Effect Of ROS-induced Mitochondrial Dysfunction In Skeletal Muscle Cell Oncosis Caused By Heat Stroke

ObjectiveSevere heatstroke(sHS)is a heat disease with a core body temperature that rises above 40℃,and is accompanied by central nervous system dysfunction,which can cause systemic inflammatory reactions and multiple organ dysfunction.It has a high incidence and mortality.Current research suggests that rhabdomyolysis is a common complication of severe heat stroke and plays an important role in the occurrence and development of severe heat stroke,but the specific physiological and pathological mechanisms are not very clear.In this study,a heatstroke human skeletal muscle(HSKMC)cell model was established to study the heat stroke-induced skeletal muscle cell injury mode,Oncosis,and to explore the possible molecular mechanism of skeletal muscle cell oncosis induced by heatstroke and the expansion of the muscle the inflammatory response,which lays a foundation for further studying the specific mechanism of heatstroke-induced skeletal muscle cell oncosis,and provides new ideas for the clinical prevention and treatment of severe heatstroke.MethodsIn vitro experiments,human skeletal muscle cell lines were used to establish a heat stroke model of skeletal muscle cells.CCK8 was used to detect cell viability after heat shock,LDH was used to detect cytotoxicity changes,cell morphology changes were detected using ordinary light microscopy,and cell ultrastructure was observed using a transmission electron microscope Changes,Annexin V-FITC/PI double staining labeling flow cytometry to detect the rate of double positive cells(Annexin V+/PI+),Western Blot Western blot to detect the expression of oncosis-related protein porimin and the expression of apoptosis-related protein caspase-3、JC-1 fluorescence staining method to detect mitochondrial membrane potential damage,luciferase method to detect intracellular ATP level change,DCFH-DA method to detect intracellular reactive oxygen species(ROS)expression,and Elisa method to detect cellular inflammatory factors(TNF-α,IL-6,IL-1β)release levels.ResultsCompared with the control group,cellular viability decreased after heatstroke to human skeletal muscle cells,LDH release increased,cell morphology changes(cell shrinkage,enlargement and roundness)under normal light microscopy,cell ultrastructure changes(cell swelling,cytoplasmic vacuole,the mitochondria swelled obviously,degranulated,the double ridges disappeared,the perinuclear space widened,the nuclei condensed and contracted,and some envelope continuity was interrupted;in the later stage,the nucleoli dissolved and disappeared,the nuclei were fragmented,chromatin edges gathered,and the cytoplasm The structure is disintegrated and granular),the rate of double positive cells(Annexin V+/PI+)is increased,the expression of porimin protein is increased,the expression of caspase-3 protein is unchanged,the mitochondrial membrane potential is decreased,the intracellular ATP level is decreased,and intracellular ROS expression is increased,increased release of cytokines(TNF-α,IL-6,IL-1β).After treatment with ATP supplement adenosine triphosphate disodium,compared with the heat shock group,the rate of double positive cells(Annexin V+/PI+)decreased,the expression of porimin protein decreased,and the release of cytokine factors(TNF-α,IL-6,IL-1β)release is reduced.Compared with the heat stroke group,the ROS scavenger NAC intervention increased cell viability,reduced mitochondrial membrane potential damage,increased intracellular ATP expression,decreased double positive cell rate(Annexin V+/PI+),and decreased expression of porimin protein.ConclusionFirstly,we verified that heatstroke induced oncosis of HSKMC cells.Secondly,heat stroke-induced oncosis is associated with mitochondrial dysfunction and ATP depletion,and is mediated by excess ROS.The heat stroke-induced oncosis of HSKMC cells can amplify the inflammatory response.Finally,intervention with ROS scavenger NAC and ATP supplement disodium adenosine triphosphate can reduce reduce oncosis caused by heat shock and protect skeletal muscle cells.In summary,our research has initially demonstrated that skeletal muscle cells undergo oncosis in a model of heat stroke in skeletal muscle cells,and the molecular mechanism of skeletal muscle cells oncosis in severe heatstroke rhabdomyolysis has been preliminary explored.