铁死亡对肌肉损伤后再生能力的影响  

Effect of ferroptosis on regeneration after muscle injury

作　　者：杜玉婷 张婧[1] 黄莺[1] 张晶[1] DU Yuting;ZHANG Jing;HUANG Ying;ZHANG Jing(Department of Pathophysiology,Key Laboratory for Cell Differentiation and Apoptosis Ministry of Education,Shanghai Jiao Tong University School of Basic Medicine,Shanghai 200025,China)

机构地区：[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

出　　处：《上海交通大学学报（医学版）》2022年第3期298-306,共9页Journal of Shanghai Jiao tong University:Medical Science

基　　金：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)。

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 hematoxylineosin(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 spe

关 键 词：铁死亡 肌肉损伤 肌肉再生 心脏毒素 

分 类 号：R363.1[医药卫生—病理学]