Figure 6.

Evaluation of the antisenescence properties of BMMSCs derived from melatonin-treated rats. BMMSCs were isolated from untreated and melatonin-treated (MT) sham-op or OVX rats and then exposed to H₂O₂ (100 μM) for 2 h. (a, b) Senescent cells were labeled by senescence-associated β-galactosidase (SA-β-gal) staining. Scale bar = 100 μm. (c) The cell viability of OVX BMMSCs was determined by CCK-8 assays. (d, e) The protein levels of SIRT1, P16, P21, and P53 were determined using Western blot assays. (f) After exposure to H₂O₂, BMMSCs were induced toward osteogenic differentiation. Matrix mineralization was determined by Alizarin Red S (ARS) staining. Scale bar = 200 μm. (g) The stained mineral layers were quantified. The values were normalized to those of the sham group. (h) The mRNA levels of osteoblast-specific marker genes, including Runx2, Sp7, and Bglap were quantified using qRT-PCR with Gapdh serving as the internal reference gene for normalization. Values are presented as the mean ± S.E.M of six independent experiments (n = 6) in SA-β-gal staining, eight independent experiments (n = 8) in cell viability assays, four independent experiments (n = 4) in ARS assays, three independent experiments (n = 3) in Western blot assays, and four independent experiments (n = 4) in qRT-PCR experiments. Statistically significant differences are indicated by ^∗p < 0.05 or ^∗∗p < 0.01 between the indicated groups; ^#p < 0.05 or ^##p < 0.01 versus the sham group.