WIN-1001X is composed of 20% ethanol extracts of three botanical drugs; Polygala tenuifolia Wildenow [Polygalaceae; Polygalae radix], Angelica tenuissima Nakai [Apiaceae; Angelicae tenuissimae radix], and Dimocarpus longan Loureiro [Sapindaceae; Longan arillus] which were purchased from Booyoung Pharmacy (Seoul, Korea). These three medicinal botanical drugs were mixed in a weight ratio of 1:1:1. The identification and authentication of these botanical drugs were verified by KGC Yebon Co., Ltd. (Cheongju-si, Korea), and specimens were stored in the Medihelpline Research Center plant specimen room (Seoul, Korea). The mixture was reflux-extracted for 3 h with 6 times the weight of the raw material in 20% ethanol (v/v) and then filtered. The residue was subsequently reflux-extracted for another 3 h with 4 times the weight of the raw material in 20% ethanol (v/v) and filtered. The combined filtrates were concentrated under reduced pressure and lyophilized to obtain the final dry extract. The drug-to-extract ratio (DER) is 3:1, meaning 1 g of the final extract (WIN-1001X) corresponds to 3 g of the initial raw botanical drug mixture. The preparation of WIN-1001X was conducted at KGC Yebon Co., Ltd. The analytical results for WIN-1001X are provided in Supplementary Figures 2–6.

To confirm chemical compositions and consistent preparation of WIN-1001X from batch to batch, the UPLC-MS analysis was performed using a single quadrupole mass spectrometer (MSD, Agilent 6120, Santa Clara, CA, USA) coupled with Waters ACQUITY ultra performance liquid chromatography (UPLC) system (Waters, Milford, USA). An ACQUITY BEH C18 column (3.0 × 150 mm, 1.7 µm) was used and the mobile phase was composed of solvent A (0.2% acetic acid in H2O) and solvent B (0.2% acetic acid in acetonitrile). The elution gradient proceeded as follows: 0–3 min, 5% B; 3–53 min, 28% B; 53–55 min, 34% B; 55–75 min, 38% B; 75–76 min, 70% B; 76–80 min, 70% B. The flow rate is maintained at 0.5 mL/min and injection volume is 10 µL. The column oven is maintained at a temperature of 30 °C. The mass spectrometry parameters were as follows: positive ion mode; gas temperature, 350 °C; drying gas, 12 L/min; capillary voltage, +4,000 V; m/z range, 200–1,500. The quantitation of each major metabolite was made using a Photodiode Array (PDA) detector set at 320 nm.

The WIN-1001X cream contained 3% (w/w) of WIN-1001X, respectively. Polyethylene glycol 400 was added as a base, and WIN-1001X was dissolved at 72∼78 °C for 20 min. Then, with the addition of other base ingredients and preservatives, the manufacturing process followed oil phase preparation, cooling, filling and packaging processes in accordance with the manufacturing method of ointment in the General Regulations of the Korean Pharmacopoeia. The 3% WIN-1001X cream was formulated with the following composition (w/w): WIN-1001X (3.0%), propylene glycol (8.0%), heavy liquid paraffin (5.0%), cetanol (2.5%), stearyl alcohol (5.0%), stearic acid (5.0%), isopropyl myristate (4.0%), sorbitan monostearate (3.0%), polysorbate 60 (3.0%), methyl parahydroxybenzoate (0.1%), and propyl parahydroxybenzoate (0.05%). The mixture was homogenized to ensure uniform dispersion. The preparation of WIN-1001X cream was conducted by Cires Pharmaceutical Inc. (Hwaseong-si, Korea).

Adult male C57BL/6J mice (7 weeks old, DBL, Korea) were maintained in a temperature-controlled room (23 °C) at 55% humidity, with a 12-h light-dark cycle. The Committee for Experimental Animal Research at Sogang University approved the animal experiments (IACUCSGU2021_11). Diabetes was induced by intraperitoneal injection of streptozotocin (ALX-380-010-G001, ENZO) for 5 days. Streptozotocin was dissolved in 0.5 M sodium citrate buffer (pH 4.5). Whole blood from the mouse tail vein was measured using a blood glucose monitoring system (Accu-CheK Rerforma, Roche). On day 25, mice with a blood glucose value ≥300 mg/dL, were defined as streptozotocin-induced diabetic mice. On day 28, the mouse was anesthetized with 2,2,2-tribromoethanol and a full-thickness excision wound was made on the shaved dorsal skin using a 6 mm biopsy punch (Kai Industries) (n = 6 mice). WIN-1001X cream was topically applied daily for 12 days. All animal experiments were indeed performed three times independently, with n = 6 mice per group in each iteration. Data analysis was performed in a blinded manner. Samples were coded, and investigators were unaware of the treatment groups during measurement and histological evaluation.

