Gabexate Mesilate Inhibits the Expression of HMGB1 in Lipopolysaccharide-Induced Acute Lung Injury

High mobility group box 1 (HMGB1) is an important late mediator of acute lung injury. Gabexate mesilate (GM) is a synthetic protease inhibitor with some anti-inflammatory action. We aimed to evaluate the effect of GM on HMGB1 in lipopolysaccharide (LPS)-induced lung injury in rats. Prior to the injection of LPS to induce lung injury, rats were administered saline or GM. Injury to the lung and expression of HMGB1, plasminogen activator inhibitor-1 (PAI-1), and protease-activated receptor-2 (PAR-2) were examined. In an accompanying in vitro study, we performed LPS stimulation under GM administration in a mouse macrophage cell line and measured the quantity of HMGB1 and cytokines in the supernatant, and cell signal in the cells.

Histologic examination revealed that interstitial edema, leukocytic infiltration, and HMGB1 protein expression were markedly reduced in the GM + LPS group compared wih the LPS group. Furthermore, LPS-induced increases in PAI-1 and PAR-2 activity and in plasma HMGB1 concentrations were lower in the rats given both GM and LPS than in the rats given LPS alone. Release of HMGB1 and cytokines from the cell after the administration of LPS were decreased by GM. Phosphorylation of IκB was inhibited by GM. GM may have inhibited PAI-1 and PAR-2, thereby indirectly inhibiting HMGB1 and reducing tissue damage in the lung. This indicates that GM can inhibit lung injury induced by LPS in rats. GM is a candidate for use in novel strategies to prevent or minimize lung injury in sepsis.

Introduction

Sepsis is a serious condition with significant morbidity and mortality. Despite recent advances in therapeutic methods, sepsis remains the major cause of death in intensive care units with a mortality rate of nearly 30% 1, 2. Sepsis generates an excessive inflammatory response, which may result in coagulopathy, acute respiratory distress syndrome (ARDS), and multiple organ failure. As such, numerous clinical trials of anti-inflammatory strategies for the treatment of sepsis have been conducted over the past several decades. Unfortunately, due to variation in immune system activity over the course of the disease, an anti-inflammatory strategy may not be helpful, and could even be harmful in certain patients [3]. Consideration of these facts prompted us to identify novel mediators of lethality in sepsis, such that an efficient treatment method may be developed.

Recently, it has been demonstrated that the high mobility group box 1 (HMGB1) protein plays a key role as a late-phase mediator of lipopolysaccharide (LPS) lethality and systemic inflammation [4]. HMGB1 is constitutively expressed in many cell types and is stored in the nucleus due to the presence of two lysine-rich nuclear localization sequences [5]. Extracellular HMGB1 plays a pathogenic role in infection- or injury-elicited inflammatory diseases. HMGB1 is first detectable in the circulation 8 h after onset of lethal endotoxemia and sepsis, and subsequently increases to plateau levels from 16 to 32 h [6]. HMGB1 can bind to receptors for advanced glycation end-products (RAGE), Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), and signaling occurs in part through these receptors. It is activated by the intracellular signaling pathway, such as NF-kappaB (NF-κB), and activates downstream cytokine release [7].

HMGB1 can stimulate the release of cytokines [6] and, conversely, cytokines can control the further release of HMGB1 into the extracellular space [8]. As such, HMGB1 enhances the inflammatory response in septic shock. Elevated concentrations of HMGB1 have been observed in the serum of patients who were septic, with the highest concentrations in nonsurvivors [6]. HMGB1 has also been shown to stimulate cell migration [9] and activate innate immune cells [10], as well as increase acute lung injury [11]. These observations demonstrate why HMGB1 has been categorized as an “alarmin,” but also why extracellular HMGB1 might be a potential novel therapeutic target.

The synthetic serine protease inhibitor gabexate mesilate (GM) shows various modes of biological activity, and is capable of blocking a number of steps in the inflammatory process and the coagulation system 12, 13, 14. Its primary activity is as an anticoagulant via the blockade of thrombin and active coagulation factors, but GM may also improve the disease profile during shock. Indeed, Murakami et al. showed that GM attenuates LPS-induced coagulation abnormalities and pulmonary vascular injury by inhibiting TNF-α production in rats, suggesting that GM might reduce both DIC and ARDS in patients with sepsis [12]. Although these findings suggest the protective effect of GM on lung injury, whether GM inhibits the sepsis-associated increase in the expression of HMGB1 is not known. To further investigate the mechanism by which GM reduces endotoxin-induced lung injury, we examined the effect of GM on HMGB1 production in the lungs of rats exposed to endotoxin.