We purchased KFX from Xinglin Pharmacy (Chengdu, China).

After disinfecting the skin with 75% alcohol, we dislocated the necks of suckling female BALB/C mice (3 days old). Mouse thenar-skin samples were excised, tissues were rinsed with phosphate-buffered saline (PBS), and subcutaneous fat and blood vessels were carefully removed. Then, we incubated the tissues in 0.25% trypsin–EDTA and 2 mg/ml dispase (volume ratio, 1:1; MilliporeSigma, St. Louis, MO, United States) at 37°C for 1 h. After dispase treatment, we separated the epidermis, cut the dermal tissue into pieces of approximately 1.0 mm³ using surgical scissors, and digested these pieces with 0.25% collagenase II at 37°C for 2 h. The cell complex was centrifuged at 500g for 5 min. Finally, we collected the dermal cells and washed them three times in PBS to acquire dermal MSCs.

MSCs from suckling mice were cultured in MedGro Stem Cell Medium supplemented with 10% fetal bovine serum (FBS; GIBCO) in a 37°C, 5% CO₂ incubator.

We purchased wild-type (WT) female BALB/c mice (6–8 weeks old, 23–27 g) from Cheng Du Dossy Animal Technology Co., Ltd. (Chengdu, China). They were maintained at the Experimental Animal Center of Basic Medical College of Chengdu University of TCM. Animals were treated in accordance with the regulations of the Experimental Animal Ethics Committee of Chengdu University of TCM (TCM-LAEC2020074). The mice drank water and ate freely, and the room was kept at a temperature of 22°C ± 2°C and humidity of 40% ± 10%.

Cells treated with different concentrations of KFX in 96-well plates at a density of 1 × 10⁴ cells per well. After 2 h of incubation, 10 μL CCK-8 solution were added to each well. Cells were further incubated for 1 h in a 37°C incubator. We obtained data using a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States) with absorbance at 450 nm. ImagePro Plus statistical-analysis and plotting software version 6.0 (Media Cybernetics, Rockville, MD, United States) was used to analyze the experimental data and calculate cell viability and proliferation rates. After calculating the difference between the drug administration group and the blank control group, we compared the proliferation percentages of the two groups. The experiment was repeated three times, with five replicates per sample.

We performed a scratch wound assay by seeding target cells on 6-well plates, growing them to a cell density of 90%, and then starving the cells for 48 h in serum-free medium with or without different concentrations of KFX. The medium was removed, and the cells were scratched with a 200-µl pipette to form a uniform cell-free wound area. Cell debris was gently cleaned away with PBS buffer. At 0, 24, and 48 h, we observed and photographed cell migration to the wound area under a bio-electron microscope (ECLIPSE Ti-E; Nikon, Tokyo, Japan). Picture magnification was ×100. We used ImageJ software (National Institutes of Health [NIH], Bethesda, MD, United States) to analyze scratch widths at 0, 24, and 48 h of culture, and percentages of scratch width reduction were compared to assess cell mobility. The experiment was repeated three times with three replicates per sample.

All mice were anesthetized by inhalation of avertin. We established a classic cutaneous excision wound model on the back of each mouse after hair removal and disinfection with 70% alcohol. Wounded mice were randomly divided into two groups (20 per group) and treated with normal saline and KFX three times per day. At 0–8 days, we photographed and measured the mice’s wound areas using ImageJ software. The wound healing rate was calculated as follows: wound reduction rate  ( % )   =   ( A 0 - l t ) / A 0  ×  100 % where A₀ was the initial wound area (T₀) and l _t was the wound area at the time interval (t ≥ 1). We used ImageJ software in accordance with the instructions offered by Schneider (Nature Methods, 2012).

We fixed the traumatized cutaneous tissues in 4% paraformaldehyde for 24 h and then prepared tissue paraffin slides. The slides were dewaxed, rehydrated, and washed with different reagents before staining with hematoxylin and eosin (H&E; Beyotime Biotech). We obtained pictures under a bio-electron microscope (DP80; Olympus Corp., Tokyo, Japan). Picture magnification was ×100. We analyzed wound size, granular-tissue thickness, and hair follicle regeneration.

We fixed the traumatized cutaneous samples in 4% paraformaldehyde for 24 h and then prepared tissue paraffin slides. After dewaxing and dehydration, antigen retrieval was performed at 95°C for 10 min. We blocked the slides with 5% BSA at room temperature (RT) for 1 h. Anti-vimentin primary aB was diluted to 1:1000, anti-K5 primary aB to 1:1500. After rinsing them with PBS, we incubated the slides with the secondary aB for 1 h. The slides were then counterstained with DAPI to stain the nuclei. Finally, we photographed them under a bio-electron microscope (BX53; Olympus Corporation, Tokyo, Japan).