Total RNA was extracted from mouse skin wounds using Tri-RNA Reagent (TR118, Favorgen). First-strand cDNA synthesis was performed with PrimeScript RT master mix (RR036A, Takara). The resulting cDNAs were subjected to real-time PCR using qPCR 2x Premix SYBR (RT500M, Enzynomics) with a QuantStudio 1 Real-Time PCR System (Applied Biosystems). The PCR conditions were 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 64 °C for 40 s. Expression data were calculated from the cycle threshold (Ct) value using the ΔCt method of quantification. 18s rRNA was used for normalization. Oligonucleotides are listed in Supplementary Table 1.

Mouse dorsal skin samples were immediately fixed with 10% neutral buffered formalin (0144, Medilab, Korea) and left overnight at 4 °C. The samples were dehydrated, embedded in paraffin, and sectioned at 5 μm. The tissue sections were deparaffinized with xylene twice for 10 min each. Rehydration of sections was serially performed with 100%, 95%, 70%, 50% ethanol, followed by tap water. Tissue sections were stained with hematoxylin and eosin. Mast cells and collagen were stained with toluidine blue and Masson’s trichrome stain, respectively.

Rehydrated tissue sections were autoclaved in sodium citrate buffer (pH 6.0) for 10 min. After cooling, sections were washed in PBST (0.1% Triton X-100 in PBS) for 5 min and blocked with 5% BSA in 0.1% PBST for 1 h. For immunofluorescence, tissue sections were incubated with anti-arginase1 (66129-1-Ig, Proteintech), anti-PCNA (ab15497, Abcam), and anti-cytokeratin 17 (sc-393002, Santa Cruz Biotechnology) antibodies overnight at 4 ℃ , respectively . A secondary antibody conjugated to Cy5 (ab6563, ab6564, Abcam) was used. For DAB staining immunohistochemistry, tissue sections were incubated with anti-MPO (PA5-16672, Invitrogen), anti-iNOS (BD 610329, BD Bioscience), anti-VEGF (sc-152, Santa Cruz Biotechnology), and anti-αSMA (sc-32251, Santa Cruz Biotechnology) antibodies. HRP/DAB (ABC) detection IHC kit (ab64264, Abcam) was used. For DAB immunostaining, tissue sections were incubated with anti-MPO (PA5-16672, Invitrogen) and anti-iNOS (BD 610329, BD bioscience), anti-VEGF (sc-152, Santa Cruz Biotechnology), anti-αSMA (sc-32251, Santa Cruz Biotechnology) antibodies overnight at 4 ℃ . Then, the HRP/DAB (ABC) detection IHC kit (ab64264, Abcam) was used according to the manufacturer’s instructions. Expression was quantified using ImageJ software (NIH, Bethesda, MD, USA).

All animal experiments were indeed performed independently three times, with n = 6 mice per group in each experiment. All quantitative data were presented as the mean ± standard error of the mean (S.E.M.) from three independent experiments. Statistical differences among multiple groups were analyzed by one-way or two-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Statistical significance was defined as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005. All p-values indicated in the figures are listed in Supplementary Table 2.

We previously found that topical application of WIN-1001X reduces skin inflammation (manuscript in preparation). Thus, we further tested whether WIN-1001X cream alleviates chronic skin wounds that exhibit prolonged and extensive inflammation (Rosique et al., 2015; Zhao et al., 2016; Burgess et al., 2021). We used streptozotocin (STZ)-induced diabetic mice with fasting blood glucose levels exceeding 300 mg/dL as a chronic skin wound model (Figures 1A,B). For topical application, different percentages of WIN-1001X cream (WNX) were prepared and tested the efficacy of WIN-1001X cream in chronic skin wound healing (Supplementary Figure 1). We finally selected 3% WIN-1001X cream for further study. Although diabetic mouse treated with placebo cream (STZ placebo) showed delayed skin wound healing compared with normal skin wounds treated with placebo cream (control placebo), topical application of 3% WIN-1001X cream (STZ WNX) accelerated skin wound healing in diabetic mice (Figure 1C).

