mTOR Inhibition Delays Replicative-induced Senescence of Human Gingival Fibroblasts

Objectives: This study investigated whether the mammalian target of rapamycin (mTOR) inhibition resultes in delay or reversal of cellular senescence on the human gingival fibroblasts (hGFs).

Methods: hGFs undergoing replicative-induced senescence were treated with 20nmol/L rapamycin for 3d and 30d. The levels of phosphorylated-S6 were measured by western blotting. Cell cycle, apoptosis and intracellular reactive oxidative species (ROS) were detected by flow cytometry. Rapamycin was withdrawn after 3day and 30day treatment, and then the senescence-associated beta-galactosidase (SA-β-Gal) staining and immunofluorescence analysis for Ki-67 were performed. The expression of cyclin-dependent kinase inhibitor (p16^INK4a, p21^CIP1a), senescence-associated cytokines (interleukin (IL)-1β, IL-6, IL-8 and VEGF) and antioxidant components (Cat, Sod2 and Prdx3) were quantitatively assessed by real-time PCR, western blotting and enzyme-linked immunosorbent assay (ELISA). All the data were statistically analyzed (ANOVA, Dunnett’s, p<0.05).
Results: 20nmol/L rapamycin could completely inhibit the mTOR signaling. There was no effect on cell viability and apoptosis, but rapamycin resulted in a modest increase of G1 fraction and an insignificant decrease in cell proliferation. After 3day and 30day treatment, the experimental groups leaded to fewer SA-β-Gal-positive cells (p<0.001) and preserved a higher capability of proliferation (indicated by the Ki-67 labeling index) comparing with untreated hGFs(p<0.05). This was consistent with the significant decrease in the expression of senescence-associated markers(p16^INK4a, p21^CIP1a, IL-1β, IL-6, IL-8 and VEGF) (p <0.05). On the other hand, rapamycin-treated hGFs showed higher mRNA levels of antioxidant components (Cat, Sod2 and Prdx3) (p <0.05) and the lower intracellular level of ROS.
Conclusions: mTOR inhibition may rejuvenate the aging hGFs through arresting the intracellular ROS formation and oxidative stress.