上海交通大学学报(医学版) ›› 2022, Vol. 42 ›› Issue (3): 298-306.doi: 10.3969/j.issn.1674-8115.2022.03.006

• 论著 · 基础研究 • 上一篇    下一篇

铁死亡对肌肉损伤后再生能力的影响

杜玉婷(), 张婧, 黄莺, 张晶()   

  1. 上海交通大学基础医学院病理生理学系,细胞分化与凋亡教育部重点实验室,上海 200025
  • 收稿日期:2021-12-24 出版日期:2022-03-28 发布日期:2022-05-09
  • 通讯作者: 张晶 E-mail:duduyuting@sjtu.edu.cn;jingzhang@shsmu.edu.cn
  • 作者简介:杜玉婷(1997—),女,硕士生;电子信箱:duduyuting@sjtu.edu.cn
  • 基金资助:
    国家自然科学基金(32070734);上海市自然科学基金(20ZR1430800);上海市浦江人才计划(20PJ1409500);上海交通大学医学院高水平地方高校创新团队(SHSMU-ZDCX20212000)

Effect of ferroptosis on regeneration after muscle injury

Yuting DU(), Jing ZHANG, Ying HUANG, Jing ZHANG()   

  1. Department of Pathophysiology, Key Laboratory for Cell Differentiation and Apoptosis Ministry of Education, Shanghai Jiao Tong University School of Basic Medicine, Shanghai 200025, China
  • Received:2021-12-24 Online:2022-03-28 Published:2022-05-09
  • Contact: Jing ZHANG E-mail:duduyuting@sjtu.edu.cn;jingzhang@shsmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32070734);Natural Science Foundation of Shanghai(20ZR1430800);Shanghai Pujiang Talent Plan(20PJ1409500);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20212000)

摘要:

目的·探讨铁死亡对心脏毒素(cardiotoxin,CTX)诱发肌肉损伤后肌肉再生能力的影响。方法·在15只8周龄C57BL/6J雄性小鼠胫骨前肌(tibialis anterior,TA)上取上、中、下3个点注射CTX,在第0、3、7日分别取5只小鼠的TA做苏木精-伊红染色(hematoxylin-eosin,H-E染色)观察肌肉损伤程度;采用实时荧光定量PCR(quantitative real-time PCR,qPCR)和蛋白质印迹法分别从RNA和蛋白水平检测肌肉再生相关指标及铁死亡相关指标的表达水平。同时,将45只8周龄C57BL/6J雄性小鼠分为生理盐水对照组、铁离子螯合剂去铁胺(deferoxamine,DFO)处理组和铁死亡抑制剂UAMC-3203处理组,每组15只。通过分别提前1周腹腔注射生理盐水或DFO,以及提前1 d腹腔注射UAMC-3203对小鼠进行预处理。随后在小鼠TA部位注射CTX,分别于第0、3、7日取TA组织,利用RNA测序(RNA sequencing,RNA-seq)技术以及生物信息学分析铁死亡抑制剂预处理后对CTX诱导的肌肉损伤后再生能力的影响,通过H-E染色观察及qPCR检测铁死亡抑制剂对肌肉再生相关基因RNA表达水平的影响。结果·H-E染色显示CTX注射后肌肉损伤再生模型构建成功。实验观察到铁死亡相关指标酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,Acsl4)和血红素加氧酶1(heme oxygenase-1,Hmox-1)在RNA和蛋白水平上明显升高,提示肌肉损伤及再生过程中发生了铁死亡。在注射CTX后第3日肌肉组织的损伤情况较为严重,伴随着肌肉再生相关基因成肌分化抗原(myogenic differentiation antigen,Myod)、肌细胞生成素(myogenin,Myog)、固生蛋白(tenascin-c,Tnc)的明显上调,第7日有所恢复。通过对RNA-seq差异基因的京都基因与基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析发现,与生理盐水组比较,UAMC-3203处理组中性粒细胞的脱颗粒化,活性氧(reactive oxygen species,ROS)的生成以及吞噬作用中的磷脂等信号通路发生了明显的改变;并且组织蛋白酶S(cathepsin S,Ctss)的表达水平明显升高。更重要的是,注射UAMC-3203和DFO可抑制肌肉再生相关基因的表达。结论·抑制铁死亡在一定程度上减慢了肌肉的再生过程,提示铁死亡的发生可能促进了肌肉的再生。

关键词: 铁死亡, 肌肉损伤, 肌肉再生, 心脏毒素

Abstract:

Objective·To investigate the role of ferroptosis in muscle regeneration after injury induced by cardiotoxin (CTX).

Methods·CTX was injected into the tibialis anterior (TA) of fifteen 8-week-old male C57BL/6J mice at the upper, middle and lower points. After injection, TA tissue of the mice was collected at 0 d, 3 d and 7 d respectively (n=5) to detect injury by hematoxylin-eosin (H-E) staining. Meanwhile, quantitative real-time PCR (qPCR) and Western blotting were used respectively to detect the expression levels of muscle regeneration-related indexes and ferroptosis-related genes from RNA and protein levels, respectively. At the same time, forty-five 8-week-old C57BL/6J male mice were divided into 3 groups before CTX injection: saline control group, iron chelator deferoxamine (DFO) treatment group and ferroptosis inhibitor UAMC-3203 treatment group (n=15). CTX was injected into TA, and muscle tissue was collected at 0 d, 3 d and 7 d respectively. RNA sequencing (RNA-seq) technology and bioinformatics were used to analyze the effect of ferroptosis inhibitor pretreatment on muscle injury and regeneration after CTX injection. H-E staining and qPCR were utilized to analyze the effect of ferroptosis inhibitor on the expression levels of muscle regeneration-related genes.

Results·The muscle injury and regeneration model was successfully established by CTX injection, as revealed by H-E staining. The increase of ferroptosis-related genes including acyl-CoA synthetase long chain family member 4 (Acsl4) and heme oxygenase-1 (Hmox-1) at both RNA and protein levels was observed, suggesting the occurrence of ferroptosis during muscle injury. There was severe muscle injury at day 3, which was detected by the up-regulation of myogenic differentiation antigen (Myod), myogenin (Myog), and tenascin-c (Tnc), followed by declines at day 7. According to the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of RNA-seq differential genes, it was found that UAMC-3203 treatment group had significant changes in neutrophil degranulation, production of reactive oxygen species (ROS) and phospholipids in phagocytosis compared with CTX injection alone. And the expression of cathepsin S (Ctss) was much higher in the UAMC-3203 treatment group. More importantly, the expression of muscle regeneration-related genes were dramatically inhibited by both UAMC-3203 and DFO injection.

Conclusion·Inhibition of ferroptosis slows down the process of muscle regeneration to a certain degree, suggesting that ferroptosis may play a key role in facilitating muscle regeneration.

Key words: ferroptosis, muscle injury, muscle regeneration, cardiotoxin (CTX)

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