上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 3: 298 - 306

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.03.006

论著 · 基础研究

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
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  • 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
杜玉婷（1997—），女，硕士生；电子信箱：duduyuting@sjtu.edu.cn。
张晶，电子信箱：jingzhang@shsmu.edu.cn。

收稿日期: 2021-12-24

  网络出版日期: 2022-05-09

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)

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Effect of ferroptosis on regeneration after muscle injury

  • Yuting DU ,
  • Jing ZHANG ,
  • Ying HUANG ,
  • Jing ZHANG
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  • Department of Pathophysiology, Key Laboratory for Cell Differentiation and Apoptosis Ministry of Education, Shanghai Jiao Tong University School of Basic Medicine, Shanghai 200025, China
ZHANG Jing, E-mail: jingzhang@shsmu.edu.cn.

Received date: 2021-12-24

  Online published: 2022-05-09

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)

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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

本文引用格式

杜玉婷 , 张婧 , 黄莺 , 张晶 . 铁死亡对肌肉损伤后再生能力的影响[J]. 上海交通大学学报（医学版）, 2022 , 42(3) : 298 -306 . DOI: 10.3969/j.issn.1674-8115.2022.03.006

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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