For immunofluorescence (IF), fluorescein isothiocyanate (FITC)–labeled fluorescent primary aBs against PCNA (dilution, 1:500) and vimentin (dilution, 1:1000) were incubated overnight at 4°C. On day 2, we rewarmed and washed the sections. Sections were incubated with Alexa Fluor 488 fluorescent secondary antibodies (dilution, 1:1000) in the dark for 1 h. After counterstaining them with DAPI, we observed the slides under a fluorescence microscope and photographed them. Three fields of view were analyzed; picture magnification was ×100. ImagePro Plus v6.0 was used to analyze the experimental data and calculate average optical density (OD). We estimated the percentage of double-fluorescent–labeled cells among ECs or MSCs using different fluorescence-labeled cell counting methods and fluorescence intensity statistics in ImageJ. In addition, we anonymously shuffled image order statistics to prevent statistical preferences. We estimated the percentage of double-fluorescent–labeled cells among either epithelial or stromal cells using ImagePro Plus v6.0.

Total traumatized cutaneous-tissue RNA was extracted using an RNA Fast Kit (Yeasen Biotechnology, Shanghai, China). We synthesized cDNA using a HiScriptII Reverse Kit (Roche). Real-time polymerase chain reaction (RT-qPCR) was performed using FastStart Universal SYBR Green PCR Mix on an IQ5 PCR System (BioRad Laboratories, Hercules, CA, United States). The reaction procedure was as follows: 40 cycles of 94°C for 1 min, 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s. Primers used are listed in Table 1. We calculated mRNA expression of related target genes using the classic formula 2^−xpre. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal gene.

The samples used in the transcriptomics experiment were the suckling mouse MSCs treated with different concentrations (0, 0.05, 0.1, 0.2, and 0.25 mg/ml) of KFX for 24 h. Transcriptomics sequencing was performed by Majorbio Co., Ltd. (Shanghai, China). Briefly, RNA quality and integrity were assessed using a NanoDrop 2000 system (Thermo Fisher). We used 2 µg RNA per sample as the input control for transcriptomics sequencing. A clustering analysis was performed using a cBot Cluster Generation System and a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, United States). Genes with p < 0.05 were considered to be differentially expressed.

We collected wound tissues for total protein extraction and analyzed STAT3 and p-STAT3 levels after 3 days of cutaneous-wound healing in vivo. Traumatized cutaneous tissues from mice were scraped and resuspended in 0.5 ml RIPA; tissue lysates were placed on ice for 1 h. After centrifugation at 10,000g for 20 min, we determined total protein concentration using a Bicinchoninic Acid (BCA) Protein Determination Kit (Yeasen). We electrophoresed 20-ct samples via 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; Yeasen) and transferred them onto the PVDF membrane. Samples were blocked with blocking buffer at RT for 2 h and then probed with STAT3 (dilution, 1:500; Invitrogen), p-STAT3 (dilution, 1:1000; Invitrogen), and β-actin (dilution, 1:10000; AB clonal Technology, Woburn, MA, United States) at 4°C overnight. Secondary aBs used for detection included horseradish peroxidase (HRP)–conjugated anti-rabbit immunoglobulin G (IgG; dilution, 1:10000; ABclonal Technology). We detected target protein expression using an Enhanced Chemiluminescence (ECL) Detection Kit (Boster).

We used the classic scientific software SPSS version 17.0 (IBM Corp., Armonk, NY, United States) for statistical-data analysis. Data were uniformly represented as mean ± standard deviation (SD). We compared different groups using Student’s t test and one-way analysis of variance (ANOVA). p < 0.05 was considered to indicate a statistically significant difference. We plotted the analyzed data using GraphPad Prism software version 8.0 (GraphPad Software, Inc., San Diego, CA, United States).

Cell proliferation and migration are necessary to restore cutaneous integrity and accelerate the growth of ECs at the cutaneous-wound site via epidermal-cell migration and proliferation from the wound tissue edge (Qian et al., 2016). We used a CCK-8 assay to confirm cutaneous-EC proliferation in the presence of KFX at 48 h. Compared with the control group, the results showed that KFX could stimulate proliferation of cutaneous ECs in a dose-dependent manner. A low dose (0.5 g/ml, 5%) induced optimal growth in cutaneous ECs at 48 h (Figures 1A,B). Conversely, high doses of KFX (5 and 2.5 mg/ml) inhibited cutaneous-EC proliferation and showed a steep, dose-dependent dominant toxicity. Furthermore, KFX treatment caused evident cell proliferation after 48 h. We then used a scratch wound assay to assess cutaneous-EC migration in vitro. Figures 1C,D shows that, compared with the control group, KFX significantly improved cell wound closure migration in the KFX-treated group (p < 0.05). Taken together, the above results indicated that KFX could promote the proliferation and migration of cutaneous ECs in vitro.

