The coagulation status was evaluated through the measurement of plasma protein C and TAT concentrations. After heat exposure, plasma protein C levels were significantly suppressed compared with group NC, and recombinant soluble TM improved this suppression (Fig. 9A). TAT levels in group HS significantly increased compared with group NC. Recombinant soluble TM treatment before heat exposure suppressed increased TAT levels after heat exposure (Fig. 9B).
In this study, we showed that plasma levels of inflammatory mediators, such as TNF-α, IL-6, and HMGB1, increased after heat exposure in an experimental mouse model. Heat exposure induces liver dysfunction and coagulopathy. Recombinant soluble TM treatment before heat exposure improved the inflammatory process, liver injury, coagulopathy, and mortality after heat exposure. Recombinant soluble TM treatment decreased the plasma HMGB1 levels and mortality even with delayed treatment.
Previous studies demonstrated that plasma inflammatory cytokine level increases in patients with heatstroke. Elevated cytokine concentrations were correlated with the severity of heatstroke.5,6,12 The plasma levels of inflammatory cytokines such as TNF-α, IL-6, and IL-8 are increased in heatstroke patients.13 In addition, plasma IL-1β and IL-6 levels increased in experimental heatstroke mice.14 Our results also showed that these cytokines increase after heat exposure. Furthermore, in our experimental heatstroke model, plasma HMGB1 levels increased after heat exposure. A previous study demonstrated that HMGB1 is released from macrophages and monocytes which are activated by inflammatory cytokines.7 HMGB1 is also leaked from necrotic or damaged cells.15–19 In our results, TNF-α and IL-6 levels had already increased by the time plasma HMGB1 levels increased. In addition, immunohistochemical and histopathological examination showed that HMGB1 was observed in the hepatic macrophages and hepatic nuclei in heat-exposed mice. These results suggested that the increase of plasma HMGB1 after heat exposure was caused by the active secretion from activated macrophages and monocytes. Recent studies showed elevated plasma HMGB1 levels in experimental animal heatstroke models and in patients with heat stroke.20–22 Tong et al.21 suggested that the HMGB1 level is an indicator of the severity of illness and a useful mortality predictor in heatstroke patients. In this study, we demonstrated that soluble TM treatment decreased the plasma HMGB1 levels even with delayed treatment. This result suggested that recombinant soluble TM treatment may be a beneficial treatment for heatstroke patients.
After heatstroke, multiple organ failure, such as respiratory dysfunction, cardiovascular failure, renal dysfunction, and coagulopathy, occurred and finally lead to death.1,2 There are some studies regarding heat exposure–induced liver dysfunction.23–25 Liver injury is characterized by the accumulation of activated neutrophils in the liver.26,27 These activated neutrophils produce proinflammatory cytokines, such as IL-1 and TNF-α, and release reactive oxygen species as well as other mediators contributing to tissue injury. Liver levels of HMGB1 are increased in the setting of liver injury, and HMGB1 itself can produce liver injury by the accumulation of activated neutrophils.28–30 Therefore, regulating HMGB1 levels may be a strategy to suppress liver injury from activated neutrophils. In this study, we demonstrated that TM suppresses plasma HMGB1 levels and MPO activity in the liver of a mouse heatstroke model. Our results suggest that heat exposure induces neutrophil accumulation which results in liver injury; however, recombinant soluble TM has a protective effect on liver injury via suppression of neutrophil accumulation in the liver. Our results also demonstrated the anti-HMGB1 mechanism of recombinant soluble TM. Recombinant soluble TM administration decreases immunostaining reaction in the hepatocyte nuclei of HMGB1. Therefore, recombinant soluble TM has an effect on the intranuclear expression of HMGB1. Our results are supported by recent studies by Tong et al.25 and Dehbi et al.23 which demonstrated that inhibition of HMGB1 activity with the HMGB1 antibody or DNA-binding A Box protects the liver from heatstroke.
Many treatments have been reported as the potential HMGB1-inhibiting therapeutic agents in experimental endotoxemia or sepsis models.43–45 However, most of these agents are not effective in the clinical setting for humans. These agents suppress the systemic HMGB1 accumulation by attenuating HMGB1 release from damaged cells. Therefore, these agents cannot regulate HMGB1 already in the extracellular space. Previous reports demonstrated that TM inhibits the active secretion of HMGB1 by suppressing the activation of macrophages and monocytes.11 In addition, human soluble TM (ART-123) binds to HMGB1 protein and inhibits HMGB1 activity at the level of D1 (N-terminal lectin-like domain) of TM.46 Our results showed recombinant soluble TM’s protective effect on heat exposure–induced plasma HMGB1 level elevation even with delayed treatment. Therefore, recombinant soluble TM seems to have the ability to suppress HMGB1 directly.
There are some limitations in this study. First, we evaluated the inflammatory process, liver function, and coagulant status. Therefore, other vital organs such as heart, lungs, kidneys, or vasculature could be affected after heat exposure and soluble TM treatment. We are planning future studies of these functions and outcomes after heat exposure. Second, we demonstrated that soluble TM can reduce the levels of plasma TNF-α and IL-6 as well as HMGB1, however, considering HMGB1 as the factor that induces liver injury associated with heat exposure. Therefore, it is possible that TNF-α and IL-6 also contribute to liver injury. In this study, we focused on HMGB1; however, further studies are required to identify the major factor in heat exposure–induced organ injuries and mortality. Third, we did not show the direct effects of HMGB1 on heat exposure. We need to perform more mechanistic studies to support the hypothesis that HMGB1 is the main contributor to the development of liver injury after heat stroke.
In conclusion, this study demonstrated that recombinant soluble TM treatment improves the development of inflammatory processes, liver injury, coagulopathy, and mortality after heatstroke. Recombinant soluble TM treatment decreases plasma HMGB1 levels and mortality even with delayed treatment. These results suggest that recombinant soluble TM treatment may be a beneficial treatment for heatstroke patients.
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