Heliox Preconditioning Exerts Neuroprotective Effects on Neonatal Ischemia/Hypoxia Injury by Inhibiting Necroptosis Induced by Ca²⁺ Elevation

Our previous studies have indicated that heliox preconditioning (HePC) may exert neuroprotective effects on neonatal hypoxic-ischemic encephalopathy (HIE). The present study was to investigate whether HePC alleviates neonatal HIE by inhibiting necroptosis and explore the potential mechanism. Seven-day-old rat pups were randomly divided into Sham group, HIE group, HIE + HePC group, HIE + Dantrolene (DAN) group, and HIE + Necrostatin-1 (Nec-1) group. HIE was induced by common carotid artery ligation and subsequent hypoxia exposure. The neurological function, brain injury, and molecular mechanism were evaluated by histological staining, neurobehavioral test, Western blotting, Ca²⁺, immunofluorescence staining, co-immunoprecipitation (Co-IP), and transmission electron microscopy (TEM). Results supported that the expression of necroptosis markers and p-RyR2 in the brain increased significantly after HIE. HePC, DAN, or Nec-1 was found to improve the neurological deficits after H/I and inhibit neuronal necroptosis. Interestingly, both HePC and DAN inhibited the increases in cytoplasmic Ca²⁺ and CaMK-II phosphorylation in the brain secondary to HIE, but Nec-1 failed to affect Ca²⁺. In conclusion, our results suggest HePC may alleviate cytoplasmic Ca²⁺ overload by regulating p-RyR2, which inhibits the necroptosis in the brain, exerting neuroprotective effects on HIE.