A Preliminary Study On The Effects And Mechanism Of Ferroptosis In Rhabdomyolysis Induced By Exertional Heat Stroke

Objective:To investigate the role and molecular mechanism of ferroptosis in rhabdomyolysis induced by EHS.Methods:1.Establishment of a mouse model of rhabdomyolysis induced by exertional heat stroke through four relatively higher temperature and humidity conditions(37.5℃ 65%RH;37.5℃ 70%RH;39.5℃65%RH;39.5℃70%RH).Ambient condition of mortality rate of close to 50%was selected as experiment condition for EHS modeling.Meanwhile,a higher core temperature(Tc=42℃,Tc=42.5℃,Tc=43℃)was set for heatstroke.Core temperature of survival rate close to 50%was selected as endpoint core temperature for EHS mice.2.temperature curves,organ injury indicators and histological analysis for skeletal muscle and intravital 2-photon microscopy were used to examine calcium overload to verify the mouse model of EHS and skeletal muscle injury 3.Detection of relative makers of ferroptosis in skeletal muscle with EHS,such as expression level of GPX4 and ACSL4,the activity of GPX4,ultramicromorphologic structure of mitochondria by TEM,and the metabolites of lipid peroxidation of MDA,HETEs.4.Reverse inhibition experiment in vivo and in vitro.Ionomycin HS was used as a cell model for EHS in vitro.Ferroptosis was verified and lipid peroxidation products were detected.5.ACSL4 inhibitor Rosiglitazone and knock-down ACSL4 were performed in vivo and in vitro for ferroptosis validation and lipid peroxidation product level detection.6.ACSL4 molecular mechanisms of transcriptional expression.Transfection of YAP plasmid and YAP siRNA was carried out to test the expression of YAP,meanwhile,ACSL4 expression was detected.Results:l.This study successfully optimized a animal models of EHS-induced RM.2.Expression of GPX4 in gastrocnemius was down-regulated and ACLS4 expression was up-regulated in EHS mice.At the same time,the product of lipid peroxidation increased obviously,the mitochondrial crest disappeared and the outer membrane ruptured,and the free iron ions accumnulated.3.Ferrostain-1 could effectively improve skeletal muscle damage and decreased lipid peroxidation in mice with EHS.4.ACSL4 inhibitor Rosiglizatone and or knock down of ACSL4 could significantly improve skeletal muscle injury and in vitro cell damage and decrease lipid peroxidation in EHS mice.5.Transcription factor YAP could regulate the expression of ACSL4 under the condition of Ionomycin+hs.Conclusion:The model of EHS-induced RM was optimized in this study,which is consistent with the high mortality and high incidence of rhabdomyolysis in human.This study demonstrated that ferroptosis mediates RM induced by EHS for the first time.upregulation of ACSL4 expression promotes ferroptosis in the EHS-induced RM.transcription factors YAP could regulate ACSL4 transcription under Ionomycin HS conditions.This study was conducted from the perspecitve of programmed cell death.It is found that ferroptosis mediates rhabdomyolysis under the condition of EHS,which may provide a new strategy for clinical treatment of EHS with rhabdomyolysis.