Proteomic Identification Of Hippocalcin And Its Anti-appotosis Role In Heatstroke-induced Hypothalamic Injury In Mice

ObjectiveHeatstroke is a devastating condition that is characterized by severe hyperthermia and central nervous system dysfunction.Severe heat stroke can lead to multiple organ failure,accompany with high mortality and disability.Current research suggests that heatstroke is a hypothalamic thermoregulatory disorder caused by an imbalance between heat production and heat dissipation.Hypothalamic injury plays a key role in the development of severe heat stroke,but the specific mechanism is still unclear.In this study,a severe heatstroke mice model was established,and the histopathology,fluorescence two-dimensional differential gel electrophoresis analysis,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to identify hypothalamic lesions and to identify differentially expressed proteins in the hypothalamus.The chromoblastoma-12(PC12)cell line was used to establish a heatstroke neuron cell model to study the effects and its specific mechanism of hippocalcin on apoptosis to reveal the pathological process of severe heatstroke neurons.Finally,the role of ulinastatin and its specific possible mechanism in heat stroke was studied,in which ulinastatin was pretreated the in vivo and in vitro before heat stress.Aims to provide the basis and ideas for the clinical treatment and basic research of severe heat stroke.MethodsThe heatstroke mice model was established using a climate chamber with specific temperature and humidity.Histopathology was used to detect hypothalamic injury in mice with different severity of heat stroke.TUNEL was used to detect neuronal apoptosis in hypothalamic slices of severe heatstroke mice.Fourteen differentially expressed proteins in the hypothalamus were detected and identified by fluorescence two-dimensional differential gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,and hippocalcin(HPAC)was selected.In order to clarified the changes in its down-regulation and its role in hypothalamic injury in heat stroke.A rat pheochromocytoma cell line was used to establish a neuronal apoptosis detection system in vitro after heat stress.The cell viability after heat stress was detected by MTT,ANNEXIN V combined with 7-AAD double staining flow cytometry.The changes of HPAC and apoptosis-related protein caspase-3 were detected by Western blotting.The effects of HPAC on heat stress PC12 cells apoptosis were studied by transfecting cells with HPAC plasmid and HPAC targeting siRNA.Ulinastatin(UTI)is a cytoprotective drug widely used in heatstroke patient as a clinical treatment.The effects of UTI on heat stroke mice and heat stress PC12 cells are studied by UTI pretreatment in vitro and in vivo.ResultsBoth heatstroke mice and PC12 cells showed that heat stress significantly aggravated neuronal damage in the hypothalamus,showing a higher pathological score in hypothalamus tissue.In addition,cell viability decreased,with increased caspase-3 activity and apoptosis rate of flow cytometry after heat stress,suggesting that heat stress promotes neuronal cells apoptosis.Fourteen differentially expressed proteins in the hypothalamus were analyzed by fluorescence two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The results showed that these proteins were involved in energy and substance metabolism,cytodynamics,cell proliferation and apoptosis.The expression of HPAC was found to be decreased in hypothalamic of heatstroke mice and heat-stressed PC12 cells by immunochemistry and Western blotting.Further,HPAC overexpression and siRNA-HPAC transfection found that HPAC plays an anti-apoptotic role in neurons injury after heat stress.Finally,pretreatment with ulinastatin in vitro and in vivo found that it protected hypothalamic neurons and PC12 cells from heat-induced apoptosis by inhibit heat-induced down-regulation of HPAC expression,and also increase the heat tolerance of heatstroke animals.ConclusionHPAC is down-regulated in the hypothalamus of heatstroke mice and heat-stressed PC12 cells with anti-apoptotic function.Intervention with ulinastatin pretreatment inhibits the heat induced down-regulation of HPAC and reduces cell apoptosis.Ulinastatin can protects neurons from heat injury and increases the heat tolerance of heatstroke animals.In summary,our study reveals that HPAC has anti-apoptosis function and plays a protective role in heat stroke hypothalamic injury.