Fig 1: Virtual screening of an RSAD2‑targeted small‑molecule compound accelerates skin‑wound repair. (A) Workflow of virtual screening and experimental validation for RSAD2‑targeting compounds. (B) Chemical structure of PSB06126. (C) 3D binding model of PSB06126 with RSAD2 (left) and detailed analysis of the interaction interface (right). PSB06126 is shown as a rose‑colored stick representation; carbon atoms of mouse RSAD2 are shown in yellow, oxygen atoms in red, and hydrogen atoms in blue. (D) Schematic of drug administration. Mice received daily intraperitoneal injections of 10 mg/kg PDK1 inhibitor, PSB06126, LCA, or equivalent vehicle for 3 days before modelling. Western blot of NLRP3, CMPK2, and RSAD2 protein expression at 1 day after injury, normalized to the sham group. (E, F) Quantification of mtDNA levels by qPCR in F4/80+ cells magnetically isolated from wound areas 1 day after injury among different drug‑treatment groups. Data were analyzed by one‑way ANOVA followed by Tukey's post‑hoc test (n = 5). (G, H) SPR analysis of PSB 06126 binding to immobilized RSAD2. (G) Representative sensorgrams showing binding responses at increasing concentrations of PSB 06126. (H) Steady‐state binding curve of PSB 06126 interaction with RSAD2. The KD derived from affinity fitting was 1.66 um. (I) ELISA quantification of IL‐1β levels in skin wounds from vehicle‐ and PSB 06126‐treated mice at 1 d post‐injury (n = 3). (J) Representative H&E staining images and quantification of inflammatory‑cell infiltration in wounded areas 1 day after injury in vehicle and PSB06126 groups. (scale bar, 100 µm). (K) Representative H&E staining and CD31 immunofluorescence of granulation tissue in the vehicle and PSB 06126‐treated groups at day 7 post‐injury. Black triangles indicate the leading edge of the epithelial tongue; black arrows indicate the origin of the epithelial tongue (scale bar, 200 µm). (L–N) Quantification of epithelialization length, CD31+ vessel density, and granulation tissue thickness in the vehicle and PSB 06126‐treated groups at day 7 post‐injury. (O) Representative Masson's trichrome staining of granulation tissue 14 days after injury in vehicle and PSB06126 groups (scale bar, 200 µm). (P) Representative wound images and simulated healing curves of vehicle and PSB06126 ‑treated group. (Q) Immunoblot analysis of NLRP3 and CMPK2 protein expression in Lepr db/db mice 1 day after skin injury, normalized to the vehicle group. (R) Quantitative PCR analysis of mtDNA in F4/80+ cells from Lepr db/db mice treated with vehicle or PSB 06126 1 day after injury (n = 5; unpaired t test). (S) Representative H&E staining of wounded areas from Lepr db/db mice 1 day after injury (scale bar, 100 µm). (T) Representative H&E staining and CD31 immunofluorescence of granulation tissue in the vehicle and PSB 06126‐treated groups at day 7 post‐injury. Black triangles indicate the leading edge of the epithelial tongue; black arrows indicate the origin of the epithelial tongue (scale bar, 200 µm). (U–W) Quantification of epithelialization length, CD31+ vessel density, and granulation tissue thickness in the vehicle and PSB 06126‐treated groups at day 7 post‐injury. (X) Representative Masson's trichrome staining of granulation tissue from Lepr db/db mice 21 days after injury in vehicle and PSB06126 groups (scale bar, 200 µm). (Y) Representative wound images and simulated healing curves of vehicle and PSB06126 ‑treated Lepr db/db mice. Within‐group comparisons were analyzed using unpaired t tests, * p <0.05; NS, not significant.