Royal jelly protein hydrolysates accelerate in vitro diabetic wound healing by enhancing keratinocytes functions and suppressing macrophage-mediated inflammation

The pathophysiology of diabetic wounds is underpinned by a vicious cycle of persistent inflammation and impaired re-epithelialization, posing a substantial challenge to clinical management. Therapeutic strategies capable of simultaneously targeting these interconnected pathologies are urgently needed. Royal jelly from Castanea mollissima Bl. (CmRJ) has previously demonstrated wound healing properties, yet the activity of its protein hydrolysates against diabetic complications has not been reported. This study focused on investigating the effects and mechanisms of the protein hydrolysates from CmRJ (CmRJPH) on the in vitro diabetic wounds, particularly on the functions of keratinocyte and macrophages. CmRJPH were trypsinized and then analyzed using MALDI-TOF MS and RPLC-MS/MS. The biological activities and the relevant mode of action of CmRJPH were assessed on the advanced glycation end products (AGEs)-damaged human keratinocytes (HaCaT) and lipopolysaccharide (LPS)-activated macrophages (Raw 264.7). CmRJPH was found to not only enhance the proliferation and migration of keratinocytes, but significantly rescue the impaired migratory function of an in vitro diabetic wound model induced by AGEs. Concurrently, CmRJPH exerted potent anti-inflammatory effects on LPS activated macrophages. It markedly suppressed the activation of the NF-κB signaling pathway, thereby curtailing the expression of the downstream pro-inflammatory effectors, including INOS, COX-2, IL-6, and TNF-α. Besides, CmRJPH exhibited strong antioxidant properties by scavenging DPPH and hydroxyl free radicals and reducing intracellular ROS levels in macrophages. These findings suggested that CmRJPH had the potential to promote the healing of diabetic wounds by restoring the migratory capacity of impaired epithelial cells and concurrently resolving pathological inflammation. It offers new insights into the molecular mechanisms of RJ in promoting wound healing, positioning CmRJPH as a promising therapeutic candidate for the treatment of intractable diabetic wounds.