Animals

This study received approval from the Ethics Committee the Tongji Medical College, Huazhong University of Science and Technology. Male C57BL/6 J mice (Vital River Laboratories, China) were housed in a specific pathogen-free environment at Tongji Medical College Animal Center. After a 4-week high-fat diet, mice underwent an overnight fast. Streptozotocin (STZ) (Biosharp, China) was dissolved in citrate buffer (0.1 mmol/L citric acid: 0.1 mmol/L sodium citrate at a 1:1.32 ratio) to form a 0.01 mg/μL solution (Prepared immediately before use). Mice received a single intraperitoneal injection of STZ based on weight (0.1 mg/g) to induce a type 2 diabetes mouse model. On the fourth day, we measured fasting blood glucose levels, consistently exceeding 11.1 mM, indicating successful modeling [14, 15].

To establish a murine model of diabetes with full-thickness skin incisions, we anesthetized STZ mice (50 mg/kg) using 1% pentobarbital, removed the dorsal hair, and disinfected the corresponding skin area with 75% alcohol. After delineating two 6mm-diameter-circular areas on each side of the back, we used the iris to remove the skin and subcutaneous tissue, creating a full-thickness skin wound on the dorsal area. Post-surgery, the wounds were cleansed with sterile physiological saline solution, thereby completing the modeling process. To investigate the effects of intermittent fasting and PRFE on diabetic wound healing, STZ mice were randomly divided into four groups based on body weight, each containing 10 mice: 1) Control group: Diabetic wounds received no specific treatment; 2) PRFE group: Mice received PRFE treatment alone, twice daily, with each session lasting 30 min; 3) Intermittent fasting group: Mice fasted for 24 h on the 2nd and 6th days post-wound creation; 4) Intermittent fasting and PRFE group: Mice fasted for 24 h on the 2nd and 6th days post-wound creation, while also receiving PRFE treatment twice daily, with each session lasting 30 min.

After wound creation, photographs of the wound were captured on days 0, 2, 4, 6, and 8 to document the healing progress. The wound closure was calculated using Image J and subjected to statistical analysis.

$$}=(}-})/}\times 100 \% .$$

On the 8th day, skin samples were obtained from the modeled region. Half of the tissues were fixed in 4% paraformaldehyde solution for histopathological analysis, while the remaining half were promptly frozen in liquid nitrogen and stored at −80 °C for subsequent experiments involving western blotting (WB) and quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Cell culture and treatments

HUVECs (ATCC, USA) and rat SCs (China Center for Type Culture Collection, China) were cultured in high-glucose Dulbecco’s Modified Eagle’s medium (DMEM, PM150210, Procell, China) supplemented with 1% Penicillin-Streptomycin Solution (PB180120, Procell, China) and 10% premium fetal bovine serum (164210, Procell, China). HDFs (ScienCell, USA) were cultured in Fibroblast Medium (2301, ScienCell, USA) with 25 mM D-Anhydrous Glucose (BS099, Biosharp, China). All cells were cultured at 37 °C in 5% CO2.

In order to assess the mechanistic impact of combined intermittent fasting and PRFE therapy on skin cells, we partitioned the cells into four distinct groups: 1) Control group: Cells were cultured in high-glucose medium with serum for 48 h; 2) PRFE group: Cells were cultured in high-glucose medium with serum for 48 h and received 30 min of PRFE treatment every 24 h; 3) Intermittent fasting group: Cells were cultured in serum-free high-glucose medium for 24 h followed by culture in high-glucose medium with serum for another 24 h; 4) Intermittent fasting and PRFE group: Cells were cultured in serum-free high-glucose medium for 24 h followed by culture in high-glucose medium with serum for another 24 h. Additionally, cells in this group received 30 min of PRFE treatment every 24 h. Following the principle of synchronizing with animal experiments, PRFE treatment was administered prior to changing the culture medium.

Histological and immunohistochemical staining

After fixation in 4% paraformaldehyde, the skin tissue was subsequently embedded in paraffin and sectioned into 4 μm-thick slices for further histological and IHC staining.