To further determine the effects of KFX on cutaneous ECs, we performed transcriptomics sequencing analysis on cutaneous ECs treated with KFX. The results showed that, compared with the saline group, KFX could upregulate 805 genes and downregulate 932 (Figure 2A). The top 20 most up- and downregulated genes were as follows: Upregulated: matrix metalloproteinase 1 (Mmp3), prostaglandin reductase 1 (Ptgr1), heme oxygenase 1 (Hmox1), decorin (Dcn), glucose-6-phosphate-dehydrogenase (G6pd), aryl hydrocarbon receptor repressor (Ahrr), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)–inducible poly (adenosine diphosphate [ADP]–ribose) polymerase (Tiparp), thioredoxin reductase 1 (Txnrd1), solute carrier family 48 member A1 (Slc48a1), cystatin 6 (Cst6), KIT ligand (Kitlg), Fosl1, vascular endothelial growth factor delta (Vegfd), arginine vasopressin receptor 1A (Avpr1a), Mmp13, scavenger receptor class A member 5 (Scara5), glutamate–cysteine ligase catalytic subunit (Gclc), chemokine (C-C motif) ligand 2 (Ccl2), glutaredoxin (Glrx), and cyclin-dependent kinase inhibitor 1a (Cdkn1a). Downregulated: calponin 1 (Cnn1), myosin-11 (Myh11), actin alpha 2 (Acta2), secreted phosphoprotein 1 (Spp1), glypican 4 (Gpc4), Acta1, T-box transcription factor 18 (Tbx18), coiled-coil domain containing 80 (Ccdc80), serine protease 23 (Prss23), transforming growth factor-beta 3 (Tgfb3), actin filament–associated protein 1l2 (Afap1l2), gamma-actin 2 (Actg2), lymphocyte cytosolic protein 1 (Lcp1), Slc6a6, keratin 7 (Krt7), angiomotin (Amot), growth arrest specific 6 (Gas6), tropomyosin 2 (Tpm2), and Myh10. Our analysis found that upregulated expression was mainly concentrated in genes related to cell adhesion and angiogenesis, as shown in Figure 2B. Therefore, KFX significantly promoted the expression of genes related to these processes.

We evaluated the beneficial activities of KFX in cutaneous-wound healing in a cutaneous-perforation wound healing animal model. Compared with the untreated group, the KFX formulation promoted cutaneous healing 3 days after initial wound information. On days 3–6 after cutaneous-perforation wounding, the wound area in the KFX treatment group was obviously smaller than that in the saline group (Figure 3A). Interestingly, the KFX group achieved complete wound healing within 8 days after perforation wounding, indicating that this group experienced a far better subsequent wound healing effect than the saline group (Figures 3A,B). Furthermore, H&E staining of cutaneous-wound tissues (Figure 3C) showed that epidermal tissues were remarkably thicker in the KFX group than in the saline group at days 3 and 14 after cutaneous-perforation wounding. In summary, KFX could promote cutaneous-wound healing in vivo.

To better understand the effect of KFX treatment on wound healing, we analyzed cutaneous-wound tissues using IHC and IF staining. Cytokeratin 5 (CK5) is an epidermal-cell molecular marker (Park et al., 2019). In our study, IHC staining was used to study epidermal regeneration of healed skin. We identified epidermal cells stained with green fluorescence as CK5 epidermal cells, while PCNA was labeled with red fluorescence to show total proliferating cells (Pan et al., 2021) (Figures 4A–D). Compared with the saline group, the KFX group saw a significant increase in K5⁺ cell count, and its PCNA⁺ cell count was also greater than that of the control group on days 3 and 14 after cutaneous-perforation wounding. These results indicated that KFX could promote epidermal-cell proliferation, differentiation, and formation in wounds.

Furthermore, cutaneous-wound healing is related to the proliferation and differentiation of mesenchymal cells, of which vimentin is a biomarker (Paulin et al., 2022). Therefore, we further analyzed the effect of KFX on the proliferation of cutaneous mesenchymal cells in wounds, using IF staining to indicate vimentin⁺ cells. Cells labeled green were considered vimentin⁺, and those co-labeled red and green indicated total proliferating-cell count among vimentin⁺ cells. As seen in Figures 4E,F, our results showed that compared with the saline group, the counts of vimentin– and PCNA–double-positive cells in the KFX group were significantly increased. Vimentin anchors and supports organelles in the cytoplasm of mesenchymal cells; its expression is upregulated during epithelial–mesenchymal transformation (EMT), which occurs during wound healing. The increase in vimentin⁺–cell count in the KFX group might be a biomarker of mesenchymal-origin cells or EMT-producing cells. In addition, we quantified mRNA levels of genes related to cell proliferation and differentiation at day 3 after cutaneous-perforation wounding using RT-qPCR. The mRNA expression of related target genesis is shown in Figure 4G. The results indicated that, relative to GAPDH, expression of vimentin was higher on day 3 after cutaneous-perforation wounding followed by KFX treatment. This indicated that KFX could promote MSC proliferation and differentiation in the wound and contribute to wound healing, and that it could also promote proliferation of cutaneous cells in vivo.

To better understand the molecular mechanism of KFX-mediated regulation in the cutaneous-wound healing model, we assessed the signaling pathway associated with the proliferation and migration. Expression levels of the STAT3 signaling pathway–related proteins STAT3 and p-STAT3 were markedly upregulated in our mouse model after KFX treatment (Figures 5A,B), indicating that KFX could promote cutaneous-wound healing by enhancing the STAT3 signaling pathway in vivo.