Background: Ischemic stroke remains a leading cause of disability and mortality worldwide, with limited therapeutic options beyond acute reperfusion. Promoting angiogenesis and collateral circulation in ischemic penumbra is crucial for neurological recovery, yet the underlying mechanisms are not fully understood. This study investigates the regulatory role of hydrogen sulfide (H₂S) in angiogenesis and its associated molecular mechanisms in ischemic penumbra following transient focal cerebral ischemia in rats, with particular focus on examining the potential involvement of the Sonic hedgehog (SHH) signaling pathway.

Methods: Focal cerebral ischemia was induced via middle cerebral artery occlusion, with 90-minute reperfusion, in Sprague–Dawley rats. The rats were grouped as sham-7d, sham-14d, model-7d, model-14d, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137)-7d, and GYY4137-14d, according to the treatment method and length of observation. Neurofunctional assessments included Longa scale and beam walking tests. Plasma H₂S levels, cerebral blood flow, infarct volume, microvessel density, and expression of relevant proteins were measured using microassay, laser speckle imaging, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Ki67/CD31 co-immunofluorescence, double immunofluorescence, and Western blotting, respectively.

Results: In GYY4137-treated rats, neurobehavioral performance improved significantly and plasma H₂S levels also significantly increased, compared to those of the model group (p < 0.05). Furthermore, cerebral blood flow showed substantial recovery, infarct volumes were significantly reduced, and microvessel density was augmented (p < 0.05). Immunofluorescence analysis revealed colocalization of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and angiopoietin 1 (ANG1) with CD31-positive endothelial cells. Western blot analysis demonstrated that ANG1, FGF2, VEGF, and SHH proteins were upregulated while cystathionine-β-synthase was downregulated in response to GYY4137 treatment (p < 0.05).

Conclusion: H₂S upregulates the expression of VEGF, ANG1, and FGF2 proteins in the peri-infarct tissue of rats, accompanied by activation of the SHH signaling pathway, promoting angiogenesis and collateral circulation, thereby significantly improving neurological function. These findings suggest that the SHH signaling pathway may be involved in H₂S-mediated pro-angiogenic effects, although further mechanistic validation is warranted.