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.

Exposure to probiotics in early life contributes to host intestinal development and prevention of necrotizing enterocolitis (NEC). Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen, can cause NEC, bacteremia, and meningitis in neonates, but the research of probiotics against C. sakazakii is limited relative to other enteropathogens. Here, the protective effect and mechanism of a novel probiotic Ligilactobacillus salivarius (L. salivarius) YL20 isolated from breast milk on C. sakazakii-induced intestinal injury were explored by using two in vitro models, including an C. sakazakii-infected intestinal organoid model and intestinal barrier model, as well as an in vivo experimental animal model. Our results revealed that L. salivarius YL20 could promote epithelial cell proliferation in intestinal organoids, rescue budding-impaired organoids, prevent the decrease of mRNA levels of leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), zonula occludens-1 (Zo-1) and Occludin, and reverse C. sakazakii-induced low level of Mucin 2 (MUC2) in intestinal organoids. Additionally, YL20 could inhibit C. sakazakii invasion, increase the expression of ZO-1 and occludin in C. sakazakii-infected HT-29 cells, and reverse TEER decrease and corresponding permeability increase across C. sakazakii-infected Caco-2 monolayers. Furthermore, YL20 administration could alleviate NEC in C. sakazakii-infected neonatal mice by increasing the mice survival ratio, decreasing pathology scores, and downregulating pro-inflammatory cytokines. Meanwhile, YL20 could also enhance intestinal barrier function in vivo by increasing the number of goblet cells, the level of MUC-2 and the expression of ZO-1. Our overall findings demonstrated for the first time the beneficial effects of L. salivarius YL20 against C. sakazakii-induced NEC by improving intestinal stem cell function and enhancing intestinal barrier integrity.