Mechanism and Component Study of Scorpion Peptides Regulation of Macrophage Polarization in Diabetic Wounds

Background: The incidence of diabetes-related chronic wounds has been increasing annually, characterized by delayed wound healing due to persistent inflammatory responses. To alleviate patient suffering, there is an urgent need to discover novel wound-healing agents that reduce local inflammatory reactions. Animal peptides emerge as promising candidates due to their low molecular weight, low toxicity, low resistance potential, high sensitivity, potent activity, and ease of transmembrane absorption. However, the precise mechanism by which animal peptides accelerate skin wound healing remains unclear. Method: Scorpion peptides were extracted using ultrafiltration, followed by de novo analysis after library searching with Peaks 8 software and combined LC-MS/MS detection to identify peptide components in scorpions. A full-thickness skin defect model in diabetic mice was established to evaluate the wound-healing promotion capacity of scorpion peptides. In vitro experiments were conducted using mouse monocyte–macrophage leukemia cells (RAW264.7 cells). Enzyme-linked immunosorbent assay (ELISA) evaluated inflammatory cytokine expression, flow cytometry analyzed macrophage phenotypes, Western blotting (WB) measured P-P65 and P65 protein levels, immunofluorescence to observe P-P65 nuclear translocation, molecular docking and molecular dynamics simulations, and the Cellular Thermal Shift Assay (CETSA) to evaluate the binding capacity of scorpion peptides to target proteins, and the TUNEL assay to detect apoptosis, among other methods, to investigate the mechanisms by which scorpion peptides promote wound healing. Result: This study successfully identified 2331 peptides from a scorpion peptide extract. In a full-thickness skin defect model in diabetic mice, treatment with scorpion peptides significantly promoted wound healing and induced M2 polarization of macrophages at the wound site. Further in vitro mechanism studies were conducted using a RAW264.7 cell inflammation model established by combining lipopolysaccharide (LPS) with high glucose. The results showed that scorpion peptide reduced the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), increased the anti-inflammatory cytokine interleukin-10 (IL-10), downregulate the P-P65/P65 protein expression ratio, and reduce the number of TUNEL-positive apoptotic cells, suggesting that the NF-κB pathway may be involved in mediating its anti-inflammatory and anti-apoptotic effects. To further identify the target, molecular docking and molecular dynamics simulations showed that the active peptide segments PPPPPP and GPPPPP adopt stable binding conformations with the P65 protein; CETSA further validated that they enhance the thermal stability of the P-P65 protein. Through repeated validation using active peptide fragments, ELISA, flow cytometry, Western blot, and immunofluorescence assays consistently confirmed that scorpion peptide, PPPPPPP, and GPPPPP not only inhibit P65 phosphorylation but also inhibit the nuclear translocation of P-P65, and exhibit anti-inflammatory, macrophage M2 polarization-promoting, and anti-apoptotic effects. Conclusions: Scorpion peptides and their active peptide fragments block the activation of the NF-κB pathway by interfering with P65 phosphorylation and nuclear translocation, effectively inducing M2 polarization of macrophages, reducing inflammation and apoptosis, and ultimately promoting the healing of diabetic wounds.