[Role and mechanism of Wnt9a in human and mouse chronic wound healing]

Objective: To investigate the role and mechanism of Wnt9a in human and mouse chronic wound healing. Methods: This study was a case series combined with group-designed basic study. The chronic wound tissue and its adjacent normal skin tissue were collected from 8 patients with diabetic foot ulcers, who received debridement surgery in the First Affiliated Hospital of Air Force Medical University from June to September 2023, including 5 males and 3 females, aged 45-72 years. The expression of Wnt9a was detected by enzyme-linked immunosorbent assay (ELISA) method and immunofluorescence method. Eight male C57BL/6 mice aged 6-8 weeks were used to establish a full-thickness skin resected wound on the back. They were divided into control group received no additional treatment and chronic wound group subcutaneously injected with mouse M1 macrophage derived exosomes at the wound edge to establish a chronic wound model using a random number table method (the same grouping method below), with 4 mice in each group. At 7 days after modeling, the Wnt9a expressions in the wound tissue of mice in two groups was detected by ELISA method. Additional 16 male C57BL/6 mice aged 6-8 weeks were used to establish the chronic wound model as before and were divided into empty control group and Wnt9a overexpression group, with 8 mice in each group, which were injected subcutaneously at the wound edge with enhanced green fluorescent protein empty adenovirus (AV-eGFP) and Wnt9a gene recombinant adenovirus expressing enhanced green fluorescent protein (AV-Wnt9a-eGFP), respectively. At 3, 7, and 14 days after modeling, the percentages of residual wound area were calculated. At 14 days after modeling, the expressions of type Ⅰ and type Ⅲ collagen were detected by Western blotting, and the arrangement of collagen fibers was observed after Masson staining. The normal human skin tissue collected in the abovementioned experiment was used to isolate fibroblasts (Fbs), which were divided into empty control group infected with AV-eGFP and Wnt9a overexpression group infected with AV-Wnt9a-eGFP. The protein expression of Wnt9a at 72 h after infection was detected by Western blotting; at 48 h after infection, the cell migration rate at 48 h after scratching was detected by scratch test. Additional normal human skin Fbs were collected and divided into Wnt9a specific small interfering RNA (siRNA-Wnt9a) group and negative control small interfering RNA (siRNA-NC) group, which were transfected with corresponding small interfering RNA, respectively. At 24 h after transfection, the cell migration rate at 48 h after scratching was detected by scratch test. Additional normal human skin Fbs were taken and divided into empty control group and Wnt9a overexpression group treated as before. At 72 h after infection, transcriptome sequencing was performed to screen for differentially expressed genes (DEGs), and the gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis were performed. The sample number in all cell experiments was 3. Results: The results of both ELISA method and immunofluorescence method showed that the expression level of Wnt9a in human chronic wound tissue was significantly lower than that in normal skin tissue (with t values of 7.68 and 10.25, respectively, P<0.05). At 7 days after modeling, the expression level of Wnt9a in the wound tissue of mice in chronic wound group was significantly lower than that in control group (t=5.12, P<0.05). The percentages of residual wound area of mice in Wnt9a overexpression group were significantly lower than those in empty control group at 3, 7, and 14 days after modeling (with t values of 3.90, 6.62, and 5.73, respectively, P<0.05). At 14 days after modeling, the expression levels of type Ⅰ and type Ⅲ collagen in the wound tissue of mice in Wnt9a overexpression group were significantly lower than those in empty control group (with t values of 6.25 and 5.48, respectively, P<0.05). At 14 days after modeling, the collagen fibers in the wound tissue of mice in Wnt9a overexpression group arranged more orderly than those in empty control group. At 72 h after infection, the protein expression level of Wnt9a in cells in Wnt9a overexpression group was significantly higher than that in empty control group (t=6.96, P<0.05). At 48 h after infection, the cell migration rate in Wnt9a overexpression group was (71.6±6.4)% at 48 h after scratching, which was significantly higher than (38.5±2.4)% in empty control group (t=8.31, P<0.05). At 24 h after transfection, the cell migration rate in siRNA-Wnt9a group was (15.4±3.2)% at 48 h after scratching, which was significantly lower than (31.9±3.6)% in siRNA-NC group (t=5.93, P<0.05). At 72 h after infection, compared with that in empty control group, the significantly downregulated DEGs in cells in Wnt9a overexpression group included multiple collagen family genes, and the genes in cells in Wnt9a overexpression group were significantly enriched in the non-classical Wnt signaling pathway. Conclusions: Wnt9a expression is downregulated in chronic wound tissue of human and mice, and the overexpression of Wnt9a may promote migration of Fbs and collagen remodeling through non-classical Wnt signaling pathway, thereby accelerating chronic wound healing.