铁死亡对肌肉损伤后再生能力的影响

doi:10.3969/j.issn.1674-8115.2022.03.006

摘要/Abstract

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)

中图分类号:

R363.1

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.

DU Yuting, ZHANG Jing, HUANG Ying, ZHANG Jing. Effect of ferroptosis on regeneration after muscle injury[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(3): 298-306.

图/表 5

表1 qPCR引物序列（5′→3′）

Tab 1 Primer sequences for qPCR （5′→3′）

Gene	Forward primer	Reverse primer
Myod	CCACTCCGGGACATAGACTTG	AAAAGCGCAGGTCTGGTGAG
Myog	GAGACATCCCCCTATTTCTACCA	GCTCAGTCCGCTCATAGCC
Tnc	TTTGCCCTCACTCCCGAAG	AGGGTCATGTTTAGCCCACTC
Hmox-1	ACAGAGGAACACAAAGACCAG	GTGTCTGGGATGAGCTAGTG
Acsl4	CTCACCATTATATTGCTGCCTGT	TCTCTTTGCCATAGCGTTTTTCT
Ctss	CCATTGGGATCTCTGGAAGAAAA	TCATGCCCACTTGGTAGGTAT
Gapdh	CCCCAATGTGTCCGTCGTG	GCCTGCTTCACCACCTTCT

图1 CTX注射诱导肌肉组织损伤后再生Note： A. Representative H-E staining (×20) of TA muscle cross-sections from injured mice at different time points (0, 3 and 7 d after CTX injection, respectively). B. qPCR analysis of muscle regeneration factors Myod, Myog, and Tnc mRNA levels in TA isolated from injured mice (0, 3 and 7 d after CTX injection). The mRNA level of Gapdh was used as the internal controls. ①P=0.005, ②P=0.040, ③P=0.000, ④P=0.000, ⑤P=0.003, ⑥P=0.000. C. Protein expression level of MYOD in TA muscle isolated from injured mice (0, 3 and 7 d after CTX injection). The protein level of GAPDH was used as the internal controls.

Fig 1 Muscle injury and regeneration induced by CTX

图2 CTX注射后铁死亡相关指标RNA和蛋白的表达Note： A. qPCR analysis of ferroptosis marker gene Acsl4 and Hmox-1 expression levels in TA muscle isolated from mice (0, 3 and 7 d after CTX injection). The mRNA level of Gadph was used as the internal control. ①P=0.042, ②P=0.001, ③P=0.042, ④P=0.015. B. Protein expression level of ACSL4 and HMOX-1 in TA muscle.

Fig 2 RNA and protein expression of ferroptosis-related indicators after CTX injection

图3 铁死亡抑制剂UAMC-3203预处理之后的转录组分析Note： A. Volcano plot of differentially expressed genes in the TA muscle of CTX＋saline and CTX+UAMC-3203 injection mice 3 days after CTX-injection. B. KEGG pathway of RNA-seq analysis between the CTX＋saline group and CTX＋UAMC-3203 group. C. qPCR analysis of Ctss which was highly up-regulated in GO analysis in TA muscle. ①P=0.016, ②P=0.000.

Fig 3 Effect of ferroptosis inhibitors UAMC-3203 on CTX-induced skeletal muscle regeneration at transcriptome level

图4 铁死亡抑制剂预处理后对肌肉再生能力的抑制作用Note： A. Representative of H-E staining of TA muscle cross-sections from both uninjured (0 d) and injured (3 d and 7 d) mice after CTX injection. B. Protein expression level of MYOD in TA muscle isolated from injured mice treated with saline, DFO and UAMC-3203 (comparison of 0 d and 3 d, 0 d and 7 d). Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. GAPDH served as the loading control. C. qPCR analysis of Myod, Myog, Tnc mRNA levels of TA muscle isolated from injured mice (0,3 and 7 d after CTX injection). The mRNA level of Gapdh was used as the internal control. ①P=0.006, ②P=0.000, ③P=0.000, ④P=0.000, ⑤P=0.003, ⑥P=0.024, ⑦P=0.004, ⑧P=0.040, ⑨P=0.000.

Fig 4 Inhibitory effect of ferroptosis inhibitor on the regeneration ability of skeletal muscle

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