Zhou Quanying, Lei Yi
Department of Stomatology, Wuhan Ninth Hospital, Wuhan, Hubei, 430080, China.
Heliyon. 2024 Aug 28;10(17):e37079. doi: 10.1016/j.heliyon.2024.e37079. eCollection 2024 Sep 15.
The neural differentiation of dental pulp stem cells (DPSCs) exhibits great potential in the treatment of dental pulp repair and neurodegenerative diseases. However, the precise molecular mechanisms underlying this process remain unclear. This study was designed to reveal the roles and regulatory mechanisms of the armadillo repeat-containing X-linked 3 (ARMCX3) in neural differentiation and inflammatory microenvironment in human DPSCs (hDPSCs).
We treated hDPSCs with porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to simulate the inflammatory microenvironment. Then the lentiviral vectors were introduced to construct stable cell lines with ARMCX3 knockdown or overexpression. The expression of neural-specific markers, ARMCX3 and inflammation factors were estimated by immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) assays. Additionally, we used IF assays and specific kits to investigate the regulatory role of ARMCX3 on reactive oxygen species (ROS) signaling. Moreover, a ROS inhibitor was utilized to verify whether ROS inhibition reversed the effects of ARMCX3 in Pg-LPS-treated hDPSCs.
This work illustrated that Pg-LPS treatment significantly enhanced ARMCX3 expression and inflammatory response, and inhibited neural differentiation in hDPSCs. ARMCX3 knockdown effectively accelerated neural differentiation and controlled inflammatory cytokines at a lower level in hDPSCs in the presence of Pg-LPS. Additionally, knockdown of ARMCX3 notably reduced ROS production and ROS inhibition effectively eliminated the roles of ARMCX3 overexpression in hDPSCs. Besides, all results were proved to be statistically significant.
This investigation proved that ARMCX3 affected neural differentiation and inflammation microenvironment in hDPSCs at least partly by mediating ROS signal. These findings provided a new perspective on the mechanism of neural differentiation of hDPSCs and help to better explore the therapeutic schedule of pulpitis and neurodegenerative diseases.
牙髓干细胞（DPSCs）的神经分化在牙髓修复和神经退行性疾病的治疗中具有巨大潜力。然而，这一过程背后的确切分子机制仍不清楚。本研究旨在揭示含犰狳重复序列的X连锁蛋白3（ARMCX3）在人牙髓干细胞（hDPSCs）神经分化和炎症微环境中的作用及调控机制。
我们用牙龈卟啉单胞菌脂多糖（Pg-LPS）处理hDPSCs以模拟炎症微环境。然后引入慢病毒载体构建ARMCX3基因敲低或过表达的稳定细胞系。通过免疫荧光（IF）、定量实时聚合酶链反应（qRT-PCR）和酶联免疫吸附测定（ELISA）检测神经特异性标志物、ARMCX3和炎症因子的表达。此外，我们使用IF检测和特定试剂盒研究ARMCX3对活性氧（ROS）信号的调控作用。此外，利用ROS抑制剂验证ROS抑制是否能逆转ARMCX3在Pg-LPS处理的hDPSCs中的作用。
本研究表明，Pg-LPS处理显著增强了hDPSCs中ARMCX3的表达和炎症反应，并抑制了神经分化。在Pg-LPS存在的情况下，敲低ARMCX3可有效加速hDPSCs的神经分化，并将炎症细胞因子控制在较低水平。此外，敲低ARMCX3可显著降低ROS产生，而抑制ROS可有效消除ARMCX3过表达在hDPSCs中的作用。此外，所有结果均具有统计学意义。
本研究证明，ARMCX3至少部分通过介导ROS信号影响hDPSCs的神经分化和炎症微环境。这些发现为hDPSCs神经分化机制提供了新的视角，有助于更好地探索牙髓炎和神经退行性疾病的治疗方案。
