Spermidine Retarded the Senescence of Multipotent Mesenchymal Stromal Cells In Vitro and In Vivo through SIRT3-Mediated Antioxidation

Figure 4

SPD blocked H₂O₂-induced hUCMSCs senescence. (a) A representative image showing SA-β-gal staining in hUCMSCs at PDL6 (control), cells treated with 100 μM H₂O₂, and cells precultivated with 10 μM SPD for 24 h before H₂O₂ treatment (H₂O₂+SPD);  μm. (b) The percentage of senescent cells stained in blue in each group was assessed by quantitative analysis. (c) A representative image showing intracellular ROS levels by molecular probe H2DCFDA staining in hUCMSCs with indicated treatment;  μm. (d, e) Quantification of intracellular ROS levels was performed using fluorescence-activated cell sorting (FACS) analysis of stained cells to obtain the mean fluorescence intensity. (f) Immunofluorescence staining of γ-H2AX foci appearing red indicated DNA damage, while blue showed nuclei stained with Hoechst 33342;  μm. (g, h) Representative western blotting results showing the protein expression levels of SIRT3, p-P53-ser15, P53, OCT4, SOX2, or P21 in different groups. Protein expression levels detected by western blotting were quantified using ImageJ software and normalized to the control group. Approximately 10⁵ cells per well were seeded in 6-well plates. All quantitative data were obtained from three independent experiments and presented as ; and indicate a significant difference between the groups.

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