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, Ca, 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 Ca and CaMK-II phosphorylation in the brain secondary to HIE, but Nec-1 failed to affect Ca. In conclusion, our results suggest HePC may alleviate cytoplasmic Ca overload by regulating p-RyR2, which inhibits the necroptosis in the brain, exerting neuroprotective effects on HIE.
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