吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (5): 1148-1155.doi: 10.13481/j.1671-587X.20220507

• 基础研究 • 上一篇    

牙龈卟啉单胞菌脂多糖对巨噬细胞中铁死亡相关因子表达水平的影响

杜雪纯,李保胜,乔树伟,欧燕珍,李珍,孟维艳()   

  1. 吉林大学口腔医院口腔种植科,吉林 长春 130021
  • 收稿日期:2022-01-24 出版日期:2022-09-28 发布日期:2022-11-15
  • 通讯作者: 孟维艳 E-mail:mengwy@jlu.edu.cn
  • 作者简介:杜雪纯(1995-),女,吉林省通化市人,在读硕士研究生,主要从事口腔种植学方面的研究。
  • 基金资助:
    吉林省科技厅科技发展计划项目(20200404108YY)

Effect of Porphyromonas gingivalis-LPS on expression levels of ferroptosis-related factors in macrophages

Xuechun DU,Baosheng LI,Shuwei QIAO,Yanzhen OU,Zhen LI,Weiyan MENG()   

  1. Department of Oral Implantology,Stomatology Hospital,Jilin University,Changchun 130021,China
  • Received:2022-01-24 Online:2022-09-28 Published:2022-11-15
  • Contact: Weiyan MENG E-mail:mengwy@jlu.edu.cn

摘要:

目的 探讨牙龈卟啉单胞菌脂多糖(P.g-LPS)对巨噬细胞中铁死亡相关因子表达水平的影响,阐明铁死亡相关因子在牙周炎发病机制中的作用。 方法 培养小鼠单核/巨噬细胞系RAW264.7细胞,分为实验组和对照组,实验组加入10 mg·L-1P.g-LPS 分别处理6 、12 和24 h,对照组不做任何处理。采用实时荧光定量PCR(RT-qPCR)法检测2组RAW264.7细胞中长链酯酰辅酶A合成酶4(ACSL4)、谷胱甘肽过氧化物酶4(GPX4)和转铁蛋白受体1(TfR1)mRNA表达水平,2',7'-二氯荧光素二乙酸酯(DCFH-DA)荧光探针法检测2组RAW264.7细胞中活性氧(ROS)水平,硫代巴比妥酸(TBA)法检测2组RAW264.7细胞中丙二醛(MDA)水平,亚铁离子荧光探针(FeRhonox-1)法检测2组RAW264.7细胞中亚铁离子(Fe2+)水平,Western blotting法检测2组RAW264.7细胞中ACSL4、GPX4和TfR1蛋白表达水平。 结果 与对照组比较,处理6、12和24 h后,实验组RAW264.7细胞中GPX4 mRNA表达水平降低(P<0.05),处理12和24 h后ACSL4和TfR1 mRNA表达水平升高(P<0.05);处理12和24 h后,实验组RAW264.7细胞中ACSL4和TfR1蛋白表达水平升高(P<0.05),GPX4蛋白表达水平降低(P<0.05)。与对照组比较,实验组RAW264.7细胞中ROS、MDA和Fe2+水平升高(P<0.05),且随时间增加呈上升趋势。 结论 P.g-LPS诱导下RAW264.7细胞中铁死亡相关因子ACSL4和TfR1表达水平升高,而GPX4表达水平降低,并且呈时间依赖性,提示上述铁死亡相关因子的变化可能与牙周炎发病机制有关。

关键词: 巨噬细胞, 牙龈卟啉单胞菌, 脂多糖, 铁死亡, 活性氧

Abstract:

Objective To investigate the effect of lipopolysaccharide (LPS) of Porphyromonas gingivalisP.g) on the expression levels of ferroptosis-related factors in the macrophages, and to clarify the influence of ferroptosis-related factors in the pathogenesis of periodontitis. Methods The mouse mononuclear/macrophage line RAW264.7 cells were cultured and divided into experimental group and control group. The cells in experimental group were treated with 10 mg·L-1P.g-LPS for 6, 12, and 24 h, respectively; the cells in control group were not treated. Real-time fluorescence quantitative PCR (RT-qPCR) method was performed to detect the expression levels of long chain acyl-CoA synthetase 4 (ACSL4), glutathione peroxidase 4 (GPX4), and transferrin receptor 1 (TfR1) mRNA in the RAW264.7 cells in two groups. The levels of reactive oxygen species (ROS) in the RAW264.7 cells in two groups were detected by 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence probe method. The levels of malondialdehyde (MDA) in the RAW264.7 cells in two groups were detected by thiobarbituric acid (TBA) method and the levels of ferrous ion (Fe2+) in the RAW264.7 cells in two groups were detected by Ferrousion fluorescent probe (FeRhonox-1) method. The expression levels of ACSL4, GPX4, and TfR1 proteins in the RAW264.7 cells in two groups were detected by Western blotting method. Results Compared with control group, the expression levels of GPX4 mRNA in the RAW264.7 cells in experimental group after treated for 6, 12, and 24 h were decreased (P<0.05), the expression levels of ACSL4 and TfR1 mRNA in the RAW264.7 cells in experimental group after treated for 12 and 24 h were increased (P<0.05); the expression levels of ACSL4 and TfR1 proteins in the RAW264.7 cells in experimental group after treated for 12 and 24 h were increased (P<0.05);the expression levels of GPX4 protein in the RAW264.7 cells in experimental group after treated for 12 and 24 h were decreased (P<0.05).Compared with control group,the levels of ROS, MDA, and Fe2+ in the RAW264.7 cells in experimental group were significantly increased (P<0.05), and showed an increasing trend with time prolongation. Conclusion The expression levels of ferroptosis-related factors ACSL4 and TfR1 are increased and the expression level of GPX4 is decreased in a time-dependent manner induced by P.g-LPS, suggesting that the changes of ferroptosis-related factors may be related to the pathogenesis of periodontitis.

Key words: Macrophages, Porphyromonas gingivalis, Lipopolysaccharide, Ferroptosis, Reactive oxygen species

中图分类号: 

  • R781.42