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

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.


Introduction
With the growth of the world population and the economic development of developing countries, the demand for meat has increased rapidly [1][2][3]. It is estimated that by 2050, the global population will reach 9.5 billion [4]. To meet people's demand for animal-based protein, the global meat production in 2050 is expected to increase to 169% of that in 2018 [5]. It is clear that traditional animal agriculture based on livestock and meat production methods cannot maintain the growth in the meat demand and will further exacerbate environmental stress. Recently, cultivated meat (CM), also known as in vitro meat, clean meat, cell-based meat, or cultured meat, was used as an alternative source of animal protein, providing a possible solution to these problems. In fact, meat is a set of complex muscle tissues, with a structure with specific characteristics and properties. Therefore, compared with the term "cultivated meat," "food made with cultured animal cells" could describe this food more accurately at the current stage of development. Cultured animal cell food, as an important subfield of cellular agriculture, is produced in vitro using stem cells and tissue engineering, without sacrificing animals [6,7]. According to the ex ante life cycle assessment (LCA) of commercial-scale CM production in 2030 [8], compared to the traditional production of chicken, pork, and beef, it is estimated that industrialized CM production could reduce land use by 64%, 67%, and 55-90%, respectively. The carbon of Rg2 [37]. Rg2 has been shown to have a variety of pharmacological effects, including anti-oxidant [38], anti-inflammatory [38], anti-cancer [39], cardiovascular protection [40], and neuro-protection [41,42] activities. Our previous study confirmed that Rg2 can activate autophagy in multiple mouse tissues and effectively improve cognitive impairment in mice with Alzheimer disease [41]. Recently, the other two ginsenosides, Rg1 [43] and Rg3 [44], were found to increase human BMMSC proliferation and suppress senescence in vitro. Moreover, it is reported that Rg1 is also able to improve the proliferative capacity of hematopoietic stem cells [45] and neural stem cells [46]. However, the effect of ginsenoside Rg2 on the proliferation and senescence of MSCs is unclear.
In this study, the senescence characteristics and autophagic activities of porcine MSCs during long-term culture in vitro were evaluated. Next, using a D-galactose (D-gal)induced accelerated senescence model, we investigated the effect of ginsenoside Rg2 on the proliferation, senescence, and stemness of porcine MSCs and explored its potential mechanisms. Furthermore, whether Rg2 can stimulate the proliferation and maintain the stemness of porcine MSCs during long-term culture in vitro was also assessed.

Experimental Animals
In this study, 1-3-day-old pigs were obtained from the Tianjin Fushengyuan livestock farm. Animal experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at Tianjin University of Science & Technology.

Isolation and Culture of pMSCs
pMSCs were isolated from the femur and tibia of pigs according to the reported method with minor modifications [47]. Briefly, the femur and tibia were retrieved and rinsed twice with phosphate-buffered saline (PBS) containing 3% penicillin/streptomycin. After both ends of the femur and tibia were cut, the marrow was flushed out by inserting a syringe needle into the cut surface and centrifuged for 5 min at 1000 rpm at room temperature. The cells were resuspended in Dulbecco's modified Eagle's medium/F12 (DMEM/F12; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS; AusGeneX, Gold Coast, Australia) and cultured at 37 • C in a humidified atmosphere containing 5% CO 2 . Porcine MSCs were passaged with digestion with 0.25% trypsin containing 0.02% EDTA when they reached 80-90% confluence. Cellular morphology was observed and photographed using a phase-contrast microscope (Nikon Eclipse Ti, Nikon, Tokyo, Japan).
To measure the intracellular reactive oxygen species (ROS) level, cells were incubated with 10 µM of DCFH-DA (Nanjing Jiancheng Biotechnology, Nanjing, China) at 37 • C for 1 h, followed by washing and resuspending with PBS. Fluorescence was analyzed via flow cytometry (BD Biosciences, New York, NY, USA) with excitation at 500 nm and emission at 525 nm.