The purpose of HE staining is to observe the tissue structure of the wound and measure epidermal thickness and scar width using the Slideviewer software. The steps for HE staining involve staining dewaxed sections with hematoxylin and eosin, followed by dehydration and mounting. Masson’s staining involves deparaffinization and rehydration of the sections, overnight fixation in Bouin’s solution, staining with iron hematoxylin, acid fuchsin staining, treatment with phosphomolybdic acid, and finally countersinking with aniline blue before dehydration and slide mounting. The primary objective of Masson’s staining is to visualize the presence of fibrous tissue in the examined samples. Image J software was used to measure the relative integrated optical density (IOD) of each wound site view on the stained sections.

For IHC, the procedure involved deparaffinization and rehydration of the sections, antigen retrieval, endogenous peroxidase and biotin blocking, serum blocking, primary antibody incubation, secondary antibody incubation with horseradish peroxidase conjugate, 3,3’-Diaminobenzidine staining, counterstaining with hematoxylin, dehydration and clearing, and finally slide mounting. The primary antibodies used in this study including anti-CD31 (1:2000, 28083-1-AP, Proteintech, China), anti-CGRP (1:900, A5328, Abclone, China), anti-HIF1A (1:200, 20960-1-AP, Proteintech, China), and anti-Ki67 (1:400, 9129, Cell Signaling Technology, USA). CD31 is employed for assessing angiogenesis and tube formation. CGRP is utilized to investigate sensory nerve growth. HIF1A is used to detect oxidative stress-related markers. And Ki67 is employed to ascertain proliferation. Ultimately, we performed relative IOD measurements using Image J software at the wound site view on each section.

All the aforementioned sections were scanned using a white-light scanner (3DHISTECH, PANNORAMIC SCAN, China).

Immunofluorescent staining

Immunofluorescence (IF) staining was conducted on HDFs and SCs cells. Cells were initially seeded onto coverslips in a 12-well plate. After 48 h of treatment according to the designated groups, they were removed from the incubator, washed with Phosphate Buffered Saline (PBS), fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, and subsequently blocked with 1% bovine serum albumin. Following this, they incubated in primary antibody (anti-COL1A2, 1:1000, 14695-1-AP, Proteintech, China; anti-P75, 1:100, Abclone, China) overnight, followed by secondary antibody staining with Alexa 488 (Proteintech, China). Finally, the cells were stained with 4’,6-Diamidino-2-Phenylindole The samples were then washed and observed under a fluorescence microscope (Olympus IX73; Olympus Corp., Japan). Lastly, we performed mean intensity analysis using Image J software by selecting three random fields of view on each coverslip.

Reactive oxygen species assay

To ascertain the antioxidant capacity of intermittent fasting and PRFE combined therapy, HUVECs were cultured in a 24-well plate. After 48 h of treatment according to the assigned groups, the 24-well plate was removed from the incubator and washed with PBS. The cells were then co-incubated with the DCFH-DA probe (1:1000, S0033S, Beyotime, China) and Hochest stain (C1022, Beyotime, China). Following washing, the samples were observed using fluorescence microscopy (Olympus IX73; Olympus Corp., Japan). The image J software was utilized to measure the mean intensity of ROS.

Tube formation assay

The HUVECs, treated according to the assigned groups, were seeded at a density of 2 × 104 cells per well onto a pre-coated 96-well plate containing 60 μL of matrix gel (356234, Coring, USA), and incubate the plate in 37 °C for 6 h. Subsequently, three random fields of view were observed for each well using microscopy (Olympus IX73; Olympus Corp., Japan). We use Image J software to assess tube formation efficacy [16].

CCK-8 assay

In the CCK-8 assay, HUVECs were detached from the various groups and seeded into a 96-well plate at a density of 1 × 103 cells per well, with 5 replicate wells per group. The cells were cultured in serum-free medium for 0 to 24 h. Subsequently, 10 μL of CCK-8 reagent (BS350A, Biosharp, China) was added to each well and incubated at 37 °C for 1h30min. The absorbance was then measured at 450 nm using a Multiskan FC microplate reader (ThermoFisher, USA). Finally, we utilized the absorbance growth rate as a substitute for cell proliferation rate to quantify the results.

