Clinical Application Of FOXO1 Inhibitor In Diabetic Minipig Skin Wounds

Objectives: The forkhead box O1(FOXO1) transcription factor plays an important role in pro-inflammatory gene expression in diabetic wounds. In our study we tested the hypothesis that the FOXO1 inhibitor improves diabetic skin wound healing in Type2 diabetes mellitus(T2DM) preclinical large animal models.
Methods: T2DM was induced in Göttingen minipigs by the cafeteria diet and subsequent low dose injection of streptozotocin. 1-cm full-thickness skin wounds were created in the backs of normoglycemic and diabetic minipigs and treated by local injection of FOXO1 inhibitor(experimental) or vehicle(control)(day 8, n=5). Quantitative measurement was performed with H&E and Masson’s trichrome, and immunofluorescence stain at the wound edges and center separately. Immunofluorescence stain was performed with primary antibodies to α-SMA and TNF-α as well as the appropriate isotype-matched control IgG. Statistical significance was determined by ANOVA with Scheffe's post-hoc test for multiple group comparisons(p<0.05).
Results: Local application of a FOXO1 inhibitor improved connective tissue healing in diabetic wounds. Collagen percentage significantly increased by 230% at diabetic wound center and 150% at wound edges by FOXO1 inhibitor injection compared with vehicle(control) groups(p<0.05). The number of myofibroblasts increased by 153% in diabetic wound edges and 128% in center area with FOXO1 inhibitor injection compared with vehicle groups(p<0.05). FOXO1 inhibition caused a significant decrease in TNF-αpositive cells by 83% in diabetic wound edges and 97% in wound center compared with vehicle groups(p<0.05). In normal wounds the FOXO1 inhibitor injection reduced the collagen percentage by 60%(p<0.05), decreased the number of myofibroblasts by 39-46%(p<0.05) and increased the number of TNF-α positive cells by 275-585% compared with vehicle groups(p<0.05).
Conclusions: We demonstrate for the first time that the local application of a FOXO1 inhibitor significantly improved wound healing in a preclinical T2DM animal model reflected by increased collagen formation, increased the number of myofibroblasts and reduced the TNF-α expression.