Increased hydrogen sulfide turnover serves a cytoprotective role during the development of replicative senescence

The mammalian gasotransmitter hydrogen sulfide (H₂S) is produced by enzymes such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST). Prior studies suggest that H₂S may have cytoprotective and anti-aging effects. This project explores the regulation and role of endogenous H₂S in a murine model of replicative senescence. H₂S and polysulfide levels in RAW 264.7 murine macrophages (control cells: passage 5–10; senescent cells: passage 30–40) were measured using fluorescent probes. The expression of H₂S-related enzymes and the activity of senescence marker beta-galactosidase (SA-β-Gal) were also analyzed. CBS, CSE, and 3-MST were inhibited using selective pharmacological inhibitors. Senescence led to a moderate upregulation of CBS and in a significant increase in CSE and 3-MST. H₂S degradation enzymes were also elevated in senescence. Inhibition of H₂S-producing enzymes reduced H₂S levels but increased polysulfides. Inhibition of H₂S production during senescence suppressed cell proliferation, and elevated SA-β-Gal and p21 levels. Comparing young and old mice spleens revealed downregulation of CBS and ETHE1 and upregulation of rhodanese and SUOX in older mice. The results demonstrate that increased reactive sulfur turnover occurs in senescent macrophages and that reactive sulfur species support cell proliferation and regulate cellular senescence.