解剖学报 ›› 2024, Vol. 55 ›› Issue (3): 285-294.doi: 10.16098/j.issn.0529-1356.2024.03.005

• 细胞和分子生物学 • 上一篇    下一篇

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

卢嘉茵   姚志超   郝晓静   闫艺   马佩   张慧玲   王海东*   

  1. 山西农业大学动物医学学院,山西 太谷   030801
  • 收稿日期:2023-06-28 修回日期:2023-11-20 出版日期:2024-06-06 发布日期:2024-06-11
  • 通讯作者: 王海东 E-mail:wanghaidong@sxau.edu.cn
  • 作者简介:2023-10-01
  • 基金资助:
    山西省博士毕业生来晋工作奖励资金科研项目;山西省博士来晋奖励科研项目;山西省基础研究计划青年项目

College of Veterinary Medicine, Shanxi Agricultural University, Shanxi Taigu   030801, China

LU Jia-yin YAO Zhi-chao  HAO Xiao-jing  YAN  Yi  MA  Pei  ZHANG  Hui-ling WANG Hai-dong*   

  • Received:2023-06-28 Revised:2023-11-20 Online:2024-06-06 Published:2024-06-11
  • Contact: WANG Hai-dong E-mail:wanghaidong@sxau.edu.cn

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,50 μmol/L)处理细胞模拟损伤;脂多糖(LPS,100 μg/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(30 μmol/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。

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

Abstract:

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, 50 μmol/L) to mimic injury. Lipopolysaccharide (LPS, 100 μg/L) induced RAW264.7 cell lines to mimic the inflammatory response after skeletal muscle injury, and STM2457 (30 μmol/L) was added to inhibit the effect of methyltransferase 3 (Mettl3) before LPS treatment. The expression of m6A methylation-related 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 significantly in skeletal muscle tissue cells and myocytes after DEX treatment (P<0.05). After LPS treatment, the mRNA and protein expression levels of m6A methylation-related factors and the mRNA expression levels of inflammatory factors interleukin(IL)-6 and IL-1β in macrophages increased significantly (P<0.05), while the levels of IL-6 and IL-1β mRNA in macrophages decreased significantly when the Mettl3 was inhibited (P<0.05). Conclusion  m6A methylation-related factors primarily is activated in the damaged muscle cells and inflammation response of macrophages. Inhibition of m6A methyltransferase can reduce the inflammatory response of macrophages.

Key words: N6-methyladenosine methylation-related factor, Skeletal muscle injury, Macrophage, Real-time PCR, Western blotting, Mouse

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