Adipogenic and Osteogenic Differentiation of MSCs
For adipogenic differentiation, porcine MSCs (1 × 10 5 /well) at P4 were seeded in 6-well plates until they reached 70%-80% confluence. These cells were first cultured in adipogenic induction medium for 3 days and sequentially in maintenance medium for another 3 days. Next, the two media were replaced alternately until 21 days. Adipogenic induction medium is composed of DMEM/F-12 supplemented with 10% FBS, 10 µM of dexamethasone (Solarbio, Beijing, China), 200 µM of indomethacin (Solarbio, Beijing, China), and 10 µM of insulin (Solarbio, Beijing, China), while maintenance medium is composed of basal medium supplemented with 0.2 nM of insulin. After 21 days, the cells were fixed with 4% paraformaldehyde and then stained with oil red O (Solarbio, Beijing, China).

Senescence-Associated β-Galactosidase (SA-β-Gal) Staining
To evaluate cellular senescence, β-gal activity was analyzed using a SA-β-Gal staining kit (Biyuntian, Beijing, China), following the manufacturer's instructions. Briefly, pMSCs at P5, P10, and P15 were plated in a 6-well plate, fixed with fixative solution for 15 min at room temperature, and washed three times with PBS. The cells were incubated overnight with freshly prepared staining solution at 37 • C in the absence of CO 2 . After washing with 70% ethanol, the aging cells were dyed blue. The number of these blue cells was counted under a inverted phase-contrast microscope (Nikon, Tokyo, Japan).

Cell Proliferation Assay
Cellular proliferation was detected according to the instructions of a Click-iT EdU (5-Ethynyl-2 -deoxyuridine) Cell Proliferation Kit (Meilunbio, Dalian, China). pMSCs at P5, P10, and P15 were plated in a 24-well plate and cultured overnight. For labeling cells with EdU, an equal volume of 2× EdU solution was added to the cells, and the cells were incubated at 37 • C for 2 h. The samples were then fixed and permeabilized. The nuclei were stained using the Hoechst 33342 (Meilunbio, Dalian, China) fluorescent stain. Digital images were acquired using a laser confocal microscope (OLYMPUS, Tokyo, Japan), and the number of EdU-positive cells were calculated using Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Silver Spring, MD, USA). EdU incorporation (the ratio of EdU-labeled cells to total cells) indicated the cellular proliferation rate.

Quantitative Real-Time PCR (qRT-PCR)
Total RNA was isolated from porcine MSCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription of the RNA sample to cDNA was carried out using M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed on a Applied Biosystems StepOneTM RT-PCR system (Applied Biosystems, Foster City, CA, USA) with the Fast SYBR1 Green Master Mix obtained from Applied Biosystems. Primers for each targeted mRNA were designed and are listed in Table 1. The 2 −∆∆Ct method was used to calculate the relative expression levels of target genes, and GAPDH was used as an internal control.

Drug Administration
To monitor autophagic flux, pMSCs at P5, P10, and P15 were treated with 150 nM of bafilomycin A1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h, and then, the protein samples were collected for LC3II detection. To assess the effect of Rg2 on cellular proliferation, Rg2 (Shanghai Yuanye Bio-Technology Co., Shanghai, China) was dissolved in DMSO and provided to the cells. pMSCs were treated with 25 µM, 50 µM, and 100 µM of Rg2 in DMEM/F-12 containing 1%, 5%, and 10% FBS for 24, 48, and 72 h, followed by MTT and EdU staining assays. To investigate the effect of Rg2 on D-gal-induced senescence, pMSCs were pre-treated for 24 h with 20 g/L of D-gal and then incubated with 100 µM of Rg2 in the presence/absence of D-gal for another 24 h, followed by MTT, EdU staining, SA-β-gal activity, and Western blot assays. The specific protein bands were visualized with the Odyssey Infrared Imaging System (LI-COR, Lincoln, Dearborn, MI, USA). The band density was analyzed using Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Silver Spring, MD, USA) using β-actin as an internal control and then normalized to the vehicle control.

