Fig 2: The optimal therapeutic dosage of netrin-1 in treating diabetic wounds. (A) Schematic diagram of intradermally injecting netrin-1 to treat diabetic wounds. (B) Images of the different doses of netrin-1 (4, 20 and 100 μg/mL, 50 μL per wound) treating diabetic wounds and (C) the statistical analysis of the wound healing rates (%), showing that 20 μg/mL netrin-1 (50 μL per wound) was the optimal therapeutic dosage (n = 4). Netrin-1, NTN-1. Data are shown as the mean ± SD. N.S, no significance between the 20 and 100 μg/mL netrin-1 treatment groups. # 4 μg/mL NTN-1 vs. Blank, * 20/100 μg/mL NTN-1 vs. 4 μg/mL NTN-1. ### P<0.001, #### or **** P<0.0001.

Fig 3: Netrin-1 could promote the switch of macrophages toward the M2 phenotype and modulate cellular functions in vitro. (A) Procedures for bone marrow-derived macrophage (BMDM) preparation. (B) FACS of CD11b+ and F4/80+ cell populations (%) in the induced bone marrow-derived cells. (C) Changes in macrophage morphology after treatment with netrin-1 (scale bar: 100 μm). (D) Flow cytometry analysis of CD206+ macrophages upon netrin-1 treatment. (E) Immunofluorescence staining of CD206 showing the existing M2 macrophages (scale bar: 20 μm). (F) RT-qPCR analysis of macrophage markers (M1: CD86, TNF-α, IL-1β, and IL-6; M2: CD206, ARG1, IL-10, and VEGF) in netrin-1-treated BMDMs (n = 3). Netrin-1 (125 ng/mL), N125; netrin-1 (250 ng/mL), N250; netrin-1 (500 ng/mL), N500. Fluorescence-activated cell sorting, FACS. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and N·S, no significance.

Fig 4: Netrin-1 regulated macrophage polarization via the A2bR/STAT/PPARγ signaling pathway in vitro. (A) Volcano plot of the detected genes with 640 DEGs identified using the criteria (|log2 (fold change)|>0 and P < 0.05). (B) Histogram of the DEGs. (C) Heat map of the gene expression of M1 and M2 macrophage markers from the RNA-seq library (n = 3). (D–E) Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. (F) Western blotting of CD86 and CD206 in netrin-1-treated macrophages upon exposure to GW9662 (n = 3). (G) Western blotting of the key proteins of the PPAR signaling pathway in netrin-1-treated macrophages upon MRS intervention (n = 3). (H–I) Immunofluorescence staining of CD206 showing the existing M2 phenotype (scale bar: 20 μm). (J) RT-qPCR analysis of macrophage markers (M1: CD86, TNF-α, IL-1β, and IL-6; M2: CD206, ARG1, IL-10, and VEGF) (n = 3). Netrin-1, NTN-1. Differentially expressed genes, DEGs. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 5: Netrin-1 promoted the angiogenic capability of endothelial cells in vitro. (A) Results of the CCK-8 assay in HUVECs treated with netrin-1 (n = 5). (B–D) Results of the scratch and Transwell assays with statistical analyses showing the increased migration of HUVECs upon netrin-1 treatment (n = 3). (E) In vitro Matrigel tube formation of HUVECs with an analysis of the formed meshes (n = 3). Netrin-1 (125 ng/mL), N125; netrin-1 (250 ng/mL), N250; netrin-1 (500 ng/mL), N500. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.