中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (32): 6858-6865.doi: 10.12307/2025.934

• 脊柱组织构建 spinal tissue construction • 上一篇    下一篇

叔丁基氢过氧化物可诱导髓核细胞发生铁死亡

谌  超，胡耀全，吕正品，何其聪，杨阳子久，罗皓严，吴贵帅，左乾林，王学楠，张  帆   

  1. 昆明医科大学第一附属医院骨科，云南省昆明市  650032


  • 收稿日期:2024-10-18 接受日期:2024-11-28 出版日期:2025-11-18 发布日期:2025-04-25
  • 通讯作者: 张帆，博士，副教授，昆明医科大学第一附属医院骨科，云南省昆明市 650032
  • 作者简介:谌超，男，1998年生，贵州省织金县人，汉族，昆明医科大学在读博士，主要从事脊柱外科方面的研究。
  • 基金资助:
    国家自然科学基金项目(82160428)，项目负责人：张帆；昆明医科大学第一附属医院535人才项目(2022535D10)，项目负责人：张帆；云南省“兴滇英才支持计划”医疗卫生人才培养计划项目，项目负责人：张帆；昆明医科大学中青年学科带头人及后备人选-
    “乘风人才”培养计划项目(J13397034)，项目负责人：张帆；昆明医科大学一流学科团队(2024XKTDYS05)，项目参与人：张帆

tert-Butyl hydroperoxide can induce ferroptosis in nucleus pulposus cells

Chen Chao, Hu Yaoquan, Lyu Zhengpin, He Qicong, Yangyang Zijiu, Luo Haoyan, Wu Guishuai, Zuo Qianlin, Wang Xuenan, Zhang Fan   

  1. Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China
  • Received:2024-10-18 Accepted:2024-11-28 Online:2025-11-18 Published:2025-04-25
  • Contact: Zhang Fan, MD, Associate professor, Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China
  • About author:Chen Chao, MD candidate, Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 82160428 (to ZF); 535 Talent Project of First Affiliated Hospital of Kunming Medical University, No. 2022535D10 (to ZF); Yunnan Province “Xingdian Talent Support Plan” Project for Medical and Health Talent Training (No project number available) (to ZF); The “Rising Star” Talent Training Program for Young and Middle-Aged Discipline Leaders and Reserve Candidates at Kunming Medical University, No. J13397034 (to ZF); First-class Academic Team of Kunming Medical University, No. 2024XKTDYS05 (to ZF [project participant])

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摘要：


文题释义：
叔丁基氢过氧化物(tert-Butyl hydroperoxide，TBHP)：是一种有机过氧化物，常用作氧化剂和自由基源。TBHP在生物医学研究中用于模拟氧化应激状态，以研究细胞的氧化应激反应及其相关机制。
铁死亡：是一种依赖于铁离子和活性氧生成的程序性细胞死亡形式，其特征是细胞膜上的脂质过氧化，导致细胞结构和功能受损。铁死亡与传统的细胞凋亡和坏死不同，具有特定的生物化学标志，如谷胱甘肽过氧化物酶4和铁蛋白重链1的调控。该过程与多种病理状况相关，如神经退行性疾病、癌症和组织损伤，因此被视为一个重要的研究领域。

背景：髓核细胞的退变是椎间盘退变的关键环节。铁死亡是一种新型程序性细胞死亡方式，与椎间盘退变的发生和发展密切相关，但其具体机制尚不明确。
目的：旨在通过使用叔丁基氢过氧化物(tert-Butyl hydroperoxide，TBHP)诱导髓核细胞铁死亡，构建体外氧化应激模型，探讨TBHP诱导髓核细胞铁死亡的机制，进一步理解铁死亡在椎间盘退变中的作用。
方法：采用不同浓度的TBHP(0，25，50，100，200 μmol/L)处理髓核细胞，利用荧光显微镜与CCK8检测髓核细胞形态及细胞活力水平。采用TBHP 100 μmol/L、铁死亡诱导剂RSL3 10 μmol/L及二甲基亚砜干预髓核细胞，利用EdU实验检测细胞增殖能力。通过Western blot和免疫荧光检测铁死亡相关蛋白(谷胱甘肽过氧化物酶4、铁蛋白重链1、PTGS2和ACSL4)与椎间盘退变标志蛋白(基质金属蛋白酶13和Col2A)的表达，使用活性氧检测试剂盒和C11-BODIPY探针测定活性氧及脂质过氧化水平，并通过电镜观察线粒体形态变化。
结果与结论：①TBHP处理显著降低了髓核细胞的活力和增殖水平；②TBHP诱导髓核细胞发生典型的铁死亡形态学变化；③TBHP处理导致铁死亡抑制蛋白谷胱甘肽过氧化物酶4和铁蛋白重链1表达下降，铁死亡促进因子ACSL4和PTGS2表达上升；④TBHP处理增加了髓核细胞内活性氧生成和脂质过氧化水平；⑤电镜观察显示TBHP处理后的髓核细胞线粒体表现出收缩、嵴减少和膜密度增加等铁死亡特征；⑥TBHP处理导致髓核细胞中椎间盘退变标志蛋白基质金属蛋白酶13的表达增加，Col2A的表达下降。综上，TBHP可诱导髓核细胞发生铁死亡，并促使椎间盘退变的发生，可能是椎间盘退变中的关键病理机制，该研究为开发新型治疗策略提供了潜在靶点。
https://orcid.org/0000-0001-7043-745X(谌超)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: TBHP, 铁死亡, 髓核细胞, 氧化应激, 椎间盘退变

Abstract: BACKGROUND: Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.  Ferroptosis, a novel form of programmed cell death, is closely associated with the onset and progression of intervertebral disc degeneration; however, its precise mechanisms remain unclear. 
OBJECTIVE: To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells, thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.
METHODS: Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide (0, 25, 50, 100, and 200 μmol/L), and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay. Interventions with 100 μmol/L tert-butyl hydroperoxide, 10 μmol/L 
RSL3, or dimethylsulfoxide were applied to nucleus pulposus cells, and cell proliferation was evaluated using the EdU assay. The expression levels of ferroptosis-related proteins (glutathione peroxidase 4, ferritin heavy chain 1, PTGS2, and ACSL4) and intervertebral disc degeneration marker proteins (matrix metalloproteinase 13 and Col2A) were analyzed via western blot and immunofluorescence. Additionally, reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe. Mitochondrial morphological changes were observed under transmission electron microscopy.
RESULTS AND CONCLUSION: (1) tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells. (2) tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells. (3) tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1, while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2. (4) tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells. (5) Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide, including contraction, reduced cristae, and increased membrane density. (6) tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells. In conclusion, tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells, contributing to the development of intervertebral disc degeneration. This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

Key words: tert-butyl hydroperoxide, ferroptosis, nucleus pulposus cells, oxidative stress, intervertebral disc degeneration 

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