Given the extensive and prologned inflammatory phase in diabetic skin wounds (Rosique et al., 2015; Zhao et al., 2016; Burgess et al., 2021), we next examined the infiltration of immune cells into the skin wound. The infiltration of MPO (myeloperoxidase)-positive neutrophils was increased in diabetic skin wounds compared with normal skin wounds at 2 days after wounding. However, topical application of 3% WIN-1001X cream reduced the infiltration of neutrophils in diabetic skin wounds (Figure 2A). The infiltration of CD68-positive monocytes and macrophages in diabetic skin wounds treated with 3% WIN-1001X cream was also reduced compared with diabetic skin wounds treated with placebo cream at 2 days after wounding (Figure 2B). Consistently, the topical application of 3% WIN-1001X cream suppressed the upregulation of the gene expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, IL-23α, and TNFα as well as iNOS in diabetic skin wounds at 2 days after wounding (Figure 2C).

Microbial infection delays wound healing and often results in serious outcomes such as surgical debridement (Frykberg and Banks, 2015; Negut et al., 2018). WIN-1001X has no direct antimicrobial activity against bacteria including Staphylococcus aureus and Pseudomonas aeruginosa (data not shown). The skin has antimicrobial activity as part of the early stage of the immune defense system through the expression of antimicrobial peptides (Yamasaki and Gallo, 2008; Rademacher et al., 2021). We thus tested whether 3% WIN-1001X cream regulates the gene expression of antimicrobial peptides in diabetic skin. At 2 days after wounding, the topical application of 3% WIN-1001X cream had no effect on the expression of Defb1, Defb2, Defb3, and LL-37 genes in diabetic skin wounds. However, upregulation of Defb2, Defb3, and LL-37 gene expression was observed in 3% WIN-1001X cream-treated diabetic skin wounds at 7 days after wounding (Figure 3).

Classically activated M1 macrophages, which promote inflammation, could be polarized into alternatively activated M2 macrophages for successful wound repair (Ferrante and Leibovich, 2012; Louiselle et al., 2021). The prolonged existence of M1 macrophages is one of the characteristics of chronic wound healing. Thus, we examined the existence of M1 and M2 macrophages at 7 days after wounding by immunostaining with anti-iNOS (inducible NOS) and anti-Arg1 (Arginase 1) antibodies, respectively. The increased level of iNOS-positive M1 macrophages was maintained in diabetic skin wounds compared with normal skin wounds at 7 days after wounding (Figure 4A). However, 3% WIN-1001X cream treatment restored the normal level of M1 macrophages in diabetic skin wounds (Figure 4A). Additionally, 3% WIN-1001X cream maintained the normal level of Arg1-positive M2 macrophages in diabetic skin wounds at 7 days after wounding (Figure 4B). Consistent with immunohistochemical results, 3% WIN-1001X cream induces gene expression of markers of M2 macrophages such as Arg1 and Fizzl (Figure 4C).

VEGF (vascular endothelial growth factor) plays a role in angiogenesis in wound healing through endothelial cell proliferation, migration, differentiation, and survival (Bao et al., 2009; Johnson and Wilgus, 2014). We found that topical application of 3% WIN-1001X upregulated VEGF expression in diabetic skin wounds compared with the placebo control at 7 days after wounding (Figures 5A,B). Additionally, the transition from inflammation to proliferation is also a critical process for successful wound healing (Landén et al., 2016). We examined the cell proliferation of keratinocytes and fibroblasts by immunostaining using anti-PCNA (proliferating cell nuclear antigen) antibody. Topical application of 3% WIN-1001X promoted keratinocytes and fibroblasts proliferation in diabetic skin wounds compared with placebo-treated diabetic skin wounds at 7 days after wounding (Figure 5C). Moreover, 3% WIN-1001X cream activated the gene expression of growth factors including PDGFβ, HGF, KGF and TGFβ, which are responsible for cell proliferation in diabetic skin wounds compared with diabetic skin wounds treated with placebo cream (Figure 5D). An increase in granulation tissue formation was observed in 3% WIN-1001X cream-treated diabetic skin wounds compared with placebo cream-treated diabetic skin wounds at 7 days after wounding (Figure 5E). Additionally, we observed that 3% WIN-1001X cream promoted myofibroblast formation in granulation tissue (Figure 5F).

Immunostaining with an anti-K17 (keratin 17) antibody showed that topical application of 3% WIN-1001X cream promoted re-epithelialization as a result of epithelial keratinocyte migration over the wound bed in chronic skin wounds at 7 days after wounding (Figure 6A). We further examined the expression of genes related to keratinocyte differentiation. Topical application of 3% WIN-1001X cream upregulated Filaggrin, Loricrin, Involucrin, and Keratin 1 gene expression in chronic skin wounds at 7 days after wounding (Figure 6B). We also found that topical application of 3% WIN-1001X cream promoted collagen deposition, as observed in tissue sections stained with Masson’s trichrome (Figure 6C).