Molecular hydrogen exerts neuroprotection against cerebral ischemia–reperfusion injury via inhibition of Nrf2/HO-1-dependent ferroptosis in mice

Cerebral ischemia–reperfusion (I/R) injury (CIRI) represents secondary brain damage induced by reperfusion after ischemic insult. Molecular hydrogen (H₂) is a highly permeable, high biosafe and selective antioxidant, which has emerged as a promising redox-modulating agent in various oxidative stress-related to disorders. Clinically, a balanced gas mixture composed of 66.7% (v/v) hydrogen and 33.3% (v/v) oxygen is a standardized, widely accepted inhalation formulation for H₂ delivery. This rational oxygen supplementation prevents hypoxia induced by pure high-concentration H₂ inhalation, thereby guaranteeing clinical safety. However, the precise mechanisms by which H₂ regulates the Nrf2/HO-1 signaling and neuronal ferroptosis in CIRI remaines poorly clarified. In vivo experiments were performed using C57BL/6 mice subjected to middle cerebral artery occlusion and reperfusion (MCAO/R), whereas in vitro studies were conducted using SH-SY5Y cells exposed to oxygen–glucose deprivation/reoxygenation (OGD/R). Infarct volume was assessed by TTC staining, and histopathological changes by HE and Nissl stainings. Protein expression of Nrf2‑mediated ferroptosis‑related markers was detected by Western blot and immunohistochemistry, and the mRNA levels of iron transport‑related genes were detected by qPCR. Oxidative stress, mitochondrial membrane potential (MMP), and apoptosis were measured via biochemical assays and flow cytometry. ML385 (an Nrf2 inhibitor) and ferrostatin‑1 (a ferroptosis inhibitor) were used for mechanistic validation. Inhalation of 66.7% H₂ / 33.3% O₂ markedly reduced cerebral infarction volume, neurological deficits, histopathological lesions and ferroptosis in MCAO/R mice. In SH‑SY5Y cells subjected to OGD/R, H₂ treatment improved the cell viability and MMP, while repressing cellular apoptosis and ferroptosis. H₂ also upregulated Nrf2, GPX4, and HO‑1 expression, similar to ferrostatin‑1, whereas ML385 pretreatment abolished these beneficial effects. This study first demonstrates that H₂ protects against CIRI by attenuating oxidative stress and ferroptosis via the Nrf2/GPX4/HO‑1 pathway, revealing a novel mechanism underlying the hydrogen‑mediated neuroprotection.