Effect of fecal microbiota transplantation on diabetic wound healing through the IL-17A-mTOR-HIF1α signaling axis

Diabetes is the third most common chronic disorder worldwide. Diabetic wounds are a severe complication that is costly and often results in non-traumatic lower limb amputation. Recent investigations have demonstrated that the gut microbiota as a "virtual organ" can regulate metabolic diseases like diabetes. Fecal microbiota transplantation (FMT) is an innovative therapeutic approach for promoting wound healing, but its function remains incompletely defined. A diabetes model was established by supplying mice with a high-fat diet and performing an intraperitoneal injection of streptozotocin. Diabetic wounds were then created, followed by bacterial transplantation. The relevant indexes of wound healing were evaluated to verify the promoting effect of FMT on diabetic wounds. Human skin keratinocytes were also cultured, and cell scratch experiments were conducted to further investigate the underlying mechanism. The FMT regulated the levels of specific bacteria in the diabetic mice and helped restore the balance of intestinal microbes. This transplantation also enhanced wound healing in diabetic mice by augmenting the closure rate, accelerating re-epithelialization, and boosting collagen deposition in skin wounds. Furthermore, FMT promoted the production of IL-17A, which significantly enhanced the growth and movement of human keratinocytes. Inhibiting molecules related to the IL-17A-mTOR-HIF1α signaling axis were shown to hinder wound re-epithelialization. This study clarifies the function of the IL-17A-mTOR-HIF1α signaling axis in the utilization of FMT in diabetic wound healing, providing a new therapeutic method and target for promoting the healing of diabetic wounds. Methods Diabetic wound healing experiment Once the mice were fully anesthetized through an intraperitoneal injection of 1% sodium pentobarbital at a dosage of 0.3 mg/kg, the fur on their backs was shaved and disinfected. A circular full-thickness skin wound measuring 5 mm in diameter was then made on the posterior back near the hip joint using a punch tool. After the creation of the wound, each mouse was housed separately in a single cage to prevent self-scratching. One hour prior to wound creation, as illustrated in Fig. 6, mice in the SECU group received a subcutaneous injection of 10 mg/kg of the IL-17A inhibitor secukinumab (SECU, S412013, Aladdin), while the remaining groups were administered an equivalent amount of sterile saline solution. The injection was administered once a week until sacrifice. Wound healing was assessed using Image J on days 0, 3, 7, and 14 after the wound was created. The wound closure (%) was calculated as (W0 – Wn) / W0 × 100%; W0 and Wn indicate the wound area on days 0 and n. Two weeks later, the mice were sacrificed under general anesthesia, and colon, serum, and skin specimens were gathered for corresponding tests. Hematoxylin and eosin staining and Masson’s trichrome staining Following general anesthesia, the mice were perfused through the heart with a 0.9% saline solution, followed by a perfusion of 0.1 M phosphate buffer with 4% paraformaldehyde. The colon and skin tissues were carefully excised and promptly placed in 4% paraformaldehyde for 24 hours. These tissues were subsequently embedded in paraffin and cut into 5 μm sections. The embedding medium was removed using solvents such as xylene. The sections were rehydrated with graded alcohol solutions. Hematoxylin and eosin were used for HE staining, and Weigert's iron hematoxylin staining solution was employed for Masson staining. The experimental results were then observed under an optical microscope. Fecal 16S rDNA sequencing Each mouse was placed in a separate sterile cage, and fresh fecal pellets were collected in sterile EP tubes. The samples were immediately chilled and stored at -80 °C for subsequent analysis. Total DNA was extracted from microbial community samples using the CTAB method. DNA integrity was assessed by electrophoresis, and quantification was done with a UV-Vis spectrophotometer. The V3-V4 hypervariable region of the bacterial 16S rDNA gene was selected for PCR amplification. The forward primer 341F (5'-CCTACGGGNGGCWGCAG-3') and the reverse primer 805R (5'-GACTACHVGGGTATCTAATCC-3') were used. PCR products were confirmed via 2% agarose gel electrophoresis. Purification was performed using AMPure XT beads (Beckman Coulter Genomics), followed by quantification with Qubit (Invitrogen). Purified products were recovered using an AMPure XT bead recovery kit. The refined PCR products were evaluated using an Agilent 2100 Bioanalyzer and a Kapa Biosciences Illumina library quantification kit. A qualified sequencing library, with unique index sequences, underwent gradient dilution and proportional mixing based on required sequencing amounts. The library was denatured into single strands for sequencing on a NovaSeq 6000 sequencer, with paired-end sequencing at 2×250 bp using the NovaSeq 6000 SP Reagent Kit (500 cycles), followed by data analysis. Sequencing was performed on the Illumina NovaSeq platform. Paired-end reads were assigned to samples using unique barcodes and processed by removing barcode and primer sequences. The paired-end reads were assembled with FLASH. Raw reads were filtered to generate high-quality clean tags using fqtrim (v0.94). Chimeric sequences were identified and removed with Vsearch (v2.3.4). After dereplication with DADA2, we generated an ASV feature table and extracted feature sequences. Alpha and beta diversity metrics were computed based on randomly subsampled sequences to ensure