Deng Qianyi, Liu Qin, Zhang Huini, Fan Wenguo, Li Jingzhou, Kang Jun, He Hongwen, Huang Fang
Department of Pediatric Dentistry, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China.
Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China.
J Dent Sci. 2019 Dec;14(4):370-377. doi: 10.1016/j.jds.2019.05.003. Epub 2019 Jul 23.
BACKGROUND/PURPOSE: Melatonin, at physiological concentrations, was previously found to inhibit proliferation and promote odontogenic differentiation in human dental pulp cells (hDPCs), but its effect on apoptosis is unclear. Our study aimed to investigate the effect of melatonin on the HO-mediated viability reduction and apoptosis in hDPCs.
hDPCs were treated with HO (0, 250, 500, 1000 μmol/L), melatonin (0, 10, 10, 10 mol/L), and melatonin with HO for 24 h. CCK-8 assays were performed to evaluate cell viability. Apoptosis was measured by DAPI and Annexin V/propidium iodide staining. Intracellular reactive oxygen species (ROS) were measured by CellROX® staining and mitochondrial membrane potential (ΔΨm) was examined by JC-1 staining.
HO obviously decreased the viability of hDPCs in a concentration-dependent manner and melatonin alone also reduced viability by 16-20%. Melatonin was also found to enhance HO-induced toxicity in a concentration-dependent manner, and the highest physiological concentration of melatonin (10 mol/L) had the most obvious effect ( < 0.001). Treating HO-exposed hDPCs with melatonin significantly increased the ratio of apoptotic cells with condensed and deformed nuclei ( < 0.001), as well as the percentage of Annexin V-positive cells ( < 0.01). Furthermore, melatonin significantly increased intracellular ROS levels and induced the loss of ΔΨm in HO-exposed cells ( < 0.05).
Our results indicate that melatonin, at physiological concentrations, can enhance HO-induced apoptosis in hDPCs and increase HO-mediated ROS production and ΔΨm loss. Further studies are needed to investigate whether melatonin targets the mitochondrial death pathway during the process.
背景/目的：先前发现生理浓度的褪黑素可抑制人牙髓细胞（hDPCs）的增殖并促进其牙源性分化，但其对细胞凋亡的影响尚不清楚。我们的研究旨在探讨褪黑素对hDPCs中血红素加氧酶（HO）介导的活力降低和细胞凋亡的影响。
将hDPCs分别用HO（0、250、500、1000μmol/L）、褪黑素（0、10、10、10mol/L）以及褪黑素与HO共同处理24小时。进行CCK-8检测以评估细胞活力。通过DAPI和膜联蛋白V/碘化丙啶染色检测细胞凋亡。通过CellROX®染色测量细胞内活性氧（ROS）水平，并用JC-1染色检测线粒体膜电位（ΔΨm）。
HO明显以浓度依赖的方式降低hDPCs的活力，单独使用褪黑素也使活力降低了16 - 20%。还发现褪黑素以浓度依赖的方式增强HO诱导的毒性，褪黑素的最高生理浓度（10mol/L）作用最明显（P<0.001）。用褪黑素处理暴露于HO的hDPCs显著增加了核浓缩和变形的凋亡细胞比例（P<0.001）以及膜联蛋白V阳性细胞的百分比（P<0.01）。此外，褪黑素显著增加了暴露于HO的细胞内ROS水平并诱导了ΔΨm的丧失（P<0.05）。
我们的结果表明，生理浓度的褪黑素可增强HO诱导的hDPCs凋亡，并增加HO介导的ROS产生和ΔΨm丧失。在此过程中褪黑素是否靶向线粒体死亡途径还需要进一步研究。
