Substance P promote macrophage M2 polarization to attenuate secondary lymphedema by regulating NF-kB/NLRP3 signaling pathway

Secondary lymphedema often occurs after filariasis, trauma, lymph node dissection and radiation therapy, which is manifested by infiltration of inflammatory cells and fibrosis formation in pathologically. Substance P is a widely used neuropeptide in the field of tissue repair, while the regenerative potential of the substance P has not been proven in the secondary lymphedema. In this study, animal model of secondary lymphedema was con-structed by excising the skin and subcutaneous lymphatic network in the tail of mice, and the degree of swelling in the tail of mice was evaluated after 6 weeks under the treatment with substance P. Immunofluorescence staining was also performed to assess immune cell infiltration, subcutaneous fibrosis and lymphangiogenesis. The results revealed that substance P significantly alleviated post-surgical lymphedema in mice. Furthermore, we found that substance P promoted macrophages M2 polarization, a process associated with downregulation of the NF-kB/NLRP3 pathway. After application of disodium clodronate (macrophage scavenger, CLO), the positive effect of substance P in lymphedema is significantly inhibited. In vitro experiments, we further demonstrated the polarizing effect of substance P on bone marrow-derived macrophages (BMDMs), while substance P inhibited the activation of the NF-kB/NLRP3 pathway in BMDMs after the treatment of lipopolysaccharide (LPS). In addition, polarized macrophages were demonstrated to promote the proliferation, tube-forming and migratory functions of human lymphatic endothelial cells (hLEC). In conclusion, our study provides preliminary evidence that substance P alleviates secondary lymphedema by promoting macrophage M2 polarization, and this therapeutic effect may be associated with downregulation of the NF-kB/NLRP3 pathway.