Manual Therapy Exerts Local Anti-Inflammatory Effects Through Neutrophil Clearance

Manual therapy (MT) has been widely used in China to treat local tissue inflammation for a long time. However, there is a lack of scientific evidence for using MT in anti-inflammatory therapy, and its anti-inflammatory mechanism needs further clarification. We utilized MT to treat cardiotoxin (CTX) injury-induced skeletal muscle inflammation in C57BL6/J mice. We analyzed the underlying mechanism by integrating single-cell RNA sequencing (scRNA-seq) with molecular techniques. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to assess skeletal muscle inflammation and muscle fiber cross-sectional area (CSA). scRNA-seq, immunofluorescence, and western blot were performed to determine cellular and molecular outcome changes. Compared with CTX injury-induced skeletal muscle inflammatory mice, MT intervention significantly reduced proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNF-) expression levels; scRNA-seq detected that neutrophil numbers and activity were maximum proportions increased in injured skeletal muscle among macrophage, T cells, B cells, endothelial cells, fast muscle cells, fibroblasts, and skeletal muscle satellite cells; and S100A9 gene expression was supreme in neutrophils. However, after treatment with MT, S100A9 protein expression and the numbers and activity of Ly6g+/Mpo+ neutrophils were significantly inhibited, thus reducing the inflammatory cytokine levels and exerting an anti-inflammatory effect by early clearing neutrophils. MT can mitigate localized inflammation induced by injured skeletal muscle, achieved by decreasing S100A9 protein expression and clearing neutrophils in mice, which may help advance therapeutic strategies for skeletal muscle localized inflammation.