Fig 1: Effects of CoNZ on anti-inflammation in vivo and in vitro. (A–C) In vivo effects of CoNZ in STZ-induced diabetic skin wounds: (A) Illustration depicting the diabetic skin wound model using streptozotocin (STZ) and the assays performed over 21 days. (B) ROS signal (in red) in tissue samples at day 2, and quantification of signal intensity; the intense ROS signal within the defect area was substantially reduced, particularly in the CoNZ group. (C) Immunohistochemical staining of tissue samples for iNOS (pro-inflammatory signal) and IL-10 (anti-inflammatory signal) at week 1 and 3, along with semi-quantitative data using ImageJ; CoNZ down-regulated iNOS but up-regulated IL-10. For the in vivo studies, nanoparticles of 10 µL from a concentration of 100 mg/1 mL were treated to each wound. (D–G)In vitro effects of CoNZ on high glucose- and ROS-challenged inflammatory cells: (D) Diagram of the in vitro design that involves macrophages (RAW264.7 cells) challenged with high glucose (HG) and LPS. Nanoparticle concentration used for the in vitro study was 20 µg/mL. (E) Intracellular ROS signal intensity measured at 6 h. (F) Immunostaining images of cells at 24 h, depicting pro-inflammatory (iNOS, IL-1b) and anti-inflammatory (Arg-1) signals, along with semi-quantitative data using ImageJ. (G) Western blot analysis at 24 h, along with semi-quantitative data using ImageJ, showing iNOS, Arg-1, NF-kB, and its phosphorylated form. Statistical significance was calculated between groups using Unpaired t-test and one-way ANOVA with *p < 0.05, **p < 0.01, and ***p < 0.001 denoting significance compared to the defect group (in vivo) or HG (+)/LPS (+) group (in vitro); and +p < 0.05, ++p < 0.01, and +++p < 0.001 indicating significance compared to the BGn group. All data are presented as mean ± one standard deviation and the sample size was n = 5.