Astragaloside IV and ginsenoside Rb1 combination improves diabetic foot ulcer healing by downregulating the IRF1-ZBP1 axis in keratinocytes

Abstract Background Diabetic foot ulcer (DFU) represents a prevalent and refractory complication of diabetes mellitus. Active components from Astragalus membranaceus and Panax ginseng have shown potential in treating diabetic complications. The present study aimed to investigate the combined effects of astragaloside IV (AS-IV) and ginsenoside Rb1 (GsRb1) in DFU healing. Methods An in vitro DFU model was induced by high glucose (HG) in HaCaT keratinocytes, which were treated with AS-IV and/or GsRb1. Cell viability, cell death patterns (apoptosis, pyroptosis, and necroptosis), inflammatory reactions, and oxidative stress were determined. A rat DFU model was established and treated with AS-IV and GsRb1. The binding relationship between AS-IV/GsRb1 and potential targets was verified via molecular docking, a cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). Additionally, the regulatory roles of ZBP1 and IRF1 in HaCaT keratinocytes were clarified by downregulating or upregulating ZBP1 and IRF1. Results HaCaT keratinocytes treated with HG exhibited significantly elevated apoptosis, pyroptosis, necroptosis, and oxidative stress. The combined application of AS-IV and GsRb1 at a 2:3 ratio demonstrated optimal protective effects. In the STZ-induced diabetic rat DFU model, combined administration of AS-IV and GsRb1 significantly accelerated wound healing and enhanced granulation tissue formation, angiogenesis, and collagen deposition, while notably reducing neutrophil infiltration. Molecular docking analysis revealed that AS-IV and GsRb1 potentially bind to core proteins involved in PANoptosis. CETSA and DARTS verified that AS-IV and GsRb1 could directly bind to ZBP1 and decrease its protein level. Co-administration of AS-IV and GsRb1 suppressed activation of ZBP1-mediated PANoptosis. ZBP1 knockdown showed no additional protective effects of AS-IV and GsRb1. Overexpression of ZBP1 or its upstream regulator IRF1 exacerbated HaCaT keratinocyte injuries, which were reversed by AS-IV and GsRb1. Conclusions AS-IV and GsRb1 combination synergistically promotes wound healing in DFU via targeting the IRF1-ZBP1 axis-mediated PANoptosis, providing a potential therapeutic strategy for DFU.