N6-甲基腺苷甲基化相关因子在小鼠骨骼肌损伤修复中的表达及机制  

Expression and mechanism of N6-methyladenosine methylation-related factors in the repair of skeletal muscle injury in mice

作　　者：卢嘉茵 姚志超 郝晓静 闫艺 马佩 张慧玲 王海东[1] LU Jia-yin;YAO Zhi-chao;HAO Xiao-jing;YAN Yi;MA Pei;ZHANG Hui-ling;WANG Hai-dong(College of Veterinary Medicine,Shanxi Agricultural University,Shanxi Taigu 030801,China)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801

出　　处：《解剖学报》2024年第3期285-294,共10页Acta Anatomica Sinica

基　　金：山西省博士来晋奖励科研项目(SXBYKY2022039,SXBYKY2021043);山西省基础研究计划青年项目(202103021223166)。

摘　　要：目的探讨N6-甲基腺苷(m6A)甲基化相关因子在骨骼肌损伤后修复过程中的时间动态表达及其对损伤过程中巨噬细胞炎症反应的调控作用。方法建立BaCl2损伤小鼠腓肠肌模型,对照组和损伤组每组4只小鼠。分别于小鼠损伤后第1、3、5、7、9天取腓肠肌组织进行实验;分离培养原代腓肠肌肌组织细胞,肌卫星细胞,肌细胞和培养成肌细胞系C2C12细胞,添加地塞米松(DEX,50μmol/L)处理细胞模拟损伤;脂多糖(LPS,100μg/L)处理巨噬细胞系RAW264.7细胞模拟骨骼肌损伤后的炎症反应,LPS处理前添加STM2457(30μmol/L)抑制m6A甲基转移酶3(Mettl3)的作用。利用Real-time PCR和Western blotting方法检测m6A甲基化相关因子(Writers,Erasers,Readers)的表达和炎症因子的表达。结果肌纤维随着损伤时间的延长,出现溶解后逐渐修复,单核/巨噬细胞数量先增加后减少,配对盒7(Pax7)mRNA水平随着损伤时间的变化先升高后降低。与对照组相比,腓肠肌中的m6A甲基化相关因子的mRNA水平和蛋白水平在损伤第1天时变化不显著,到损伤第3天显著升高(P<0.05);损伤后第5天与第3天相比显著下降(P<0.05);损伤后第7天和第9天与对照组相比差异无显著性。DEX降低了原代肌卫星细胞和C2C12细胞m6A甲基转移酶因子的mRNA表达水平(P<0.05),升高了甲基化识别酶因子的mRNA表达水平(P<0.05)。骨骼肌肌组织细胞和肌细胞中m6A甲基化相关因子的mRNA水平在DEX处理后均显著升高(P<0.05)。LPS导致巨噬细胞中m6A甲基化相关因子的mRNA和蛋白水平及炎症因子白细胞介素(IL)-6和IL-1β的mRNA表达水平显著升高(P<0.05),而抑制Mettl3之后,巨噬细胞炎症因子mRNA水平显著下降(P<0.05)。结论m6A甲基化相关因子在受损的肌细胞和巨噬细胞炎症反应中被激活,抑制m6A甲基转移酶能够减弱巨噬细胞炎症反应。Objective To investigate the dynamic expression with the time change of N6-methyladenosine(m6A)methylation-related factors in the repair process of skeletal muscle injury and its mechanism in the inflammatory response of macrophage in the injure process.Methods In vivo mice models of BaCl2 injury in the gastrocnemius were established.Four mice per group in the control group and injury group.Gastrocnemius tissues were harvested at day 1,3,5,7,and 9 after injury for experiments.Primary gastrocnemius muscle tissue cells,muscle satellite cells,muscle cells,and cell line C2C12 cells were treated with dexamethasone(DEX,50μmol/L)to mimic injury.Lipopolysaccharide(LPS,100μg/L)induced RAW264.7 cell lines to mimic the inflammatory response after skeletal muscle injury,and STM2457(30μmol/L)was added to inhibit the effect of methyltransferase 3(Mettl3)before LPS treatment.The expression of m6A methylationrelated factors(Writers,Erasers,Readers)and inflammation factors were detected by Real-time PCR and Western blotting.Results The muscle fibers were dissolved and then gradually repaired with the extension of injury time,the number of monocytes/macrophages increased first and then decreased,and the Pax7 mRNA level increased first and then decreased with the change of injury time.Compared with the control group,the mRNA and protein levels of m6A methylation-related factors in gastrocnemius did not change significantly on the injury-1 day.However,they were significantly increased on the injury-3 days compared with the control group(P<0.05),and then obviously decreased on the injury-5 days group compared with the injury-3 days group(P<0.05).Compared with the control group,they were no significant differences on the injury-7 days group and-9 days group.In vitro DEX decreased the mRNA levels of m6A methyltransferase factors in primary muscle satellite cells and C2C12 cells and increased the mRNA expression level of methylation-recognition enzyme factors(P<0.05).The mRNA levels of m6A methylation-related factors increased signif

关 键 词：N6-甲基腺苷甲基化相关因子 骨骼肌损伤 巨噬细胞 实时定量聚合酶链反应 免疫印迹法 小鼠 

分 类 号：Q291[生物学—细胞生物学]