Objective:

In this study, we investigated the effects of hydrogen sulfide (H₂S) and sonic hedgehog (SHH) on the proliferation, autophagy, and apoptosis of human microvascular endothelial cells (HCMEC/D3). We also explored the regulatory relationship between cystathionine-β-synthase (CBS) and the SHH pathway.

Method:

Human microglia cells (HMC3) were stimulated under hypoxia to secrete H₂S and SHH proteins, which were then co-cultured with HCMEC/D3 cells. The relationship between H₂S and SHH was investigated by inhibiting the CBS or SHH pathways. Vascular endothelial growth factor (VEGF) levels in the culture medium were detected using ELISA, while H₂S levels were detected via a micro method. The mRNA levels of hypoxia-inducible factor 1α (HIF-1 α), VEGF, Beclin-1, light chain 3 (LC3), and caspase-3 were detected using RT-PCR. Protein levels of CBS, SHH, extracellular regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (P-ERK1/2), Beclin-1, LC3, and Caspase-3 were determined by western blot.

Results:

The results indicated that HIF-1α expression increased in HMC3 under hypoxic conditions but did not change when either the SHH signaling pathway or CBS was suppressed. H₂S secretion of HMC3 increased during hypoxia, with both CBS and SHH proteins being up-regulated. The inhibition of CBS resulted in decreased levels of H₂S and SHH in HMC3. whereas SHH pathway is inhibited, the H₂S secretion level is not affected. H₂S and SHH proteins were found to enhance the expression of VEGF, P-EKR1/2, Beclin-1, and LC3, while decreasing the expression of caspase-3 in HCMEC/D3 cells.

Conclusion:

H₂S secretion of HMC3 promotes the proliferation and regeneration of HCMEC/D3 by regulating SHH protein and alleviating hypoxic injury.