MHY2233 Attenuates Replicative Cellular Senescence in Human Endothelial Progenitor Cells via SIRT1 Signaling

Figure 4

Effect of MHY2233 on oxidative stress and cell survival: (a) young and senescent EPCs were collected and stained with carboxyl H2DFFDA to determine cellular ROS levels using flow cytometry ( per group). (b) After chronic treatment of senescent EPCs with DMSO, 10 nM MHY2233, 100 nM resveratrol, or 100 nM EX527, the percentage of apoptotic cells was measured by annexin V/PI labeling and FACS assessment of live cells (annexin V⁻/PI⁻), apoptotic cells (annexin V⁺/PI⁻), and dead cells (annexin V⁺/PI⁺). (c) Quantification of percentage of apoptotic cells plotted in a bar graph ( per group). (d) Cells were treated with DMSO, 10 nM MHY2233, 100 nM resveratrol, or 100 nM EX527 for 24 h and cotreated with or without 600 μM H₂O₂ for 30 min. Cells were collected and stained with carboxyl H2DFFDA to determine cellular ROS levels using flow cytometry ( per group). (e) Percentage of apoptotic cells was calculated by annexin V/PI staining and flow cytometry. (f) Percentage of apoptotic cells presented in a bar graph ( per group). (g) RNA was isolated, and relative mRNA levels of IL-6, IL-8, IL-1α, and IL-1β between DMSO- and 10 nM MHY2233-treated groups were determined by qRT-PCR (). (h) Cell lysates were used to determine protein levels of eNOS and p-eNOS in the various groups by western blot analysis. , , and ^###, vs. DMSO (control) and indicated groups by a one-way ANOVA test and unpaired Student’s -test.

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