Supplementary file 1_Extract of Polygala tenuifolia, Angelica tenuissima, and Dimocarpus longan improve skin wound healing in streptozotocin- induced diabetic mouse.docx

ObjectiveChronic skin wounds caused by diabetes, peripheral artery disease, pressure ulcers, and venous insufficiency do not fully recover anatomically and functionally. We previously found that topical application of 3% WIN-1001X cream reduces skin inflammation. In this study, we investigated whether 3% WIN-1001X cream alleviates chronic skin wounds exhibiting prolonged and extensive inflammation using streptozotocin-induced diabetic mouse. MethodsWIN-1001X contained 20% ethanol extracts of three botanical drugs: Polygala tenuifolia Willd., Angelica tenuissima Nakai, and Dimocarpus longan Lour. Streptozotocin-induced diabetic mice were used as a chronic wound model. After making full-thickness excision wounds were made on shaved dorsal skin, 3% WIN-1001X cream was topically applied daily for 12 days. The wound area was measured, and histology was performed to detect granulation tissue and collagen deposition. Quantitative real-time PCR and immunohistochemistry were performed to measure expression of RNA and proteins related to wound healing such as pro-inflammatory cytokines, growth factors, anti-microbial peptides, cell proliferation, keratinocyte differentiation, myofibroblast formation, and macrophage infiltration. ResultsTopical application of 3% WIN-1001X cream suppressed infiltration of neutrophils and monocytes as well as pro-inflammatory cytokine gene activation in diabetic mouse skin. It also promotes M1 to M2 macrophage polarization. Interestingly, 3% WIN-1001X cream activated the gene expression of anti-microbial peptides. Furthermore, It upregulated gene expression of PDGFβ, HGF, KGF, and TGFβ, resulting in the promotion of cell proliferation, granulation tissue formation, myofibroblast formation, and keratinocyte differentiation. Conclusion3% WIN-1001X cream suppressed skin inflammation through decreased cytokine gene expression, immune cell infiltration, and increased macrophage polarization. It also promoted cell proliferation, granulation tissue formation, and myofibroblast transition. Furthermore, 3% WIN-1001X cream promoted keratinocyte re-epithelialization and differentiation as well as increased collagen deposition in chronic skin wounds. Thus, our results suggest that 3% WIN-1001X cream may help alleviate chronic skin wounds.