Study On The Mechanism Of ZBP1-Induced Programmed Cell Death In Multiple Organ Failure Induced By Heatstroke

Heat stroke refers to a collective term for symptoms caused by the dysfunction of body temperature regulation,water and electrolyte metabolism,and damage to various systems,due to prolonged exposure to high temperature and humidity.Once it develops into severe heat stroke,multiple organ dysfunction syndrome(MODS)can occur,and the mortality rate can reach up to 60%.Previous views have suggested that the "direct cellular damage caused by heat" is the cause of heatstroke combined with MODS.However,although cooling treatment is given clinically,it cannot prevent the continuous progression of organ damage,and the specific mechanism is still unclear.There is a lack of targeted and effective drugs in clinical practice.As global temperatures continue to rise,the number of deaths caused by extreme heat exceeds the total number of deaths caused by other natural disasters.Heat stroke has become a major global public health and safety issue.Therefore,it is an urgent task for the academic community to clarify the pathogenesis of heat-induced mortality,and develop specific diagnostic and therapeutic methods and drugs.Purpose: To clarify the role of programmed cell death in heatstroke induced MODS,further clarify the molecular mechanism of programmed cell death in heat stress and explore the role and mechanism of glucocorticoids in heatstroke.Method: 1.Clarifying the role of programmed cell death in heatstroke induced MODS: In overall animal studies,Ripk3 knockout,Mlkl knockout,Ripk3 KD,Mlkl and Caspase-8 double knockout mice,Zbp1 knockout,Trif knockout,Ripk1 KD kinase mutant mice and their control mice were used to construct heatstroke animal model.Survival rate,programmed cell death related indicators,organ function damage indicators were measured,and DIC formation was observed using spinning disk confocal intravital microscopy established by our research group in the early stage;Invitro cell experiments,mouse primary macrophages and L929 cell lines were stimulated at 43℃ and the cell supernatant and cell protein were collected to detect the level of cell damage and the expression level of programmed cell death-related proteins.Multiple perspectives method were used to elucidate the role of ZBP1-RIPK3 pathway-mediated programmed cell death in MODS induced by heatstroke.2.Further elucidating the molecular mechanism of programmed cell death in heat stress: In vitro experiments,multidisciplinary methods including q PCR,Western Blot,bioinformatics prediction,chromatin immunoprecipitation were used to detect the expression of ZBP1 and its specific interactions with HSF1.Secondly,a series of Zbp1 mutant plasmids,adjacent junction technology,immune coprecipitation were constructed to determine the specific domain of ZBP1 activation caused by heat stress.Finally,biochemical,molecular biological,proteomic,and transmission electron microscopy techniques were used to further elucidate the specific mechanisms underlying heat-induced ZBP1 activation in severe heatstroke.3.Exploring the role and mechanism of glucocorticoids in heatstroke:In vitro experiments were conducted to explore the protective effect of glucocorticoids on programmed cell death levels caused by heat stress and its specific mechanisms.In vivo experiments were conducted to explore the protective effects of glucocorticoids on mouse mortality and organ damage in heat stroke;Result: 1.The role of programmed cell death in heatstroke induced MODS: 1)Heat stroke significantly increased the mortality rate of wildtype mice,induced functional and pathological damage to various organs,as well as DIC formation,and activated programmed cell death related proteins such as RIPK3,MLKL,Caspase3,GSDME in tissues.Gene knockout Ripk3 significantly alleviated the formation of organ damage and programmed cell death levels induced by heat stroke.In vitro cell experiment,Heat stress can directly cause programmed cell death related protein activation such as RIPK3 and its substrate MLKL phosphorylation.Knockout of the Ripk3 gene significantly inhibited programmed cell death levels and related protein expression activation caused by heat stress;2)Ripk3 KD kinase mutant mice and Mlkl knockout mice improved the mortality rate of severe heat stroke by about 70%,while Mlkl and Caspase-8 double knockout mice blocked the mortality rate caused by heat stroke;3)Gene knockout of Zbp1 significantly improved mortality and organ function damage in heatstroke mice.Zbp1 knockout primary macrophages or L929 knockout cell lines significantly reduced the release of LDH caused by heat stress and the activation of programmed cell death indicators.2.The molecular mechanism of programmed cell death in heat stress:1)Heat stress activated HSF1 phosphorylation and trimer formation,which in turn increased ZBP1 expression by HSF1 binding to the Zbp1 promoter;2)Heat stress mainly promoted the aggregation and activation of ZBP1 through the RHIM domain rather than the Zα domain,and further promoted RIPK3 phosphorylation through interaction with RIPK3,as well as downstream programmed cell death related protein expression activation;RHIM domain mutation or deletion significantly inhibited the level of cell death induced by heat stress;3)Heat stress mainly activated ZBP1 through amyloidosis.3.Exploring the role and mechanism of glucocorticoids in heatstroke:In vitro experiments had shown that glucocorticoids significantly reduced the level of programmed cell death and related protein expression activation caused by heat stress,mainly by reducing the formation of MLKL oligomerization caused by heat stress.Glucocorticoids significantly improved the activation level of programmed cell death proteins in various tissues and organs,as well as the mortality rate caused by heat stroke.Conclusion: 1.Heatstroke can activate ZBP1-RIPK3 pathway mediated programmed cell death,leading to MODS and death.2.Heat stress upregulates the expression of ZBP1 through HSF1 and promotes ZBP1 aggregation through the RHIM domain.3.Glucocorticoids improve organ dysfunction and mortality in heatstroke by reducing programmed cell death levels.