Ginsenoside Rg2 Promotes the Proliferation and Stemness Maintenance of Porcine Mesenchymal Stem Cells through Autophagy Induction

Abstract

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

Arctigenin from Fructus arctii Exhibits Antiaging Effects via Autophagy Induction, Antioxidative Stress, and Increase in Telomerase Activity in Yeast

“…Since stressed cells also showed positivity in the SA-β-Gal activity assay, and stressed cells may either recover from stress state or undergo senescence, assaying SA-β-Gal activity alone to demonstrate the anti-aging activity of ATG has limitations [ 33 ]. It is well known that cells gradually lose their ability to proliferate after entering senescence state, so we performed a cell proliferation assay using EdU to assess whether the cells had reached the senescent state after Eto induction [ 28 ]. As shown in the results, almost half of NIH/3T3 cells were shown EdU positive with green fluorescence in control group, while the percentage decreased to no more than 1% in Eto-treated cells ( Figure 1 h,i, p < 0.001).…”

“…To determine SA-β-Gal activity [ 28 ], approximately 50,000 PC12 or NIH/3T3 cells were seeded in each well of a 24-well plate and cultured in 5% CO 2 at 37 °C for 24 h. Then, the cells were treated with different tested samples. The positive and negative controls used were 0.5% DMSO and Rapa, respectively.…”

“…For 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assays [ 28 ], BeyoClick™ EdU-488 cell proliferation kit (Beyotime Biotechnology Inc., Shanghai, China) was used, and the assay was performed according to the manufacturer’s instructions. NIH/3T3 cells were cultured in different doses of ATG (1, 3, 10, 30 µM) and 100 nM Rapa as a positive control for 24 h and then treated with 0.3 µM Eto for 2 days.…”

“…3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the cell viability [ 28 ]. In general, 5000 NIH/3T3 cells were seeded into each well of a 96-well plate and cultured in 5% CO 2 at 37 °C for 24 h. Then, cells were incubated with different concentrations of ATG and Rapa (as a positive control) for one day.…”

Ginsenosides on stem cells fate specification—a novel perspective

“…Similarly, the senescence-associated secretory phenotype (SASP) resulting from DNA damage and oxidative damage associated with the aging of MSCs can be inhibited by ginsenoside Rg1, which promotes MSC proliferation by enhancing its antioxidant capacity, the activation of Nrf2 and PI3K/Akt is required for this process to occur ( Wang et al, 2021 ). Ginsenoside Rg2 activates AMPK-mediated autophagy restores pig MSCs proliferation and inhibits oxidative stress-induced replicative senescence in pig MSCs ( Che et al, 2023 ). Ginsenoside Rg3 mainly enhances the biogenesis ability of mitochondria and antioxidant function by promoting Ca 2+ concentration properly, thus improving proliferation and differentiation potential and preventing human MSCs from aging ( Hong et al, 2020 ).…”