Glycogen synthase kinase 3 inhibitor attenuates endotoxin-induced liver injury

Abstract

Background/Aims

Endotoxin (lipopolysaccharide, LPS)-induced acute liver injury was attenuated by endotoxin tolerance (ET), which is characterized by phosphatidylinositol 3-kinase pathway/Akt signaling. Glycogen synthase kinase 3 (GSK-3) acts downstream of phosphatidylinositol 3-kinase pathway/Akt and GSK-3 inhibitor protects against organic injury. This study evaluates the hypothesis that ET attenuated LPS-induced liver injury through inhibiting GSK-3 functional activity and downstream signaling.

Methods

Sprague-Dawley rats with or without low-dose LPS pretreatment were challenged with or without large dose of LPS and subsequently received studies. Serum tumor necrosis factor-alpha, interleukin-10, alanine aminotransferase, lactate dehydrogenase, and total bilirubin levels were analyzed, morphology of liver tissue was performed, glycogen content, myeloperoxidase content, phagocytosis activity of Kupffer cells, and the expression and inhibitory phosphorylation as well as kinase activity of GSK-3 were examined. Survival after LPS administration was also determined.

Results

LPS induced significant increases of serum TNF-α, alanine aminotransferase, lactate dehydrogenase, and total bilirubin (P < 0.05), which were companied by obvious alterations in liver: the injury of liver tissue, the decrease of glycogen, the infiltration of neutrophils, and the enhancement of phagocytosis of Kupffer cells (P < 0.05). LPS pretreatment significantly attenuated these alterations, promoted the inhibitory phosphorylation of GSK-3 and inhibited its kinase activity, and improved the survival rate (P < 0.05).

Conclusions

ET attenuated LPS-induced acute liver injury through inhibiting GSK-3 functional activity and its downstream signaling.

Introduction

It has been reported that LPS-induced excessive release of cytokines from Kupffer cells (KCs) contributes to organ damage during sepsis [1], including the so-called LPS-induced liver injury. LPS-induced liver injury promoted the pathogenesis of many liver diseases, such as alcoholic liver disease, hepatic fibrosis, hepatic cirrhosis, and liver failure [2]. And the original injury of liver tissue induced by LPS was bound to the impairment of the clearance of LPS, which actually leads to further injury and forms an infernal circle. Of which, endotoxin tolerance (ET)–mediated protective effect has undergone a remarkable increase in interest and attention in recent years [3], [4], [5]. In addition, the cross-tolerance of LPS to Toll-like receptor (TLR) 2 ligands, TLR3 ligands, interleukin (IL) 1, and tumor necrosis factor-alpha (TNF-α) were also demonstrated to attenuate LPS-induced liver injury [6], [7], [8]. These investigations strongly suggested that ET attenuated LPS-induced acute liver injury. However, the precise mechanism has not yet been thoroughly elucidated.

Recently, glycogen synthase kinase 3 (GSK-3), a constitutively active downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, was demonstrated as a novel target for treatment of inflammation, shock, and sepsis [9], [10], but its role in ET in hepatocyte is fully unknown. It was thoroughly demonstrated that GSK-3 acts downstream of PI3K/Akt pathway and that the activation of PI3K/Akt pathway was induced during ET [11], which implying the modification of GSK-3 activation by PI3K/Akt signaling during ET. In fact, the initial characterization of GSK-3 as a critical enzyme involved in glycogen biosynthesis [12], which regulated the metabolism of glycogen. Interestingly, the recent discovery that GSK-3 promotes inflammation reveals a new component of its well-documented actions in several prevalent diseases that involve inflammation [13], suggesting the role of GSK-3 in this signal event. In addition, the infiltration of neutrophils (indicated by myeloperoxidase [MPO] activity) in liver tissue was indispensable in LPS-induced acute liver injury [14]. The articles show that GSK-3 inhibition has also been shown to exhibit protection against other septic associated events, that is, hemorrhage and resuscitation, by reducing both renal dysfunction and liver injury [15]. This effect was significantly reduced by using GSK-3 inhibitor [16], [17], which further suggested the role of GSK-3 in regulating the migration of neutrophils and the tissue injury of liver. Furthermore, liver macrophages, KCs, which preside in a strategic position within liver sinusoids, are continually exposed to various pathogen-associated molecular patterns, including LPS [18], but overactive KCs trigger an intracellular signaling cascade that culminates in the upregulation of proinflammatory cytokines [19]. And the phagocytosis activity of macrophage could be enhanced when GSK-3 was under the conditions of inhibitory phosphorylation and then inactivation [20], which also supported the conception that GSK-3 regulated the phagocytosis activity of KCs and associated with liver injury.

Based on these findings, it appears that LPS-induced acute liver injury is associated with the regulation of GSK-3 and that ET may attenuate LPS-induced acute liver injury by affecting PI3K/Akt and GSK-3 pathway as well as its downstream signal events, such as the metabolism of liver glycogen, the infiltration of neutrophils in liver tissue, and the phagocytic activity of KCs. The purpose of this study, therefore, was to evaluate the hypothesis that ET attenuates LPS-induced acute liver injury by affecting the functional activity and signaling of PI3K/Akt and GSK-3 pathway.