Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine-PAGE, gene ontology (GO), and LC-MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 μM) became senescent; UPla (10 μg/mL), ULu (10 μg/mL), as well as positive controls lipoic acid (10 μg/mL) and transferrin (10 μg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-β-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine-PAGE revealed prominent detectable bands between 10 and 100 kDa. LC-MS/MS identified 150-180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with "extracellular space" and "proteasome core complex". UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.
Keywords: SA-β-X-gal; cell proliferation; cellular senescence; intracellular ROS; mitochondrial ROS; mitochondrial fission; senescence marker.