Hydrogen gas inhalation alleviated cerebral ischemia/reperfusion injury by regulating mitophagy in SH-SY5Y cells and mice via PTEN-induced kinase 1/Parkin pathway

BACKGROUND: alleviates CIRI by modulating mitophagy and its upstream regulatory signaling pathways. METHODS: In vivo experiments were performed using male C57BL/6 mice subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) with mice randomly divided into three groups: Sham group, MCAO/R group, and MCAO/H₂ group. In vitro, human neuroblastoma SH-SY5Y cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), with four experimental groups: Control group, OGD/R group, OGD/R + H₂ group, and OGD/R + H₂+ML385 group (5 μM ML385, a specific Nrf2 inhibitor, pretreated for 1 h before OGD). Neurological function was assessed via neurological deficits score (zea-Longa); Cerebral infarct volume was measured by TTC staining; Neuronal histopathological damage and apoptosis were evaluated via HE, Nissl, and TUNEL staining; Cell viability was detected using CCK-8 assay; Cell apoptosis, mitochondrial reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were analyzed by flow cytometry; Protein expression levels were quantified by Western blotting. RESULTS: In vivo experiments demonstrated that H₂ inhalation markedly alleviated neurological deficits, reduced cerebral infarct volume and histopathological damage, inhibited neuronal apoptosis, and promoted mitophagy in MCAO/R mice. In SH-SY5Y cells, H₂ treatment significantly improved cell viability, attenuated oxidative stress and mitochondrial dysfunction, and enhanced mitophagy via activation of the PINK1/Parkin pathway. Mechanistically, H₂ maintained cellular redox homeostasis, cleared damaged mitochondria, upregulated the Nrf2/HO-1 antioxidant pathway, and suppressed NF-κB-mediated inflammatory signaling. Notably, inhibition of Nrf2 with ML385 significantly reversed the mitochondrial protective and anti-apoptotic effects of H₂ in OGD/R-exposed cells. CONCLUSION: exerts significant neuroprotective effects against CIRI by attenuating oxidative stress, inhibiting neuronal apoptosis, and improving mitochondrial function. These effects are closely associated with the activation of the Nrf2/PINK1/Parkin-mediated mitophagy pathway, highlighting H₂ as a potential therapeutic method for CIRI.