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

Figure 5

Silencing of SIRT3 offset the antisenescence effects of SPD on hUCMSCs. (a) The silencing efficiency of siRNA against SIRT3 (siSIRT3) or scrambled control (siScrambled) was assessed by western blotting and relative quantitative analysis. (b) A representative image showing SA-β-gal staining in siScrambled or siSIRT3 transfected hUCMSCs after SPD and H₂O₂ treatment,  μm. (c) The percentage of senescent cells stained in blue in each group was determined by quantitative analysis. (d) A representative image showing intracellular ROS levels by molecular probe H2DCFDA staining in hUCMSCs with indicated treatment;  μm. (e, f) Quantification of intracellular ROS levels was performed by fluorescence-activated cell sorting (FACS) analysis of stained cells to obtain the mean fluorescence intensity. (g) Immunofluorescence staining of γ-H2AX foci appearing red indicated DNA damage, while blue showed nuclei stained with Hoechst 33342;  μm. (h) Representative western blotting results showing the protein expression levels of SIRT3, p-P53-ser15, P53, or P21 in different groups. (i) Expression levels of SIRT3, p-P53-ser15, P53, and P21 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 signify a significant difference between the indicated groups; N.S.: not significant.

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