Fig. 5: BHB alleviates cellular senescence by promoting histone acetylation via HDAC inhibition.

a Experimental scheme of H₂O₂ (hydrogen peroxide) induced senescence, AcAc (5 mM) or BHB (5 mM) treatment and analysis. b Left: representative images of SA-β-gal staining in MLTC-1 cells of the indicated groups. Scale bar: 75 μm. Right: quantitative analysis of the SA-β-gal⁺ cells. n = 4 per group. c Left: representative images of senescence marker p21 in MLTC-1 cells of the indicated groups. Scale bar: 25μm. Right: quantitative analysis of the p21⁺ cells. n = 4 per group. d, e Quantitative RT-PCR analysis of senescence marker (p21, Cxcl10) in the indicated groups. n = 3 per group. f Progesterone production of MLTC-1 cells. n = 3 per group. g Right: Representative western blots for H3K9bhb and H3K9ac and Histone H3. Left: quantitative analysis of H3K9bhb and H3K9ac protein levels. Relative to Histone H3. MLTC-1 cells were treated with BHB (0, 1, 2, 3, 4, 5 mM) for 24 h before analysis. n = 3 per group. h Left: representative images of H3H9ac staining in MLTC-1 cells treated with BHB for 24 h. Scale bar: 20μm. Right: quantitative analysis of fluorescence intensity. n = 3 per group. i HDAC activity in MLTC-1 cells treated with BHB (5 mM) or vorinostat (1 μM). n = 3 per group. j Left: representative images of SA-β-gal staining in MLTC-1 cells of the indicated groups. Senescent cells were treated with BHB (5 mM) or vorinostat (1 μM) for 48 h before staining. Scale bar: 75μm. Right: quantitative analysis of the SA-β-gal⁺ cells. n = 3 per group. k–m Quantitative RT-PCR analysis of senescence markers (p16, p21, Cxcl10) in the indicated groups. Senescence cells were treated with BHB (5 mM) or vorinostat (1 μM) for 48 h before analysis. n = 3 per group. n Progesterone production of MLTC-1 cells. Senescent cells were treated with BHB (5 mM) or vorinostat (1 μM) for 48 h before analysis. n = 3 per group. Data were presented as mean ± SEM. Significance was determined by one-way ANOVA (b–g, j–n) or Kruskal-Wallis test (h). n represents the number of biological replicates in (b–g, j–n). Illustrations were created with BioRender. Source data are provided as a Source Data file.