equal sequence depth using QIIME2, the Chao1 estimator estimates total species richness in a community, the Shannon index is derived from the information entropy of a community. the beta diversity was visualized through principal coordinate analysis (PCoA), ANOSIM (Analysis of Similarities) is a non-parametric test that evaluates whether differences between groups, based on a Jaccard index-derived distance matrix, are statistically significant. Feature abundance was normalized by relative abundance within each sample using the SILVA classifier (release 138). Graphs were generated using R (v3.5.2). Differentially abundant taxa were identified using LEfSe with default parameters. Microbial metabolic functions were analyzed with PICRUSt2 to infer functional profiles, which were then mapped against the KEGG database for pathway abundance values. Additional diagrams were also created using R (v3.5.2). Immunofluorescence staining The sections were sequentially immersed in a series of xylene and ethanol solutions for dewaxing, followed by antigen retrieval with citric acid (pH 6.0). A 3% hydrogen peroxide solution was prepared using water, and the sections were placed in this solution in an incubator under ambient temperature for 20 minutes. Blocking was conducted with 10% goat serum at 37 °C for half an hour. The primary antibodies, IL-17A (1:200 dilution, PTG, catalog number 26163-1-AP) and HIF-1α (1:200 dilution, bioss, catalog number bs-0737R), were diluted in an antibody dilution buffer and allowed to incubate overnight at 4 °C. Goat anti-rabbit IgG-CY3 conjugated with HRP (1:300 dilution, Servicebio) was prepared in PBST and kept at 37 °C for one hour. DAPI solution was utilized to stain cell nuclei. Representative images were obtained using fluorescence microscopy (Nikon Eclipse C1, Tokyo, Japan). The number of positive cells was evaluated using Image Pro Plus version 6.0 software. Enzyme-linked immunosorbent assay The enzyme-linked immunosorbent assay (ELISA) kit (ELM-IL17-1, Raybio) was used to measure IL-17A levels in the mouse serum. The experimental procedures followed the guidelines provided by the manufacturer. Absorbance readings were taken with an ELISA reader, and a standard curve was generated based on these values. Cell culture and cell wound scratch assay Human keratinocytes (hacat) were purchased from IMMOCELL (Xiamen, Fujian, China). In the normal medium group, cells were grown in DMEM/F12 medium enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin liquid. In the high-glucose medium group, an additional 25 mM of glucose was added to the medium. In group I, 100 ng/ml exogenous IL-17A (CM018-20HP, Chamot Biotechnology Co, Ltd, China) was added. In group D, 100 ng/ml exogenous IL-17A and 40 mM glycolysis inhibitor 2-Deoxy-D-glucose(2-DG) (CD4251-1g, Coolaber) were added. In group B, 100 ng/ml exogenous IL-17A and 10 μM HIF1α inhibitor BAY87-2243 (87-2243, MCE) were incorporated. Following a 24-hour incubation period, the cells covered the six-well culture plate. Subsequently, a cell scratch assay was conducted, and the scratch patterns of the cells at 0 and 24 hours were recorded. The scratch area was calculated using Image J. The percentage of the scratch area (%) was calculated as (A0 － A24) / A0 × 100%, where A0 and A24 indicate the area of the scratch at 0 and 24 hours. The levels of pathway-related proteins were assessed using Western blot (WB). Western blot The efficient RIPA lysing buffer for tissue and cell samples (with PMSF) (SL1020-100mL, Coolaber) was used to extract cell proteins. The lysate was subsequently subjected to centrifugation at 12,000 g and 4 °C for 20 minutes to isolate the total protein. The protein concentration was measured using the BCA protein assay kit (EC0001, SparkJade). The membranes were moved and allowed to incubate overnight at 4 °C with the specified primary antibodies: mouse monoclonal AKT (1:25,000, 60203-2-Ig, Proteintech), mouse monoclonal p-AKT (1:5,000, 66444-1-Ig, Proteintech), mouse monoclonal mTOR (1:25,000, 66888-1-Ig, Proteintech), rabbit monoclonal p-mTOR (1:1,000, 5536T, CST), Rabbit polyclonal antibody anti-HIF1α (1:1000, D222477-0025, Sangon Biotech), Rabbit polyclonal antibody anti-HK2 (1:25,000; 22029-1-AP; Proteintech), mouse monoclonal HIF-1 (1:5,000, 66730-1-Ig, Proteintech), mouse monoclonal HK2 (1:10,000; 66974-1-Ig; Proteintech), and β-actin (1:20,000; T0022; Affinity). Afterward, the membranes were treated with the appropriate secondary antibodies, including HRP-conjugated goat anti-rabbit antibody (1:1000; A0208; Beyotime) and HRP-conjugated goat anti-mouse antibody (1:10, 000; SA00001-１; Proteintech) at room temperature for two hours. Visualization of the blots was performed using the LAS4000 chemiluminescence system from Fujifilm in Tokyo, Japan, and the gray values of the films were analyzed using IPP software. Statistical analysis An analysis of the statistical data was carried out utilizing SPSS 20.0. Data are presented as the mean ± standard deviation (SD). In the case of multiple comparisons, one-way analysis of variance (ANOVA) was employed and subsequently analyzed using the least significant difference (LSD) post hoc test. The nonparametric Kruskal–Wallis test was applied to evaluate the pole test performance, grip strength test results, histological scores, ZO-1 integrity scores, and cell counts, with Mann–Whitney U post hoc testing. Comparisons between the two groups were conducted using independent t-tests. Spearman correlation analysis was performed with R (version 3.5.1) to assess correlations across different experiments. The results were considered statistically significant if P ＜ 0.05.