Stereoisomer-specific ginsenoside 20(S)-Rg3 reverses replicative senescence of human diploid fibroblasts via Akt-mTOR-Sirtuin signaling

Abstract Background The replicative senescence of human dermal fibroblasts (HDFs) is accompanied by growth arrest. In our previous study, the treatment of senescent HDFs with Rg3(S) lowered the intrinsic ROS levels and reversed cellular senescence by inducing peroxiredoxin-3, an antioxidant enzyme. However, the signaling pathways involved in Rg3(S)-induced senescence reversal in HDFs and the relatedness of the stereoisomer Rg3(R) in corresponding signaling pathways are not known yet. Methods We performed senescence-associated β-galactosidase (SA-β-gal) and cell cycle assays in Rg3(S)-treated senescent HDFs. The levels of ROS, ATP, and cAMP as well as the mitochondrial DNA copy number, NAD+/NADH ratio, and NAD-dependent Sirtuins expression were measured and compared among young, old, and Rg3(S)-pretreated old HDFs. Major signaling pathways of PI3-kinase/Akt, AMPK, and Sirtuin 1/3, including cell cycle regulatory proteins, were examined by immunoblot analysis. Results Ginsenoside Rg3(S) reversed the replicative senescence of HDFs by restoring the ATP level and NAD+/NADH ratio in down-regulated senescent HDFs. Rg3(S) recovered directly the cellular levels of ROS and the NAD+/NADH ratio in young HDFs inactivated by rotenone. Rg3(S) mainly down-regulated PI3-kinase/Akt through the inhibition of mTOR by cell cycle regulators like p53/p21 in senescent HDFs, whereas Rg3(R) did not alter the corresponding signaling pathways. Rg3(S) activated Sirtuin 3/PGC1α to stimulate mitochondrial biogenesis. Conclusion Cellular molecular analysis suggests that Rg3(S) specifically reverses the replicative senescence of HDFs by modulating Akt-mTOR-Sirtuin signaling to promote the biogenesis of mitochondria.