Cell Viability Assay
For the detection of cellular viability, pMSCs (5 × 10 3 /well) were seeded in a 96well plate with 100 µL of the medium, followed by MTT assay. The cells were treated with 5 mg/mL of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Solarbio, Beijing, China) solution (10 µL per well) and then incubated for 4 h. The medium was then discarded, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance of each well was measured using a Synergy 4 plate reader (Bioteck, Winooski, VT, USA) with a wavelength of 490 nm. Absorbance was directly proportional to the number of surviving cells.

Measurement of Malondialdehyde (MDA) Contents and Superoxide Dismutase (SOD) Activities
After pre-incubation with 20 g/L of D-gal for 24 h, pMSCs were incubated with 100 µM of Rg2 in the presence/absence of D-gal for another 24 h, and then, the cells were collected and lysed. The contents of MDA and the activities of SOD were detected using commercial available kits (Solarbio, Beijing, China) according to the manufacturer's instructions.

Statistical Analysis
All data are shown as the mean ± SD, and all experiments were repeated at least three times. Statistical analysis was conducted using Microsoft Excel and GraphPad Prism 6. Two-tailed, unpaired Student's t-tests were performed to determine statistical significance when comparing two groups, and one-way ANOVA followed by a Dunnett multiplecomparison test was used when comparing more than two groups. A p-value of < 0.05 was considered statistically significant.

Isolation, Culture, and Identification of pMSCs
The primary cells isolated from porcine bone marrow were cultured in basic medium for 12 h and adhered to the wall. After 3-5 days, the cells began to fuse, and the rate of cell fusion reached 65-70% within 1 week. As shown in Figure 1A, porcine MSCs at P2 and P5 showed a spindle shape and strong proliferative capacity, while the cells after several passages gradually showed the characteristics of aging, such as a flat body, hypertrophy, and weak refraction. Almost all cells lost their ability of proliferation beyond passage 20.
To identify the immunophenotypes of the primary cells isolated, cellular surface markers CD34, CD44, CD45, and CD90 were analyzed in the cells at P3 using flow cytometry. The isolated porcine MSCs were strongly positive for CD44 (96.27 ± 0.13%) and CD90 (98.79 ± 0.05%) but negative for the hematopoietic lineage markers CD34 (0.08 ± 0.02%) and CD45 (0.04 ± 0.01%); see Figure 1B. In addition, the multi-lineage differentiation ability of MSCs to adipocytes and osteoblasts was studied. Lipid droplets and positive oil red O staining were observed in the pMSCs exposed to adipogenic differentiation medium for 21 days ( Figure 1C), while calcified nodules and positive alizarin red S staining appeared in the cells exposed to osteogenic differentiation medium ( Figure 1D). These results indicated that the primary cells isolated from porcine bone marrow were mesenchymal stem cells.

Reduced Proliferation Potential and Stemness in pMSCs after Long-Time Culture
It is known that MSCs show reduced proliferation capacity and undergo replicative senescence with cellular expansion in vitro [48]. Here, some age-related changes were observed in pMSCs at P10 and P15 compared to the cells at P5. With an increase in the number of passages, the proportion of SA-β-gal-staining-positive cells significantly scaled up (Figure 2A,B), yet the proportion of EdU-positive cells decreased notably ( Figure 2C,D). Next, we detected the mRNA levels of the stemness gene OCT4 and the proliferative marker Ki67 in pMSCs at P5, P10, and P15. The mRNA levels of OCT4 and Ki67 significantly decreased in pMSCs at higher passage numbers ( Figure 2E,F). Consistent with the change in the OCT4 mRNA level, the protein level of OCT4 was downregulated in pMSCs at higher passage numbers ( Figures 2G,H and S1). Furthermore, a prominent increase in the protein level of the aging-related marker p53 was observed in pMSCs at P10 and P15 compared to the counterparts at P5 (Figures 2G,I and S1).

