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

Wnt9a expression was significantly lower in human chronic wound tissue compared to adjacent normal skin tissue.

• Tissue collected from 8 patients with diabetic foot ulcers (5 males, 3 females, aged 45-72 years) who underwent debridement surgery.
• ELISA method showed significantly lower Wnt9a expression in chronic wound tissue vs. normal skin (t=7.68, P<0.05).
• Immunofluorescence method confirmed the same finding (t=10.25, P<0.05).
• Both detection methods yielded consistent results indicating downregulation of Wnt9a in chronic wounds.

Wnt9a expression was significantly lower in mouse chronic wound tissue compared to control wound tissue at 7 days after modeling.

• Chronic wound model was established in male C57BL/6 mice (6-8 weeks) by subcutaneous injection of M1 macrophage-derived exosomes at the wound edge.
• 4 mice per group (control group and chronic wound group).
• ELISA detection at 7 days post-modeling showed significantly lower Wnt9a in chronic wound group vs. control group (t=5.12, P<0.05).

Wnt9a overexpression significantly reduced residual wound area in mice with chronic wounds at all measured time points.

• 16 male C57BL/6 mice were divided into empty control group (AV-eGFP) and Wnt9a overexpression group (AV-Wnt9a-eGFP), 8 mice per group.
• Residual wound area percentages were significantly lower in Wnt9a overexpression group at 3 days (t=3.90, P<0.05), 7 days (t=6.62, P<0.05), and 14 days (t=5.73, P<0.05) after modeling.
• Adenovirus vectors were injected subcutaneously at the wound edge.

Wnt9a overexpression reduced type I and type III collagen expression and improved collagen fiber arrangement in mouse chronic wound tissue at 14 days.

• At 14 days after modeling, type I collagen expression was significantly lower in Wnt9a overexpression group vs. empty control group (t=6.25, P<0.05).
• Type III collagen expression was also significantly lower in Wnt9a overexpression group vs. empty control group (t=5.48, P<0.05).
• Masson staining showed more orderly arrangement of collagen fibers in Wnt9a overexpression group compared to empty control group at 14 days.

Wnt9a overexpression significantly enhanced fibroblast migration in vitro.

• Human skin fibroblasts isolated from normal skin tissue were infected with AV-eGFP (empty control) or AV-Wnt9a-eGFP (Wnt9a overexpression); n=3 per group.
• At 72 hours after infection, Wnt9a protein expression was significantly higher in the overexpression group vs. empty control group (t=6.96, P<0.05).
• Cell migration rate at 48 hours after scratching was (71.6±6.4)% in Wnt9a overexpression group, significantly higher than (38.5±2.4)% in empty control group (t=8.31, P<0.05).

Knockdown of Wnt9a via siRNA significantly reduced fibroblast migration.

• Normal human skin fibroblasts were transfected with siRNA-Wnt9a or negative control siRNA (siRNA-NC); n=3 per group.
• At 24 hours after transfection, cell migration rate at 48 hours after scratching was (15.4±3.2)% in siRNA-Wnt9a group, significantly lower than (31.9±3.6)% in siRNA-NC group (t=5.93, P<0.05).

Transcriptome sequencing revealed that Wnt9a overexpression downregulated multiple collagen family genes and enriched genes in the non-classical Wnt signaling pathway.

• Transcriptome sequencing was performed on fibroblasts at 72 hours after adenoviral infection (n=3 per group).
• Significantly downregulated differentially expressed genes (DEGs) in Wnt9a overexpression group included multiple collagen family genes compared to empty control group.
• Gene ontology and KEGG enrichment analysis showed that genes in Wnt9a overexpression group were significantly enriched in the non-classical Wnt signaling pathway.