Neutrophil Extracellular Trap Formation Suppressed by Ro 106-9920 Enhances Diabetic Wound Healing by Blocking NLRP3 Inflammasome Activation.

Abstract

Background: The excessive formation of neutrophil extracellular traps (NETs) is involved in delayed wound healing under diabetic conditions. However, potential therapeutic agents remain underexplored. Our present study aimed to explore the effects of Ro 106-9920, a specific nuclear factor kappa B (NF-κB) inhibitor, on diabetic wound healing and to elucidate the underlying mechanisms.

Methods: A diabetic mouse model was established, and full-thickness wounds were created. Ro 106-9920 was administered, and wound healing was monitored. Protein levels of NET markers and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome components were assessed via western blotting and histological analysis. Functional assays were conducted to evaluate the effect of NETs on fibroblasts, endothelial cells, and keratinocytes using conditioned media (CM) from a phorbol 12-myristate 13-acetate (PMA)-treated neutrophil-macrophage coculture model (NET-CM). CM collected from the coculture model after Ro 106-9920 treatment ((NET+Ro 106-9920)-CM) was used to determine its therapeutic potential.

Results: NET formation and NLRP3 inflammasome activation were significantly elevated in wound tissues of diabetic mice from day 7 (p < 0.001). Similar results were observed in PMA-treated neutrophils and macrophages (p < 0.001). The viability and migration of endothelial cells, fibroblasts, and keratinocytes, as well as the angiogenic potential of endothelial cells, were significantly impaired by NET-CM treatment (all p < 0.001), whereas Ro 106-9920 effectively attenuated these alterations (NET-CM vs. (NET+Ro 106-9920)-CM, cell viability, p < 0.001; cell migration, p < 0.01; tube formation, p < 0.001). In vivo, Ro 106-9920 treatment inhibited NET formation, as evidenced by the decreased citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) expression (p < 0.05). This was followed by a decrease in NLRP3 inflammasome activation (p < 0.05), an increase in angiogenesis in wound tissues (p < 0.001), and improved wound healing (p < 0.001) compared with those in Ro 106-9920-untreated mice.

Conclusions: Ro 106-9920 enhances diabetic wound healing by suppressing NET formation and inhibiting NLRP3 inflammasome activation, providing a novel therapeutic choice for improving chronic wound healing in patients with diabetes.