Impaired Autophagic Flux and Elevated ROS in pMSCs after Long-Time Culture
The relationship between MSC senescence and autophagy remains unclear and debatable [26]. To investigate the relationship between the autophagy and replicative senescence of pMSCs, the expression of microtubule-associated protein 1 light chain 3 (LC3) and cargo protein SQSTM1/p62 was tested in pMSCs at P5, P10, and P15 using Western blot. As shown in Figures 3A-C and S2, the relative protein levels of LC3-II and P62 in pBMSC significantly increased in aged pMSCs (P10 and P15) compared to young cells (P5). The increase in LC3-II indicates the combined results of increased autophagosome synthesis (activated autophagy induction) or suppressed autophagosome degradation (blockage of autophagic flux), while the accumulation of P62 indicates suppressed autophagic flux. To further distinguish between these two possibilities, BafA1 was used to block autophagosome-lysosome fusion ( Figure 3D). Treatment with BafA1 for 2 h resulted in a noticeable accumulation of LC3-II in young pMSCs at P5, suggesting activated autophagic flux. Compared with BafA1-treated cells at P5, BafA1-treated pMSCs at P10 exhibited a further increase in LC3-II levels ( Figures 3E,F and S2), suggesting that in the early stages of aging, pMSCs can promote autophagy induction to remove toxic substrates. Although increased autophagosome synthesis was observed in pMSCs at P10, the accumulation of P62 in these cells ( Figures 3A and S2) suggested a defect in the later stages of autophagy (a potential inhibition of autophagosome degradation). Combined with markedly enhanced P62 levels, these results illustrate that senescent pMSCs activate autophagy at an early stage in response to oxidative stress, but the weakened autophagic flux makes it insufficient for them to completely remove toxic substances. Importantly, there was no significant difference in LC3-II levels between P5 and P15 pMSCs along with BafA1, but a profound increase in LC3-II levels was observed in P15 pMSCs without BafA1, compared to P5 cells, indicating that in the late stages of aging, autophagic flux is further impaired in pMSCs ( Figures 3E,F and S2).
Oxidative stress can cause oxidative damage to organelles and proteins, leading to cell senescence [49]. Correspondingly, we detected the ROS levels in pMSCs at P5, P10, and P15 using flow cytometry. As shown in Figure 3G,H, compared with young cells at P5, aged pMSCs at P10 and P15 showed a marked increase in ROS levels. These results further indicate the attenuated ability of senescent cells to scavenge ROS.

Ginsenoside Rg2 Promoted the Proliferation of pMSCs
To evaluate the stimulatory effect of ginsenoside Rg2 on the proliferation of porcine MSCs, cells at P6 were treated with different concentrations of Rg2 (25, 50, and 100 µM) in DMEM/F12 containing 1%, 5%, and 10% FBS for 24 h, 48 h, and 72 h, and then, MTT assay was carried out. Our data showed that ginsenoside Rg2 at a concentration of 25-100 µM exhibits no cytotoxicity and that cellular viability increased remarkably with increasing Rg2 concentration ( Figure 4A-C). In addition, 100 µM of Rg2 showed the most significant proliferative effect under the condition of 1% serum ( Figure 4A). Furthermore, the number of EdU-staining-positive cells markedly increased in pMSCs treated with 50 and 100 µM of Rg2 ( Figure 4D,E). Our data showed that Rg2 can promote the proliferation of pMSCs in a concentration-and time-dependent manner.

Ginsenoside Rg2 Reversed D-Gal-Induced Senescence and Maintained Stemness in pMSCs
Next, the anti-senescence effect of Rg2 was assessed using a model of accelerated aging induced by D-gal. After pre-incubation with 20 g/L of D-gal for 24 h, pMSCs were treated with 100 µM of Rg2 in the presence/absence of D-gal for another 24 h and subsequently subjected to MTT, EdU staining, and SA-β-gal staining assays. Consistent with a previous study [50], we found that D-gal treatment significantly inhibited cell viability ( Figure 5A), reduced the numbers of EdU-positive cells ( Figure 5D,E) and increased the percentage of SAβ-gal-positive cells ( Figure 5B,C). These changes mediated by D-gal were reversed by the administration of 100 µM of Rg2 ( Figure 5A-E). Moreover, decreased OCT4 levels induced by D-gal were rescued by the addition of Rg2 (Figures 5F,G and S3), suggesting that Rg2 can contribute to the maintenance of pMSC stemness. Similarly, Rg2 significantly inhibited the D-gal-caused increase in the protein expression of P53 in pMSCs ( Figures 5F,H and S3). These results indicated that treatment with Rg2 effectively prevents the pro-senescence effects of D-gal on pMSCs.

