macrophages showed inhibited production ofTNF-α and transcriptional acticities of AP-1and NF-κB in response to LPS.Similiarly, the expression of TLR4/MD2was not affected and GAupregulated IRAK-M level. Using siRNA to silence IRAK-M geneexpression, the protective effect of GA was inhibited.Conclusion：Taken together, we conclude that GA can exert protectiveeffect on FHF induced by LPS/D-GalN by inhibitingmacrophages-mediated TNF-α production. The underlying mechanismsmay be related to up-regulation of IRAK-M, which in turn caused reducedinteraction between IRAK-1and TRAF, inhibited activation of MAPKs andNF-κB signaling.