二氢杨梅素通过Nrf2/HO-1信号通路抑制地塞米松诱发的成骨细胞系MC3T3-E1细胞铁死亡

Dihydromyricetin inhibits dexamethasone induced ferroptosis of osteoblast line MC3T3-E1 cells through Nrf2/HO-1 signaling pathway

关键词:  二氢杨梅素  成骨细胞  地塞米松  铁死亡  Nrf2/HO-1信号通路

Key words:  Dihydromyricetin  Osteoblast  dexamethasone  Ferroptosis  Nrf2/HO-1 signaling pathway

摘要:

目的 研究二氢杨梅素（Dihydromyricetin, DMY）对通过地塞米松（Dexamethasone, DEX）诱发的人成骨细胞系MC3T3-E1细胞铁死亡的影响，同时探讨其机制。方法 使用α-MEM培养基用来对MC3T3-E1细胞进行培养。实验分为：对照组、DEX(10 μmol·L-1)组、DMY(20 μmol·L-1)组、DEX+DMY组、铁死亡诱导剂RSL3组、DEX+DMY+RSL3组、Nrf2抑制剂ML385（5 μmol·L-1）组、DEX+DMY+ML385组。噻唑兰（Thiazolyl blue tetrazolium bromide, MTT）法用来完成检测细胞活力。采用试剂盒检测细胞内总铁和二价铁的水平，丙二醛（Malondialdehyde, MDA）、谷胱甘肽（Glutathione, GSH）和活性氧（Reactive oxygen species, ROS）的水平。通过透射电镜检测MC3T3-E1细胞的超微结构。谷胱甘肽过氧化物酶4（Glutathione peroxidase 4, GPX-4）、血红素加氧酶1（Heme oxygenase-1, HO-1）以及核因子E2相关因子-2（Nuclear factor erythroid-2-related factor 2, Nrf2）蛋白的表达通过Western Blotting检测。结果 与DEX组比较，DEX+DMY组MC3T3-E1细胞的细胞活力、GSH浓度以及GPX4蛋白的表达显著性增加（P<0.05），细胞线粒体损伤的情况明显减轻，细胞中总铁离子、二价铁离子、ROS以及MDA的水平显著性降低（均P<0.05）。与DEX+DMY组比较，DEX+DMY+RSL3组MC3T3-E1细胞的细胞活力显著性降低（P<0.05）。与DEX组比较，DEX+DMY组Nrf2在细胞核中的表达水平、Nrf2在细胞核中表达与Nrf2总蛋白表达的比值、HO-1的蛋白表达水平均显著性升高（均P<0.05）。与DEX+DMY组比较，DEX+DMY+ML385组MC3T3-E1细胞的细胞活力、GSH浓度显著降低（P<0.05），细胞中总铁离子、二价铁离子、ROS和MDA的水平均显著升高（P<0.05）。结论 DMY抑制了DEX诱发的成骨细胞的铁死亡，其机制可能是通过Nrf2/HO-1信号通路实现的。

Abstract:

Objective To observe the effect of dihydromyricetin（DMY） on dexamethasone (DEX)-induced ferroptosis in osteoblast MC3T3-E1 cells and explore its mechanism. Methods MC3T3-E1 cells were cultured in α-MEM medium. The experiment was divided into control group and DEX (10 μmol·L-1) group, DMY (20 μmol·L-1), DEX + DMY group,ferroptosis inducer RSL3 group, DEX + DMY + RSL 3 group, Nrf2 inhibitor ML385 (5 μmol·L-1) group, DEX + DMY + ML385 group. Cell viability was detected by thiazolyl blue tetrazolium bromide (MTT) method. The concentrations of total iron ions and divalent iron ions, glutathione (GSH), malondialdehyde (MDA) and reactive oxygen species (ROS) in cells were detected by kit. The transmission electron microscope was used to test the ultrastructure of the cells. The expression levels of glutathione peroxidase 4 (GPX-4), heme oxygenase 1 (HO-1) and nuclear factor E2 related factor-2 (Nrf-2) proteins were detected by Western blotting. Results Compared with the DEX group, the cell viability, GSH concentration and expression of GPX4 protein in MC3T3-E1 cells in the DEX+DMY group increased significantly (P<0.05), the mitochondrial damage of cells was significantly reduced, and the levels of total iron ions, ferric ions, ROS and MDA in the cells decreased significantly (P<0.05). Compared with the DEX+DMY group, the cell viability of MC3T3-E1 cells in the DEX+DMY+RSL3 group was significantly reduced (P<0.05). Compared with the DEX group, the expression level of Nrf2 in the nucleus, the ratio of Nrf2 expression in the nucleus to the expression of Nrf2 total protein, and the protein expression level of HO-1 in the DEX+DMY group were significantly increased (P<0.05). Compared with the DEX+DMY group, the cell viability and GSH concentration of MC3T3-E1 cells in the DEX+DMY+ML385 group were significantly reduced (P<0.05), and the levels of total iron ions, divalent iron ions, ROS and MDA in the cells were significantly increased (P<0.05).Conclusion Dihydromyricetin inhibits dexamethasone induced the ferroptosis in osteoblasts, and its mechanism may be through Nrf2/HO-1 signaling pathway.