Yuqian Liu1,2,3, Linfeng Wang1,2,3, Shengcan Li1,2,3, Tao Zhang1,2,3, Can Chen2,3, Jianzhong Hu2,3,4, Deyi Sun1,2,3, Hongbin Lu1,2,3. 1. Department of Sports Medicine, Xiangya Hospital, Central South University, Changsha, China. 2. Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, China. 3. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China. 4. Department of Spine Surgery and Orthopedics, Xiangya Hospital, Central South University, Changsha, China.
Abstract
Background: It is well known that appropriate mechanical stimulation benefits tendon-bone (T-B) healing, however, the mechanisms behind this are still uncovered completely. Here, we aimed to explore whether the IL-4/JAK/STAT signaling pathway mediated macrophage polarization was involved in mechanical stimulation induced T-B healing. Method: C57BL/6 mice rotator cuff (RC) repair model was established, and the mice were randomly allocated to the following group. 1. Mice were allowed for free cage activities after surgery (FC group); 2. Mice received treadmill running initiated on postoperative day 7 (TR group); 3. Mice only received a local injection of hydrogel containing IL-4 neutralizing antibody without postoperative intervention (FC + AF-404-SP group); 4. Mice received a local injection of hydrogel containing IL-4 neutralizing antibody and postoperative treadmill running (TR + AF-404-SP group). The expression of IL-4 within supraspinatus tendon (SST) enthesis was measured by Enzyme-linked immunosorbent assay (ELISA). In addition, the activation of JAK/STAT signaling pathway in macrophages and identification of macrophage phenotype at the RC insertion site was detected by Flow cytometry and qRT-PCR. T-B healing quality in this RC repair model was evaluated by histological staining, Micro-computed tomography (Micro-CT) scanning, and biomechanical testing. Result: In this study, using the RC repair model, we confirmed that generation of IL-4, activation of the JAK/STAT signaling pathway in macrophages, the ability of macrophages to polarize towards M2 subtype, and T-B healing quality were significantly enhanced in TR group compared to FC group. When comparing FC + AF-404-SP group with TR + AF-404-SP group, it was found that the mechanical stimulation induced this effect was depleted following the blockade of the IL-4/JAK/STAT signaling pathway. Conclusion: Our finding suggested that mechanical stimulation could accelerate T-B healing via activating the IL-4/JAK/STAT signaling pathway that modulates macrophages to polarize towards M2 subtype. The translational potential of this article: This is the first study to reveal a significant role of mechanical stimulation in the IL-4/JAK/STAT signaling pathway activation and macrophage polarization during RC T-B healing, which highlights the IL-4/JAK/STAT signaling pathway as a potential target to mediate macrophage M2 polarization and improves T-B healing for RC repair.
Background: It is well known that appropriate mechanical stimulation benefits tendon-bone (T-B) healing, however, the mechanisms behind this are still uncovered completely. Here, we aimed to explore whether the IL-4/JAK/STAT signaling pathway mediated macrophage polarization was involved in mechanical stimulation induced T-B healing. Method: C57BL/6 mice rotator cuff (RC) repair model was established, and the mice were randomly allocated to the following group. 1. Mice were allowed for free cage activities after surgery (FC group); 2. Mice received treadmill running initiated on postoperative day 7 (TR group); 3. Mice only received a local injection of hydrogel containing IL-4 neutralizing antibody without postoperative intervention (FC + AF-404-SP group); 4. Mice received a local injection of hydrogel containing IL-4 neutralizing antibody and postoperative treadmill running (TR + AF-404-SP group). The expression of IL-4 within supraspinatus tendon (SST) enthesis was measured by Enzyme-linked immunosorbent assay (ELISA). In addition, the activation of JAK/STAT signaling pathway in macrophages and identification of macrophage phenotype at the RC insertion site was detected by Flow cytometry and qRT-PCR. T-B healing quality in this RC repair model was evaluated by histological staining, Micro-computed tomography (Micro-CT) scanning, and biomechanical testing. Result: In this study, using the RC repair model, we confirmed that generation of IL-4, activation of the JAK/STAT signaling pathway in macrophages, the ability of macrophages to polarize towards M2 subtype, and T-B healing quality were significantly enhanced in TR group compared to FC group. When comparing FC + AF-404-SP group with TR + AF-404-SP group, it was found that the mechanical stimulation induced this effect was depleted following the blockade of the IL-4/JAK/STAT signaling pathway. Conclusion: Our finding suggested that mechanical stimulation could accelerate T-B healing via activating the IL-4/JAK/STAT signaling pathway that modulates macrophages to polarize towards M2 subtype. The translational potential of this article: This is the first study to reveal a significant role of mechanical stimulation in the IL-4/JAK/STAT signaling pathway activation and macrophage polarization during RC T-B healing, which highlights the IL-4/JAK/STAT signaling pathway as a potential target to mediate macrophage M2 polarization and improves T-B healing for RC repair.
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