Ginsenoside Rg2 Protected pMSCs against Oxidative Stress
To determine whether Rg2 can delay the senescence of pMSCs by reducing ROS levels, intracellular ROS levels were assessed in Rg2-treated pMSCs using flow cytometry. D-gal treatment markedly stimulated the production of ROS in pMSCs, whereas the effect was attenuated by the administration of Rg2 ( Figure 6A,B). Furthermore, MDA contents and SOD activities were detected in Rg2-treated pMSCs. The addition of Rg2 dramatically inhibited the D-gal-stimulated increase in MDA contents ( Figure 6C). SOD activities were significantly downregulated in D-gal-stimulated pMSCs, while Rg2 treatment partly reversed the D-gal-induced reduction in SOD activities ( Figure 6D). These results indicated that Rg2 prevents the senescence of pMSCs by increasing SOD activities and reducing ROS and MDA levels.

Ginsenoside Rg2 Induced Autophagy in pMSCs via the AMPK Signaling Pathway
To demonstrate whether the positive effect of Rg2 on the anti-senescence and stemness maintenance of pMSCs is related to autophagy induction, the protein expression of LC3 and P62 was tested in Rg2-treated pMSCs with/without D-gal using Western blot. Compared with the D-gal group, Rg2-treated cells showed increased LC3II expression and reduced P62 levels, indicating the activation of autophagy ( Figures 7A-C and S4). Our previous study confirmed that Rg2 can activate autophagy in multiple types of cells via the AMPK signaling pathway [41], but it is unknown whether Rg2 can activate the AMPK signaling pathway in porcine MSCs. Thus, we detected the expression of p-AMPK and AMPK in Rg2treated pMSCs with/without D-gal using Western blot. As shown in Figures 7D,E and S4, the relative protein level of p-AMPK/AMPK significantly increased in the Rg2 group compared to the D-gal group. These results further confirmed that autophagy activated by Rg2 can play a critical role in the anti-senescence and stemness maintenance of pMSCs via the AMPK signaling pathway.

Ginsenoside Rg2 Improved Longevity of pMSCs during Long-Term Culture
As Rg2 could maintain the stemness of pMSCs and stimulate proliferation, we next checked the effects of Rg2 on pMSCs during long-term culture. First, we checked the protein expression of OCT4 and P53 in pMSCs at P5, P10, and P15 in the presence/absence of Rg2. The protein expression of OCT4 significantly decreased in pMSCs at higher passage numbers, whether in Rg2-treated pMSCs or in cells without Rg2 ( Figures 8A,B and S5). However, higher OCT4 protein expression was observed in Rg2-treated cells compared to the control group. Similarly, Rg2 treatment also resulted in low expression of P53 protein ( Figures 8A,C and S5). Consistent with these results, the percentage of EdU-positive cells remarkably decreased in pMSCs at higher passage numbers, whereas the administration of Rg2 upregulated a percentage of EdU-positive cells ( Figure 8D,E). Meanwhile, we found that the administration of Rg2 downregulated the numbers of SA-β-gal-positive cells ( Figure 8D,F). Taking together, long-term culture of pMSCs with Rg2 can help maintain stemness and promote proliferation, as well as inhibit aging.

