HINT2-Mediated Mitochondrial Modulation Contributes to G-CSF Neuroprotection in Alcohol Use Disorder-Related Ischemic Stroke.

This study investigated the role of histidine triad nucleotide-binding protein 2 (HINT2) in regulating mitochondrial dysfunction and oxidative stress in the context of ischemic stroke associated with alcohol use disorder (AUD), and examined whether the neuroprotective effects of granulocyte colony-stimulating factor (G-CSF) are partially dependent on HINT2 signaling. AUD was modeled in male Sprague-Dawley rats using the two-bottle choice paradigm. Focal cerebral ischemia was induced via middle cerebral artery occlusion (MCAO) using the intraluminal filament technique. HINT2 expression was downregulated through administration of small interfering RNA. Animals were randomized into five experimental groups: Sham, AUD, MCAO, G-CSF treatment, and HINT2 inhibition. Mitochondrial membrane potential was assessed via JC-1 staining, and reactive oxygen species (ROS) levels were quantified using 2',7'-dichlorodihydrofluorescein diacetate. Protein expression of HINT2 was evaluated by western blotting. Infarct volume was measured using 2,3,5-triphenyltetrazolium chloride staining, and histopathological changes were analyzed using hematoxylin and eosin staining. Neurological function was evaluated using standardized behavioral assessments. G-CSF administration significantly reduced cerebral infarct volume and improved neurological outcomes in both the AUD and MCAO groups (p < 0.05). G-CSF also partially restored mitochondrial membrane potential and attenuated ROS production (p < 0.05). Under conditions of HINT2 suppression, the effects of G-CSF on ROS reduction and mitochondrial membrane potential were attenuated (p < 0.05). HINT2 expression was downregulated following ischemic insult but was partially restored by G-CSF treatment (p < 0.05). Neurological scores indicated improved functional recovery in G-CSF-treated animals, whereas outcomes were significantly impaired in the HINT2-inhibited group. The findings indicate that HINT2 contributes to the neuroprotective effects of G-CSF in AUD-related ischemic stroke by modulating mitochondrial function and reducing oxidative stress. The G-CSF-HINT2-mitochondrial axis may represent a potential therapeutic target for patients with ischemic stroke and comorbid AUD. Further investigation is warranted to elucidate the underlying mechanisms and therapeutic potential.