Yi Lei, Wang Kaiming, Liufu Sui, Chen Wenwu, Chen Bohe, Liu Xiaolin, Liu Caihong, Liu Jingwen, Xu Xin, Ma Haiming
College of Animal Science and Technology, Hunan Agricultural University, Changsha, PR, 410128, China.
Key Laboratory of Livestock and Poultry Resources (Pig) Evaluation and Utilization, Ministry of Agriculture and Rural Affairs, Changsha, PR, 410128, China.
BMC Musculoskelet Disord. 2025 May 9;26(1):457. doi: 10.1186/s12891-025-08703-y.
Skeletal muscle possesses a robust regenerative capacity and can effectively repair itself following injury. However, research on the metabolic changes during skeletal muscle regeneration in large animals remains relatively limited. Therefore, in this study, we used pigs as a model and applied non-targeted LC-MS/MS metabolomic technology to reveal the metabolic changes during skeletal muscle regeneration, and conducted an in-depth exploration of important signaling pathways, which can provide a reference for further research on the mechanisms promoting skeletal muscle regeneration.
In this study, we used 18 piglets aged 35 days and weighing 7.10 ± 0.90 kg to construct a skeletal muscle regeneration model. These piglets were randomly divided into three treatment groups (n = 6) and injected with cardiotoxins (CTX) in the right longissimus dorsi muscle. They were euthanized on the 1st, 4th, and 16th days post-injection to collect right longissimus dorsi muscle samples as the treatment group. Additionally, the left longissimus dorsi muscle of piglets on the 4th day post-injection was selected as the control group. Phenotypic changes in skeletal muscle regeneration were determined through H&E staining, immunofluorescence, and Western Blot analysis, and LC-MS/MS untargeted metabolomics technology was utilized to explore the differential expressed metabolites (DEMs) involved in skeletal muscle regeneration.
Phenotyping results showed that the regeneration model showed 3 stages of inflammation, regeneration and remodeling, which indicated successful model construction. Non-targeted LC-MS/MS metabolomics analysis showed significant differences in the structure of metabolites in these 3 stages. (1) In the inflammatory stage, a total of 198 DEMs were identified, which were mainly enriched in the pathways regulating the inflammatory response. (2) in the repair stage, 264 DEMs were identified, which were mainly enriched in pathways that inhibit inflammatory response and promote protein synthesis. (3) During the remodeling stage, 102 DEMs were identified, which were mainly enriched in the pathways that inhibit protein depletion and promote protein deposition. Temporal expression analysis revealed metabolites consistent with changes in the skeletal muscle regeneration process and found that these metabolite functions were mainly enriched in inhibiting inflammatory responses, alleviating myofibrillar lysis, and promoting muscle growth. Among them, (R)-Lipoic acid, 8-Hydroxyguanosine, and Uridine 5'-monophosphate maybe key metabolites associated with skeletal muscle regeneration.
The skeletal muscle regeneration mechanism was systematically explored, and the metabolite time series analysis during skeletal muscle regeneration revealed some key metabolites that reflect the degree of skeletal muscle damage.
骨骼肌具有强大的再生能力，损伤后能有效自我修复。然而，关于大型动物骨骼肌再生过程中代谢变化的研究相对有限。因此，在本研究中，我们以猪为模型，应用非靶向液相色谱 - 串联质谱代谢组学技术揭示骨骼肌再生过程中的代谢变化，并对重要信号通路进行深入探索，可为进一步研究促进骨骼肌再生的机制提供参考。
本研究选用18头35日龄、体重7.10±0.90 kg的仔猪构建骨骼肌再生模型。这些仔猪随机分为三个处理组（n = 6），在右侧背最长肌注射心肌毒素（CTX）。在注射后第1天、第4天和第16天对其实施安乐死，采集右侧背最长肌样本作为处理组。此外，选取注射后第4天仔猪的左侧背最长肌作为对照组。通过苏木精 - 伊红染色、免疫荧光和蛋白质免疫印迹分析确定骨骼肌再生的表型变化，并利用液相色谱 - 串联质谱非靶向代谢组学技术探索参与骨骼肌再生的差异表达代谢物（DEM）。
表型分析结果显示，再生模型呈现炎症、再生和重塑三个阶段，表明模型构建成功。非靶向液相色谱 - 串联质谱代谢组学分析显示这三个阶段代谢物结构存在显著差异。（1）在炎症阶段，共鉴定出198种DEM，主要富集于调节炎症反应的通路。（2）在修复阶段，鉴定出264种DEM，主要富集于抑制炎症反应和促进蛋白质合成的通路。（3）在重塑阶段，鉴定出102种DEM，主要富集于抑制蛋白质消耗和促进蛋白质沉积的通路。时间表达分析揭示了与骨骼肌再生过程变化一致的代谢物，发现这些代谢物功能主要富集于抑制炎症反应、减轻肌原纤维溶解和促进肌肉生长。其中，（R）-硫辛酸、8 - 羟基鸟苷和尿苷5'-单磷酸可能是与骨骼肌再生相关的关键代谢物。
系统探索了骨骼肌再生机制，骨骼肌再生过程中的代谢物时间序列分析揭示了一些反映骨骼肌损伤程度的关键代谢物。
