Department of Stomatology, PuRen Hospital, Wuhan University of Science and Technology, Wuhan, 430081, Hubei, China.
Biological Cell Therapy Research Center, PuRen Hospital, Wuhan University of Science and Technology, Wuhan, 430081, Hubei, China.
Cell Biochem Biophys. 2024 Sep;82(3):2787-2795. doi: 10.1007/s12013-024-01394-7. Epub 2024 Jul 11.
The potential therapeutic benefits of human dental pulp stem cells (HDPSCs) in dental regenerative medicine have been demonstrated. However, little is known about the molecular mechanisms regulating the biological characteristics of HDPSCs. The experiment aims to explore whether VEGF activates signaling pathways such as FAK, PI3K, Akt, and p38 in HDPSCs, and to investigate the molecular mechanisms by which VEGF influences proliferation and migration of HDPSCs. Normal and inflamed human dental pulp (HDP) samples were collected, and the levels of VEGF in HDP were assessed. HDPSCs were cultured and purified. HDPSCs were stimulated with lipopolysaccharide (LPS) at gradient concentrations, and real-time quantitative polymerase chain reaction (qPCR) was used to assess changes in VEGF mRNA. Gradient concentrations of VEGF were used to stimulate HDPSCs, and cell migration ability was evaluated through scratch assays and Transwell chamber experiments. Phosphorylation levels of FAK, AKT, and P38 were assessed using Western blotting. Inhibitors of VEGFR2, FAK, AKT, P38, and VEGF were separately applied to HDPSCs, and cell migration ability and phosphorylation levels of FAK, AKT, and P38 were determined. The results indicated significant differences in VEGF levels between normal and inflamed HDP tissues, with levels in the inflamed state reaching 435% of normal levels (normal: 87.91 ng/mL, inflamed: 382.76 ng/mL, P < 0.05). LPS stimulation of HDPSCs showed a significant increase in VEGF mRNA expression with increasing LPS concentrations (LPS concentrations of 0.01, 0.1, 1, and 10 μg/mL resulted in VEGF mRNA expressions of 181.2%, 274.2%, 345.8%, and 460.9%, respectively, P < 0.05). VEGF treatment significantly enhanced the migration ability of HDPSCs in Transwell chamber experiments, with migration rates increasing with VEGF concentrations (VEGF concentrations of 0, 1, 10, 20, 50, and 100 ng/mL resulted in migration rates of 8.41%, 9.34%, 21.33%, 28.41%, 42.87%, and 63.15%, respectively, P < 0.05). Inhibitors of VEGFR2, FAK, AKT, P38, and combined VEGF stimulation demonstrated significant migration inhibition, with migration rates decreasing to 8.31%, 12.64%, 13.43%, 18.32%, and 74.17%, respectively. The migration rate with combined VEGF stimulation showed a significant difference (P < 0.05). The analysis of phosphorylation levels revealed that VEGF stimulation significantly activated phosphorylation of FAK, AKT, and P38, with phosphorylation levels increasing with VEGF concentrations (P < 0.05). The VEGF/VEGFR2 signaling axis regulated the migration ability of HDPSCs through the FAK/PI3K/AKT and P38MAPK pathways. This finding highlighted not only the crucial role of VEGF in injury repair of HDPSCs but also provided important clues for a comprehensive understanding of the potential applications of this signaling axis in dental regenerative medicine.
牙髓干细胞（HDPSCs）在牙科再生医学中的潜在治疗益处已得到证实。然而，人们对调节 HDPSCs 生物学特性的分子机制知之甚少。本实验旨在探讨 VEGF 是否能激活 HDPSCs 中的 FAK、PI3K、Akt 和 p38 等信号通路，并研究 VEGF 影响 HDPSCs 增殖和迁移的分子机制。收集正常和炎症牙髓（HDP）样本，评估 HDP 中的 VEGF 水平。培养和纯化 HDPSCs。用梯度浓度的脂多糖（LPS）刺激 HDPSCs，用实时定量聚合酶链反应（qPCR）评估 VEGF mRNA 的变化。用梯度浓度的 VEGF 刺激 HDPSCs，通过划痕实验和 Transwell 室实验评估细胞迁移能力。用 Western blot 检测 FAK、AKT 和 P38 的磷酸化水平。分别用 VEGFR2、FAK、AKT、P38 和 VEGF 的抑制剂处理 HDPSCs，测定细胞迁移能力和 FAK、AKT 和 P38 的磷酸化水平。结果表明，正常和炎症 HDP 组织之间的 VEGF 水平存在显著差异，炎症状态下的水平达到正常水平的 435%（正常：87.91ng/ml，炎症：382.76ng/ml，P<0.05）。LPS 刺激 HDPSCs 显示，随着 LPS 浓度的增加，VEGF mRNA 表达显著增加（LPS 浓度为 0.01、0.1、1 和 10μg/ml 时，VEGF mRNA 表达分别为 181.2%、274.2%、345.8%和 460.9%，P<0.05）。VEGF 处理显著增强了 Transwell 室实验中 HDPSCs 的迁移能力，随着 VEGF 浓度的增加，迁移率增加（VEGF 浓度为 0、1、10、20、50 和 100ng/ml 时，迁移率分别为 8.41%、9.34%、21.33%、28.41%、42.87%和 63.15%，P<0.05）。VEGFR2、FAK、AKT、P38 的抑制剂和联合 VEGF 刺激均显示出显著的迁移抑制作用，迁移率分别降至 8.31%、12.64%、13.43%、18.32%和 74.17%。联合 VEGF 刺激的迁移率有显著差异（P<0.05）。磷酸化水平分析表明，VEGF 刺激显著激活了 FAK、AKT 和 P38 的磷酸化，磷酸化水平随 VEGF 浓度的增加而增加（P<0.05）。VEGF/VEGFR2 信号轴通过 FAK/PI3K/AKT 和 P38MAPK 途径调节 HDPSCs 的迁移能力。这一发现不仅突出了 VEGF 在 HDPSCs 损伤修复中的关键作用，也为全面了解该信号轴在牙科再生医学中的潜在应用提供了重要线索。
