屎肠球菌 L15 胞外多糖通过 p38MAPK 通路促进人牙髓干细胞的成骨分化。
Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway.
机构信息
Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute, Seoul National University School of Dentistry, Seoul, 08826, Korea.
Biomedical Research Institute, NeoRegen Biotech Co., Ltd., Gyeonggi-do, 16641, Korea.
出版信息
Stem Cell Res Ther. 2022 Sep 2;13(1):446. doi: 10.1186/s13287-022-03151-0.
BACKGROUND
Bone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms.
METHODS
hDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria.
RESULTS
The calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture.
CONCLUSIONS
The stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.
背景
骨骼在人体中有重要功能。有几位研究人员报告称，微生物来源的多糖和脂多糖可促进干细胞的成骨分化。屎肠球菌是一种乳酸菌（LAB），可产生多种生物活性代谢产物，已广泛应用于食品和营养保健品行业。LAB 的胞外多糖（EPS）也因其后生元作用及其在体内和体外的功能而被广泛研究。然而，利用 LAB 多糖促进骨分化的研究还很少。因此，本研究旨在探讨粪肠球菌 L15 提取物和 EPS 对人牙髓干细胞（hDPSCs）成骨分化的影响，并探讨其潜在机制。
方法
从牙髓组织中获得 hDPSCs，并从 L15 中分离 L15 提取物和 EPS。通过 qPCR 和 Western blot 分析成骨分化标志物的基因和蛋白表达，并通过 Western blot 研究可能的信号通路。通过碱性磷酸酶（ALP）染色和茜素红 s（ARS）染色检测成骨分化潜能。此外，还在新生鼠颅骨的离体培养中探索了 L15 EPS 的成骨分化潜能。
结果
添加 L15 提取物或 EPS 可增强钙沉积和 ALP 活性。L15 提取物或 EPS 处理后的 hDPSCs 中 RUNX2、ALP 和 COL1A1 mRNA 的表达水平以及 RUNX2、ALP 和 BMP4 的蛋白表达水平均升高。L15 EPS 处理可增强丝裂原活化蛋白激酶（MAPK）p38 的磷酸化。p38 MAPK 抑制剂 SB203580 可抑制 L15 EPS 诱导的 RUNX2、ALP 和 BMP4 表达增加。L15 EPS 对成骨分化的促进作用不仅见于 hDPSCs，也见于成骨前体细胞。L15 EPS 处理可增加成骨前体细胞中的 ALP 活性以及 RUNX2、ALP 和 COL1A1 的表达。此外，L15 EPS 可增加新生鼠颅骨离体培养中的骨厚度。
结论
L15 提取物和 EPS 对成骨分化的刺激作用是通过 p38 MAPK 通路发生的，L15 EPS 可增强新生鼠颅骨的新骨形成。这些数据表明，L15 EPS 具有可应用于骨再生的治疗潜力。
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