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Glycyrrhizin, a biological active compound isolated from the liquorice root, has been used as a treatment for chronic hepatitis. We have examined the involvement of matrix metalloproteinase (MMP)9 in the development of lipopolysaccharide (LPS) and D-galactosamine (GalN)-induced liver injury in mice. We also investigated the effect of glycyrrhizin on expression of MMP-9 in this model. Levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased after LPS/ GalN treatment. Expression of MMP-9 mRNA and protein was markedly up-regulated in liver tissues 6-8 h after LPS/GalN treatment. Pretreatment with glycyrrhizin (50 mg kg(-1)) and the MMP inhibitor (5 mg kg(-1)) suppressed increases in serum levels of ALT and AST in mice treated with LPS/GalN. Furthermore, glycyrrhizin inhibited levels of both mRNA and protein for MMP-9. Immunohistochemical reaction for MMP-9 was observed in macrophages/monocytes infiltrated in the inflammatory area of liver injury. Glycyrrhizin reduced the infiltration of inflammatory cells and immunoreactive MMP- 9 in liver injury. The results indicated that MMP-9 played a role in the development of LPS/GalN- induced mouse liver injury, and suggested that an inhibition by glycyrrhizin of the acute liver injury may have been due to a down-regulation of MMP-9.

Summary. The inhibition of apoptosis by glycyrrhizin (GL) in hepatic injury induced by injection of lipopolysaccharide (LPS)/D-galactosamine (D-GalN

HMGB1 is a nuclear component involved in nucleosome stabilization and transcription regulation, but extracellularly it is able to serve as a potential late mediator of lethality. In the present study, we explored inflammation-promoting activity of HMGB1 and blockade of extracellular release of HMGB1 by glycyrrhizin (GL) in LPS/GalN-triggered mouse liver injury. At 1 to 10 h after LPS/GalN-treatment, mice were anesthetized to collect blood from heart puncture, and serum transaminase and HMGB1 were evaluated. Administration of LPS/GalN precipitated tissue injury associated with time-dependent alteration in HMGB1 serum levels. At 8 h nuclear immunoreactive products were remarkably reduced and extracellular HMGB1 expression was found exclusively in the pericentral foci. The treatment with GL significantly down-regulated the serum levels of ALT, AST, and HMGB1 in addition to the strong inhibition of tissue injury and extracellular immunoreactivity to HMGB1 and to acetylated-lysine. Furthermore, GL brought about a significant decrease in the number of apoptotic hepatocytes labeled with TUNEL-method. On the basis of these results, three apoptosis-associated genes were identified with microarray analysis and real-time PCR. The ChIP-assay revealed the binding of HMGB1 protein to Gsto1 promoter sequence in LPS/GalN-treated mice and the remarkable decrease in combined HMGB1 protein by GL. The current findings claim that a single injection of LPS/GalN might stimulate apoptosis of hepatocytes through the binding of HMGB1 protein to Gsto1 promoter region and that GL-treatment might prevent the apoptosis and inflammatory infiltrates caused with LPS/GalN-injection by disturbing the binding of HMGB1 protein to Gsto1 promoter sequence.

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