Discussion
MSCs have the potential of self-renewal and multi-directional differentiation, including adipocytes and muscle cells [11,[51][52][53][54], and thus are considered one of the most advantageous seed cells for cultured animal cell food [55]. However, the replicative senescence of porcine MSCs during in vitro expansion limits their application in the large-scale industrial production of cultured animal cell food [56]. Therefore, it is of great significance to explore an effective method to promote the proliferation and delay the senescence of porcine MSCs.
An increasing amount of evidence indicates that basal autophagy serves as a key mechanism to regulate the proliferation, differentiation, and stemness maintenance of adult stem cells, including MSCs [57,58], muscle stem cells (MuSCs) [29], and hematopoietic stem cells (HSCs) [59]. Human MSCs have been demonstrated to possess constitutive autophagic flux due to the observed LC3 conversion (LC3-I to LC3-II) [57,58]. Accumulation of cellular damage during senescence activates stem cell autophagic flux to remove toxic material and maintain their stemness. Emerging evidence has revealed that the activation of autophagy can eliminate ROS and oxidative proteins in aged MSCs, thus keeping their stemness and genomic integrity [28,60,61].
The role of autophagy in MSC aging seems puzzling due to some contrary reports. Zheng et al. observed the increased expression of autophagy-related protein, including LC3-II, ATG7, and ATG12, in aging rat MSCs, thus considering that autophagy is activated during cellular senescence [30]. However, the increase in LC3-II is the result of the combination of autophagosome formation and blockage of autophagic degradation. Thus, it is necessary to analyze autophagic flux by blocking autophagy with bafilomycin A1. Contrary to activated autophagy in aged MSCs [30], more studies support that autophagy activity is impaired during aging [31][32][33]. Compared with young BMMSCs, aged cells showed reduced expression of Atg7, Beclin1, and LC3II/I and the accumulation of P62, as well as fewer autophagosomes [32]. After chloroquine (CQ) treatment, young BMMSCs possessed more LC3 dots compared to aged cells, indicating that aged BMMSCs might be characterized by impaired autophagy [32]. In addition, autophagy markedly decreased in aged BMMSCs under normoxic and hypoxic conditions [31]. Here, we found that although the expression of LC3II increased in aged pMSCs compared to young counterparts, p62 proteins accumulated, suggesting the potential blockage of autophagic flux. Accordingly, the number of autophagosomes first increased and then decreased during pMSC senescence, confirmed by the addition of bafilomycin A1. Autophagic flux is significantly impaired due to the blockage of autophagic degradation in P15 pMSCs compared with P5 cells. These results indicate that in the early stage of senescence, pMSCs need to activate autophagy in response to oxidative stress and damaged proteins, while in the late stage of senescence, cells display a decline in autophagy function, thus leading to reduced clearance ability. In line with the impaired ROS clearance during senescence, increased ROS levels were observed in aged pMSCs. Our data indicate that the ability of aged pMSCs to remove toxic substrates might be defective.
The activation of autophagy could protect MSCs from oxidative stress, thus resisting aging and promoting proliferation. The autophagic agonist rapamycin has been reported to alleviate the senescent features of aged MSCs [32,62]. Hypoxia can promote the self-renewal and proliferation of MSCs by activating autophagy [63,64]. Contrarily, the inhibition of autophagy could promote aging in MSCs. The autophagic inhibitor 3-methyladenine (3-MA) aggravates the aging of MSCs [32,62]. It is reported that blocking autophagy with kynurenine accelerates senescence in mice BMMSCs via the aryl hydrocarbon receptor pathway [65].
Our previous study confirmed the positive effect of ginsenoside Rg2 on autophagy induction [41]. However, it is not clear whether Rg2 has a retarding effect on MSC aging. Here, we demonstrated that Rg2 promotes the proliferation of porcine MSCs and slows down senescence by activating autophagy. Similar to our results, a number of natural and synthetic compounds that can activate autophagy have been demonstrated to inhibit the senescence of MSCs and increase their proliferative potential [62,66,67]. Autophagy induced by curcumin protects canine BMMSCs against replicative senescence during in vitro expansion, defined by the increased colony-forming unit-fibroblastic (CFU-F) capacity and decreased SA-β-gal activities [62]. A combination of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sir-tuin1 (SIRT1), showed the protective effect of anti-senescence and proliferation promotion in MSCs [61]. Camphorquinone [67] and licochalcone D [66] have been reported to be able to activate autophagy via the adenosine-monophosphate-activated protein kinase (AMPK) signal pathway, therefore alleviating the H 2 O 2 -induced senescence of human BMMSCs in vitro and also inhibiting D-gal-induced aging in mice in vivo.
