巨噬细胞剔除调节肌再生因子表达、抑制AKT/mTOR蛋白质合成信号通路激活损害损伤骨骼肌再生

Object：The objective of this study is to explore the roles of macrophages and the mechanisms involved in the regeneration of injured skeletal muscle. Methods：Eighty C57BL/6 mice were randomly divided into muscle contusion (S, n=32), muscle contusion control (Scon, n=8), macrophages depleted (T, n=32) and macrophages depleted control groups (Tcon, n=8). Their gastrocnemius muscles were harvested at the time points of 1, 3, 5, 7 and 14d post-injury. The changes of skeletal muscle morphology were assessed by hematoxylin and eosin (HE) stains. The gene and protein expression was analyzed by real-time polymerase chain reaction and western blotting. Results：1. The HE results showed that skeletal muscle fibers were significantly impaired and the injured fibers had almost degenerated at 1d and 3d post-injury in both groups. At 7d after injury, the damaged muscle area in the group S had been replaced mostly by newly formed muscle fibers, whereas numerous necrotic myofibers and inflammatory cells dominated the injured muscle regions of group T. The size of regenerating myofibers in macrophage depletion mice was significantly smaller than those observed in the contusion group at 14d post-injury. 2. The results showed that MyoD and myogenin mRNA increased significantly post-injury (p<0.01). As compared to the contusion group, macrophage depletion significantly inhibited MyoD mRNA level and increased myogenin mRNA level post-injury (p<0.05). 3. The data showed that regulatory factors of muscle regeneration (except MGF) mRNA increased significantly post-injury. However, macrophage depletion significantly decreased regulatory factors of muscle regeneration mRNA levels post-injury. 4. HIF-1α and Angpt1 mRNA increased significantly post-injury. Macrophage depletion significantly increased the expression of HIF-1α and Angpt1 mRNA in the later stage of muscle regeneration. 5. Western blotting analysis showed that p-AKT/AKT, p-mTOR/mTOR, p-p70S6K/p70S6 and p-4EBP1/4EBP1 increased significantly post-injury. However, there were no significantly change in the expression of p-AKT/AKT, p-mTOR/mTOR, p-p70S6K/p70S6 and p-4EBP1/4EBP1 in the macrophage depletion group after injury. Conclusion：1. The change of markers of satellite cell proliferation differentiation, muscle regeneration regulatory factors, angiogenesis factors and AKT/mTOR pathway were activated in skeletal muscle after injury, and they may play important roles in the regeneration of muscle contusion. 2. Macrophages depletion impaired muscle regeneration, the mechanism may be involved of inhibiting the expression of muscle regeneration regulatory factors, angiogenesis factors and AKT/mTOR pathway activated. In addition, macrophage plays an important role in muscle regeneration after injury.