Activation of Ca 2+ ‐AMPK‐mediated autophagy by ginsenoside Rg3 attenuates cellular senescence in human dermal fibroblasts

Abstract

Dear Editor, Cellular senescence is a multifaceted process where permanent cell cycle arrest occurs under stresses, indicating the hallmark of aging decline in organisms. 1 Ginsenoside Rg3 (Rg3) has been reported to promote rejuvenation of replicatively aged human dermal fibroblasts (HDFs) via regulation of reactive oxygen species (ROS) and Akt-mTOR-Sirtuin signaling, respectively. 2,3 However, little study has examined the relationship between the regulation of senescence by Rg3 and autophagy, even though an increasing number of reports have assessed the role of Rg3 in autophagy regulation. 4 Therefore, the present study investigated the role of autophagy in Rg3-induced senescence retention in HDFs. As reported previously, 3 Rg3 reversed cellular replicative senescence in HDFs, exhibiting reduced SA-β-gal activity and attenuated TP53 and CDKN1A expression levels (Fig-ures 1A, B and S1A-C). To examine whether Rg3 can attenuate cellular senescence in the skin tissue in vivo, where aging skin negatively affects tissue repair, wound healing was analyzed in 4-month-old (young) and 18-month-old (old) mice. Immunohistochemistry staining of proliferating cell nuclear antigen and Ki-67 did not reveal a significant difference between young mice given either treatment ; however, old Rg3-treated mice exhibited remarkably more positively stained cells than old control mice (Fig-ure 1C, D). In addition, based on the antioxidant activity of Rg3 previously reported in HDFs, 2 we investigated the role of the Rg3-induced antioxidant effect in cellular senes-cence in HDFs. Rg3 treatment not only reduced intracellu-lar ROS level (Figure 1E), but also reversed H 2 O 2-induced senescence (Figures 1F and S2), suggesting that the antiox-idant activity of Rg3 can attenuate cellular senescence. Since activation of the NRF2 pathway has been identified as a major antioxidant mechanism, we explored the relevancy of Rg3's antioxidant effect to NRF2 signal-ing. Rg3-treated HDF cells exhibited increased levels of This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. NRF2 protein and its downstream target gene, HMOX1 (Figure 2A). Moreover, Rg3-treated aging cells showed more sensitive response against H 2 O 2-derived oxidative stress with increased HMOX1 level, whereas nontreated senescent cells exhibited poor response (Figures 2B and S3). Recent studies have revealed that autophagy can enhance NRF2 stability, through promoting SQSTM1-sequestration-induced KEAP1 degradation. 5 Therefore, we investigated the role of autophagy in Rg3-induced NRF2 activation. Notably, HMOX1 induction in Rg3-primed HDFs was abolished upon treatment with the autophago-some formation inhibitor 3-methyladenine (Figure 2C). In addition, given that the SQSTM1 sequestration of KEAP1 is triggered after SQSTM1 phosphorylation, 6 the phosphory-lation state on serine 351 of SQSTM1 was explored, resulting in increased phosphorylation upon Rg3 treatment (Fig-ure 2D). Moreover, the abolished efficacy of Rg3 on the downregulation of SA-β-gal activity upon autophagy inhibition indicated the pivotal role of autophagy in Rg3-mediated senescence regulation (Figure S4A). Similar to the results of many studies reporting decreased autophagy in aged cells, 7 HDF cells in this study exhibited the inhibition of autophagy flux when under replicative senescence (Figure S4B). A recent study revealed that AMPK induces the phos-phorylation of SQSTM1 at serine 351 to enhance NRF2 activation. 8 As we previously identified AMPK activation by Rg3, the possible role of AMPK in Rg3-mediated NRF2 activation was investigated. AMPK knockdown abolished HMOX1 expression level upon Rg3 treatment (Figure 3A). Notably, Rg3-induced SQSTM1 phosphory-lation was also diminished by the pharmacological inhibition of AMPK using Compound C (CC) (Figure 3B). Moreover, Rg3 treatment induced both BECN1 phospho-rylation at serine 93 and LC3 conversion, whereas AMPK inhibitor treatment reverted (Figure 3C-E). In addition , tandem fluorescence-tagged LC3 (mRFP-EGFP-LC3) Clin. Transl. Med. 2021;11:e521. wileyonlinelibrary.com/journal/ctm2 1 of 7 https://doi.