Reactive oxygen species are known to be important risk factors affecting the aging of mesenchymal stem cells [68][69][70]. It is known that ROS production increases with age, leading to oxidative DNA damage and decreased proliferation of stem cells [71]. D-gal induced accelerated senescence has been used as a conventional experimental model to study cell senescence [72,73]. Previous studies have also shown that D-gal can significantly induce senescence in MSCs by promoting ROS production [74]. Here, we also found that the percentage of SA-β-gal-positive cells significantly increased and the number of EdU-positive cells remarkably decreased in D-gal-treated pMSCs, whereas the changes were reversed with Rg2. Rg2 treatment also inhibited D-gal-induced upregulation of P53 expression and downregulation of OCT4 expression, suggesting that Rg2 prevents D-gal-mediated senescence in porcine MSCs. A recent report showed that Rg2 could delay D-gal-induced brain aging and recover impaired memory function in mice by increasing mitochondrial autophagy flux and relieving oxidative stress [42]. Similarly, ginsenoside Rg1 has been shown to have protective effects in multiple tissues of mice with D-galinduced aging through attenuating oxidative stress [75][76][77]. Upregulating autophagy with by rapamycin has been shown to inhibit ROS generation and attenuate senescence caused by D-gal in rat BMMSCs [78]. Furthermore, our data demonstrated that Rg2 can protect porcine MSCs against the oxidative stress signal triggered by D-gal, as evidenced by the enhanced SOD activity and reduced MDA and ROS levels. This result was coincident with a previous finding that Rg2 effectively inhibits oleic acid and palmitic acid (OA&PA)-induced ROS generation in mouse primary hepatocytes [79]. The combined treatment of Rg2 and Rh1 has been found to significantly suppress LPS-induced excessive ROS accumulation in HepG2 cells [38].
In addition, we found that 100 µM of Rg2 can significantly enhance the proliferative capacity of porcine MSCs and inhibit replicative senescence during long-term culture in vitro. A recent study focused on the positive effect of Rg2 on the proliferation of induced-pluripotent-stem-cell-derived endothelial cells (iPSC-ECs) for clinical application [91]. Similar to the concentration of Rg2 used in our study, 10-200 µM of Rg2 was found to remarkably upregulate the EdU-positive cellular number of iPSC-ECs after three passages [91]. Mechanically, the stimulatory effect of Rg2 on iPSC-EC proliferation depends on mTOR-independent AMPK/ULK1-mediated autophagy. Furthermore, two recent studies on the use of Rg2 in the development of functional foods reported that the working concentration of Rg2 in the cells is approximately 80 µM [35,89], which is similar to the concentration of Rg2 (25-100 µM) used in our study.

Conclusions
Taken together, our findings suggest that in the early stage of senescence, pMSCs enhance autophagosome formation in respond to oxidative stress, while in the late stage, aged cells display impaired autophagic flux, thus leading to reduced clearance ability. Furthermore, ginsenoside Rg2 improves the longevity of porcine MSCs by inducing AMPKmediated protective autophagy. Ginsenoside Rg2 may be an effective protector of MSC senescence induced by oxidative stress. These findings highlight the positive role of Rg2 in porcine MSC expansion in vitro.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12051075/s1, Figure S1: Original Images of figure 2 for Blots; Figure S2: Original Images of figure 3 for Blots; Figure S3: Original Images of figure 5 for Blots; Figure S4: Original Images of figure 7 for Blots; Figure S5: Original Images of figure 8 for Blots.
Author Contributions: L.C., methodology, investigation, writing-original draft, and writingreview and editing; C.Z., methodology and data curation; L.H., investigation and software; H.X., investigation and data curation; X.M., investigation and data curation; X.L., methodology and data curation; H.H., methodology and data curation; T.Z., writing-review and editing and project administration; N.W., methodology, writing-original draft, funding acquisition, writing-review and editing, and project administration. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.