Hydrogen Sulfide Protects Against Cerebral Ischemia–Reperfusion Injury in Rats via S-Sulfhydrating NAMPT to Enhance Mitochondrial Function and Autophagy in Cerebrovascular Endothelial Cells

Objective: Cerebral ischemia–reperfusion (I/R) injury constitutes a pivotal pathological driver in cerebrovascular disorders such as stroke, yet effective therapeutic interventions remain scarce. This study explored whether hydrogen sulfide (H2S) mitigates endothelial cell damage in the cerebral vasculature during I/R by modulating nicotinamide phosphoribosyltransferase (NAMPT) activity and its S-sulfhydration status, consequently restoring mitochondrial integrity and energetic homeostasis. Methods: Primary cerebrovascular endothelial cells (ECs) were subjected to hypoxia/reoxygenation (H/R) conditions in vitro, while rats experienced middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo. The H2S donor sodium hydrosulfide (NaHS) was administered, and outcomes were evaluated through Western blot analysis, S-sulfhydration assays, mitochondrial functional tests, autophagy profiling, and neurobehavioral assessments. The contributions of NAMPT and S-sulfhydration were validated using FK866 and dithiothreitol (DTT), respectively. LC-MS/MS was employed to identify candidate S-sulfhydration sites on NAMPT triggered by H2S. Results: In cellular models, NaHS substantially boosted NAMPT enzymatic activity, elevated NAD+ and ATP levels, and enhanced cell survival. These protective benefits were nullified upon NAMPT inhibition with FK866 or reversal of S-sulfhydration via DTT. In animal studies, NaHS treatment significantly diminished infarct volume and ameliorated neurological deficits in MCAO/R rats; however, pretreatment with FK866 or DTT attenuated these benefits. Mechanistic investigations revealed that NaHS promoted S-sulfhydration of NAMPT, thereby activating autophagy of dysfunctional mitochondria. LC-MS/MS analysis confirmed enhanced S-sulfhydration at Cys39 and Cys397 residues of NAMPT following H2S exposure. Conclusions: H2S exerts neuroprotection against cerebral I/R injury in rats through S-sulfhydration-mediated activation of NAMPT, which improves mitochondrial performance and stimulates autophagy in cerebrovascular ECs.