生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (3): 6-.doi: 10.3969/j.issn.2095-1736.2023.03.006

• 研究报告 • 上一篇    下一篇

促凋亡蛋白Bax和Bak通过Nrf2/GPX4信号通路调控铁死亡

韩　靖1,2， 赵国平1   

  1. 1. 中国科学院合肥物质科学研究院 强磁场科学中心， 合肥 230031；
    2. 中国科学技术大学 研究生院科学岛分院， 合肥 230026
  • 出版日期:2023-06-18 发布日期:2023-06-19
  • 通讯作者: 赵国平，博士，研究员，研究方向为生物物理学，E-mail:gpz@ipp.ac.cn
  • 作者简介:韩靖，硕士研究生，研究方向为生物物理学，E-mail:hj19@mail.ustc.edu.cn
  • 基金资助:
    安徽省重点研发计划项目（202104a07020006）

Bax and Bak regulate ferroptosis via Nrf2/GPX4 signaling pathway

HAN Jing1,2， ZHAO Guoping1   

  1. 1. High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences,
    Hefei 230031, China; 2. Science Island Branch of Graduate School, University of Science and Technology of China,
    Hefei 230026, China
  • Online:2023-06-18 Published:2023-06-19

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摘要： 为探究促凋亡蛋白Bak/Bax对erastin诱导铁死亡的调控机制，以野生型（WT）、Bak/Bax双敲除（Bak/Bax-DKO）、Bax敲除（Bax-KO）和Bak敲除（Bak-KO）的小鼠成纤维细胞（MEFs）为实验对象，利用CCK-8和流式细胞术检测细胞存活率和活性氧（ROS）的生成量，利用试剂盒测定GSH/GSSG水平，采用实时荧光定量PCR（RT-qPCR）和蛋白质免疫印迹（Western Blot）检测相关基因和蛋白的表达水平。结果显示：与野生型细胞相比，敲除Bak和Bax显著抑制erastin诱导的铁死亡，RT-qPCR和Western Blot检测发现，相比WT细胞，Bak/Bax-DKO细胞中核因子NF-E2相关因子2（Nrf2）和谷胱甘肽过氧化物酶4（GPX4）蛋白、GPX4 mRNA表达水平明显升高。进一步研究发现：与Bak/Bax双敲除类似，敲除Bax也显著抑制erastin诱导的铁死亡，促进GPX4的蛋白表达水平；而敲除Bak对erastin诱导的铁死亡及GPX4的蛋白表达水平无明显影响。结果表明，促凋亡蛋白Bax和Bak通过Nrf2/GPX4信号通路抑制erastin诱导的铁死亡，且Bax在其中起主导作用。

关键词: Bak/Bax, 铁死亡, 活性氧, 谷胱甘肽过氧化物酶4, 核因子NF-E2相关因子2

Abstract: Immortalized mouse embryonic fibroblasts (MEFs) with wild-type (WT), Bak/Bax double knockout (Bak/Bax-DKO), Bak knockout (Bak-KO) and Bax knockout (Bax-KO) were used to investigate the role and possible mechanisms of pro-apoptotic protein Bak/Bax on the erastin-induced ferroptosis. The survival rates and reactive oxygen species (ROS) production were determined by CCK-8 and flow cytometry, the levels of GSH/GSSG were measured by testing kits. In addition, the expression levels of target genes and proteins were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western Blot. The results showed that knockout Bak and Bax inhibited erastin-induced ferroptosis significantly, and the expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) protein, glutathione peroxidase 4 (GPX4) protein and mRNA were increased significantly in Bak/Bax-DKO cells. Further studies showed that the absence of Bax also inhibited erastin-induced ferroptosis and promoted the expression levels of GPX4. However, no significant changes on erastin-induced ferroptosis and GPX4 expression were found in Bak-KO cells. These results indicated that Bak and Bax promoted erastin-induced ferroptosis via Nrf2/GPX4 signaling pathway and Bax rather than Bak played a key role.

Key words: Bak/Bax, ferroptosis, reactive oxygen species, glutathione peroxidase 4, nuclear factor-erythroid 2-related factor 2

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