Absorbance growth rate = (Absorbance at the specified time point - Mean absorbance of blank wells) / The mean absorbance of the previous time point after correction with the absorbance of the blank well of previous time point * 100%.

Scratch wound healing assay

For scratch wound healing assay, HUVECs were uniformly seeded into a 6-well plate and treated according to the specified groups. After the the treatment is completed and cell reach 95% confluency, a sterile pipette tip (1 mL) was utilized to create a straight scratch across the center of each well. Subsequently, the wells were rinsed with PBS and supplemented with serum-free medium for appropriate culture. Microscopic images of the cells were captured at 0-, 24-, and 48-hours post-scratch using an Olympus IX73 microscope (Olympus Corp., Japan). The closed area was quantified using Image J software. The rate of cell migration was determined using the formula:

$$}=(}-})/}\times 100 \% .$$

qRT-PCR analysis

According to the protocol, we used Trizol (R411-01, Vazyme, China) to extract the total RNA of respective cells and tissues. For the extraction of total RNA from skin tissue, we weigh and use 20 mg of skin tissue which stays in a 1.5 ml EP tube and add 1 ml of Trizol and grinding beads. The mix is then ground using a homogenizer until the tissue is completely dissolved. The extracted mRNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (#R323, Vazyme, China). qRT-PCR analysis was performed using HiScript II One Step qRT-PCR SYBR Green Kit (Q221-01, Vazyme, China) on the T100 Thermal Cycler (BIO-RAD, USA) detection system. The normalization of mRNA expression was accomplished through the utilization of the housekeeping gene Tubulin. Analysis was conducted using the 2^-ΔΔCT method. The purpose of this experiment was to investigate the impact of intermittent fasting and PRFE on the gene expression level. The primer sequences used are provided in Table 1.

Table 1 The sequences of primer pairs used in qPCR.Western blotting

Protein samples were extracted from the corresponding groups of cells and skin tissue using RIPA lysis buffer (BL504A, Biosharp, China) containing Phenylmethylsulfonyl fluoride (G2008-1ML, Servicebio, China) and Protease Inhibitor Cocktail (HY-K0010, MCE, USA), as protocol. For the extraction of total protein from skin tissue, we take 30 mg of skin tissue, use 300 µl of lysis buffer in a 1.5 ml EP tube, add a steel grinding ball, and grind it for 20 min using a grinder set at 70 Hz. The extracted protein samples were separated using 10% or 12.5% SDS-PAGE gel (PG112, epizyme, China), then transferred onto PVDF membranes (IPVH00005, Millipore, USA). The membrane was blocked at room temperature for 1 h using NcmBlot blocking buffer (P30500, NCM, China), followed by overnight incubation with the appropriate primary antibody at 4 °C. The primary antibody includes antibodies targeting VEGFA (1:1000, A12303, Abclone, China), COL1A2 (1: 2000, 14695-1-AP, Proteintech, China), HIF1A (1:1000, HY-P80704, MCE, USA), CGRP (1:1000, A5542, Abclone, China), P75 (1:1000, A19127, Abclone, China), α-Tubulin (1:10000, A19127, Abclone, China). After washing with Washing with Tris Buffered Saline (G0001-2L, Servicebio, China) containing 0.1% Tween-20 (GC204002, Servicebio, China) (TBST), the membrane was incubated at room temperature with the corresponding rabbit or mouse secondary antibody (Proteintech, China) for 1 h. Following another round of TBST washing, the membrane was immersed in an ECL chemiluminescence substrate (BL520A, Biosharp, China) and observed using the ChemiDoc XRS+ System (BIO-RAD, USA).

Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 software, and calculations were based on at least three independent experiments. All data are presented as mean ± SD. Four-group comparisons were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni post hoc test. Statistical significance was denoted as follows: P < 0.05 indicated by *, #, or †; P < 0.01 indicated by **, ##, or ††; P < 0.001 indicated by ***, ###, or †††; P < 0.0001 indicated by ****, ####, or ††††.