Mechanisms Of Necroptosis Induced Pathological Coagulation Response In Heatstroke

Objective: As global warming continues,heatstroke has become a major public health concern threatening human health worldwide,with a mortality rate of 23.3%.We have previously identified for the first time that the ZBP1-RIPK3-MLKL pathway-mediated Necroptosis plays an important contributory role in the development of heatstroke,but the exact pathophysiological mechanisms remain unclear.Heatstroke is associated with severe coagulation disorders,particularly disseminated intravascular coagulation(DIC),in which DIC causes extensive microthrombosis,tissue ischaemia and hypoxia,secondary multi-organ failure and subsequent bleeding tendencies.In recent years,numerous clinical studies have shown that the presence of DIC significantly increases the mortality rate of critical illnesses such as heatstroke,sepsis,severe pancreatitis,amniotic fluid embolism,malignancy and severe burns,and is an important factor in poor prognosis of patients.Therefore,the aim of this study was to investigate in depth whether necroptosis induces a pathological coagulation response in heatstroke and the associated mechanisms.Methods: 1.We first explored the effects of pathological coagulation induced by heatstroke on the organism in terms of overall function.On the one hand,We propose to use necroptosis-deficient mice(RIPK3knockout mice,MLKL knockout mice and ZBP1 knockout mice)and their related control mice to construct a heatstroke model,A comprehensive exploration of the impact of ZBP1-mediated necroptosis in the generation of pathological coagulation pathways from three different dimensions: circulating biomarkers of DIC(D-dimer,thrombin-antithrombin complex TAT,fibrinogen activator inhibitor PAI-1),histopathological injury and survival;On the other hand,we intervened with anticoagulants in the coagulation pathway of mice with heatstroke,and then observed the effect of pathological coagulation response caused by the above-mentioned signaling pathway on the development of DIC,organ damage and mortality.2.As tissue factor(TF)plays a crucial role in DIC,We aimed to investigate whether necroptosis facilitates TF release.On the one hand in animal experiments we used TF low expression mice,bone marrow transplanted mice(RIPK3 knockout,MLKL knockout bone marrow transplanted into WT mice)as well as in vivo macrophage clearance mice and their control mice to construct the aforementioned heatstroke model to detect TF release,circulating biomarkers of DIC(D-dimer,TAT,PAI-1),histopathological injury and survival rates.On the other hand,in cellular assays,we used necroptosis-deficient cells(RIPK3 knockout,MLKL knockout and ZBP1 knockout mouse peritoneal macrophages and bone marrow macrophages)and a human-derived mononuclear macrophage cell line(THP-1)given in vitro heatstress to detect the release of TF from cell supernatants and the exposure of phosphatidylserine(PS).Results: 1.Necroptosis-deficient mice(RIPK3 knockout mice,MLKL knockout mice and ZBP1 knockout mice)significantly reduced the elevation of circulating biomarkers of DIC(D-dimer,PAI-1,TAT),histopathological injury and mortality induced by heatstroke;Intervention of the coagulation pathway with anticoagulants in mice with heatstroke also significantly reduced the elevation of circulating biomarkers of DIC(D-dimer,PAI-1,TAT),histopathological injury and mortality.2.In animal studies TF low expression mice,bone marrow transplantation mice(RIPK3 knockout,MLKL knockout bone marrow transplanted into WT mice)and in vivo macrophage clearance mice significantly reduced TF release,elevated circulating biomarkers of DIC(D-dimer,PAI-1,TAT),histopathological injury and mortality induced by heatstroke.In cellular assays,necroptosis-deficient cells(RIPK3knockout,MLKL knockout and ZBP1 knockout mouse peritoneal macrophages and bone marrow macrophages)significantly reduced TF release as well as PS exposure under heatstress in vitro.Conclusions: 1.ZBP1-RIPK3-MLKL signaling-mediated necroptosis is a critical link in promoting pathological coagulation in heatstroke.2.ZBP1-RIPK3-MLKL signaling mediates heatstroke-induced pathological coagulation by promoting PS exposure,TF release.