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: Cerebral ischemia-reperfusion injury (CIRI) causes severe neuronal damage following restoration of cerebral blood flow, and mitochondrial dysfunction acts as a core pathological driver of this process. Molecular hydrogen (H₂) has exhibited promising neuroprotective effects in multiple neurological disease models, yet it remains unclear whether H2 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: Our findings revealed that H2 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.