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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 reactive oxygen species (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 and cell cycle assays in Rg3(S)-treated senescent HDFs. The levels of ROS, adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP) as well as the mitochondrial DNA copy number, nicotinamide adenine dinucleotide (NAD)+/1,4-dihydronicotinamide adenine dinucleotide (NADH) ratio, and NAD-dependent sirtuins expression were measured and compared among young, old, and Rg3(S)-pretreated old HDFs. Major signaling pathways of phosphatidylinositol 3-kinase/Akt, 5' adenosine monophosphate-activated protein kinase (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 downregulated 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 downregulated phosphatidylinositol 3-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. Keywords: Akt-mTOR-sirtuin signaling, Ginsenoside Rg3(S), Human dermal fibroblast, Reversal, senescence

Background: The cellular senescence of primary cultured cells is an irreversible process characterized by growth arrest. Restoration of senescence by ginsenosides has not been explored so far. Rg3(S) treatment markedly decreased senescence-associated β-galactosidase activity and intracellular reactive oxygen species levels in senescent human dermal fibroblasts (HDFs). However, the underlying mechanism of this effect of Rg3(S) on the senescent HDFs remains unknown. Methods: We performed a label-free quantitative proteomics to identify the altered proteins in Rg3(S)-treated senescent HDFs. Upregulated proteins induced by Rg3(S) were validated by real-time polymerase chain reaction and immunoblot analyses. Results: Finally, 157 human proteins were identified, and variable peroxiredoxin (PRDX) isotypes were highly implicated by network analyses. Among them, the mitochondrial PRDX3 was transcriptionally and translationally increased in response to Rg3(S) treatment in senescent HDFs in a time-dependent manner. Conclusion: Our proteomic approach provides insights into the partial reversing effect of Rg3 on senescent HDFs through induction of antioxidant enzymes, particularly PRDX3. Keywords: Ginsenoside Rg3(S), Human dermal fibroblast, Label-free quantitative proteomics, Restoration, Senescence