BACKGROUND Magnesium (Mg2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium's function in the above process and to explore the associated signaling pathway. METHODS DPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl2. Intracellular Mg2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium's role and exploring ERK/BMP2/Smads signaling. RESULTS Mg2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg2+ increase. Consistently, the positive effect of high extracellular Mg2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg2+. These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK. CONCLUSIONS Mg2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. This study revealed that Mg2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.

中文翻译：

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富含镁的微环境通过激活ERK / BMP2 / Smads信号传导，促进人牙髓干细胞的牙源性分化。

背景技术富含镁（Mg2 +）的微环境可促进人牙髓干细胞（DPSC）中的牙源性分化，但调控机制仍未确定。这项工作的目的是评估上述过程中镁的功能，并探索相关的信号传导途径。方法DPSC在牙源性培养基中添加0、1、5或10 mM MgCl2进行培养。使用Mag-Fluo-4-AM通过流式细胞仪评估DPSC中的细胞内Mg2 +水平。Mg2 +进入被TRPM7抑制剂2-氨基乙氧基二苯硼酸酯（2-APB）抑制。进行RNA测序以评估在与高细胞外Mg2 +相关的牙源性分化过程中DPSC中转录组的变化。进行KEGG通路分析，以确定与检索到的差异表达基因（DEG）相关的通路。进行了免疫印迹，以评估镁的作用并探索ERK / BMP2 / Smads信号传导。结果富含Mg2 +的微环境通过细胞内Mg2 +的增加促进了DPSC中的牙源性分化。一致地，2-APB阻断了高细胞外Mg2 +对DPSC中牙源性分化的积极作用，这减少了Mg2 +的进入。RNA测序在高细胞外Mg2 +存在下鉴定了734个与DPSC中的牙源性分化有关的DEG。这些DEG参与了许多级联反应，例如MAPK和TGF-β途径。一致地，在高细胞外Mg2 +处理的DPSC中，ERK和BMP2 / Smads途径被激活。同意 U0126对ERK信号的抑制减弱了高细胞外Mg2 +对DPSC中矿化和牙源性分化的影响。有趣的是，U0126显着降低了BMP2，BMPR1和磷酸化Smad1 / 5/9，表明BMP2 / Smads充当ERK的下游。结论富含Mg2 +的微环境通过激活ERK / BMP2 / Smads信号通过细胞内Mg2 +的增加来促进DPSC中的牙源性分化。这项研究表明，富含Mg2 +的微环境可以用作牙髓再生的新策略。结论富含Mg2 +的微环境通过激活ERK / BMP2 / Smads信号通过细胞内Mg2 +的增加来促进DPSC中的牙源性分化。这项研究表明，富含Mg2 +的微环境可以用作牙髓再生的新策略。结论富含Mg2 +的微环境通过激活ERK / BMP2 / Smads信号通过细胞内Mg2 +的增加来促进DPSC中的牙源性分化。这项研究表明，富含Mg2 +的微环境可以用作牙髓再生的新策略。