HSP70 preserves brain microvascular endothelial integrity under heat stress associated with suppressed JNK-mediated apoptosis and mitophagy

Heat stress profoundly disrupts blood-brain barrier (BBB) integrity and brain homeostasis in both livestock and humans; yet, the underlying molecular mechanisms remain poorly understood. In this study, we used in vivo models (chickens and mice) and an in vitro brain microvascular endothelial cell model (bEnd.3 cells) to elucidate the mechanisms underlying heat stress-induced endothelial injury. Our results demonstrated that heat stress produced pronounced perivascular edema and endothelial oxidative injury, downregulated tight junction proteins, and impaired barrier function. Heat stress also induced severe mitochondrial structural damage, promoted mitochondrial fission, and triggered sustained activation of c-Jun N-terminal kinase (JNK) signaling, with consequent caspase-dependent Apoptosis and enhanced PTEN-induced putative kinase 1 (PINK1)/Parkin RBR E3 ubiquitin protein Ligase (Parkin)-mediated Mitophagy. Pharmacological intervention with 3-methyladenine and mdivi-1 indicated that heat stress-induced Autophagy and Mitophagy contributed to endothelial cell injury rather than providing cytoprotection. In addition, heat stress markedly increased heat shock protein 70 (HSP70) expression in both brain tissue and endothelial cells. Functional studies with HSP70 overexpression and knockdown, together with chemical activation and inhibition of JNK, revealed that HSP70 alleviated heat stress-induced endothelial oxidative damage, mitochondrial Apoptosis, and Mitophagy by suppressing JNK activation, thereby preserving BBB integrity. Finally, Co-immunoprecipitation and immunofluorescence analyses confirmed interaction between HSP70 and JNK. Collectively, this study advances our knowledge of heat stress-induced BBB dysfunction and identifies HSP70-mediated regulation of JNK as a potential therapeutic intervention, with important implications for reducing heat stress-induced mortality in poultry and mitigating central nervous system injury in humans with heatstroke.

Apoptosis; Blood–brain barrier; Heat shock protein; Heat stress; Mitophagy.