Proteomic identification of hippocalcin and its protective role in heatstroke‐induced hypothalamic injury in mice

Abstract Heatstroke is a devastating condition that is characterized by severe hyperthermia and central nervous system dysfunction. However, the mechanism of thermoregulatory center dysfunction of the hypothalamus in heatstroke is unclear. In this study, we established a heatstroke mouse model and a heat‐stressed neuronal cellular model on the pheochromocytoma‐12 (PC12) cell line. These models revealed that HS promoted obvious neuronal injury in the hypothalamus, with high pathological scores. In addition, PC12 cell apoptosis was evident by decreased cell viability, increased caspase‐3 activity, and high apoptosis rates. Furthermore, 14 differentially expressed proteins in the hypothalamus were analyzed by fluorescence two‐dimensional difference gel electrophoresis and identified by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Expression changes in hippocalcin (HPAC), a downregulated neuron‐specific calcium‐binding protein, were confirmed in the hypothalamus of the heatstroke mice and heat‐stressed PC12 cells by immunochemistry and western blot. Moreover, HPAC overexpression and HPAC‐targeted small interfering RNA experiments revealed that HPAC functioned as an antiapoptotic protein in heat‐stressed PC12 cells and hypothalamic injury. Lastly, ulinastatin (UTI), a cell‐protective drug that is clinically used to treat patients with heatstroke, was used in vitro and in vivo to confirm the role of HPAC; UTI inhibited heat stress (HS)‐induced downregulation of HPAC expression, protected hypothalamic neurons and PC12 cells from HS‐induced apoptosis and increased heat tolerance in the heatstroke animals. In summary, our study has uncovered and demonstrated the protective role of HPAC in heatstroke‐induced hypothalamic injury in mice.