After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass. Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-β1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill. Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

中文翻译：

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外源GM-CSF瞬时表达产生的M1样巨噬细胞的骨骼肌愈合

骨骼肌外伤后，肌肉愈合通常不完全，并产生广泛的纤维化。M1和M2巨噬细胞积累的顺序以及受伤区域每种亚型的持续时间可能有助于指导愈合过程中纤维形成和肌生成的相对程度。我们假设在创伤性肌肉损伤后早期增加M1巨噬细胞的数量将产生更多的用于肌发生的细胞和分子底物，以及更少的纤维化底物，从而导致更好的肌肉愈合。为了验证这一假设，我们用质粒载体转染了骨骼肌，在损伤后不久即瞬时表达GM-CSF，从而将巨噬细胞的极化驱动向M1亚群。C57BL / 6小鼠胫前肌（TA）受到挫伤，并用uP-mGM电穿孔，它是瞬时表达GM-CSF的质粒载体。通过组织学，免疫组织化学和RT-qPCR评估肌发生，血管生成和纤维化。通过流式细胞仪检测巨噬细胞亚群；跑步机上的最大跑步速度和肌肉质量的恢复可以使肌肉发挥功能。肌肉损伤在第4天增加了局部M1样巨噬细胞的数量，并减少了M2样巨噬细胞的数量，而uP-mGM治疗增强了这种变异。uP-mGM治疗在第4天降低了TGF-β1蛋白表达，Sirius Red阳性面积从第30天的35.93±15.45％（未治疗）降至2.9％±6.5％（p <0.01）。uP-mGM电穿孔还增加了Hgf，Hif1α和Mtor基因的表达；小动脉密度 和再生过程中的肌纤维数量。用uP-mGM处理后肌肉组织质量的改善影响了TA肌肉质量的增加和跑步机上的最大跑步速度。总体而言，我们的数据表明，创伤性肌肉损伤后立即增加M1样巨噬细胞的数量可促进肌肉恢复，纤维化程度降低，这可以通过瞬时表达GM-CSF来实现。