Preparation of FA embedded carbomer hydrogel
The three concentrations (5%, 10%, and 15%) of FA embedded carbomer hydrogels were prepared as follows. Firstly, 0.75 g of carbomer 940 (#C832684, Shanghai Macklin Biochemical Technology Co., Ltd, Shanghai, China) was dispersed in 85 mL, 80 mL, and 75 mL of distilled water, respectively, and allowed to swell for 24 h. Glycerol (5 g) and ethyl p-hydroxybenzoate (0.001 g) were added to the carbomer 940 gels at different concentrations. After thorough stirring and mixing, 5 g, 10 g, and 15 g of FA (#F809521, C10H10O4, purity > 99.99%, Shanghai Macklin Biochemical Technology Co., Ltd, Shanghai, China) were added to the carbomer 940 gels of different concentrations, respectively. The pH value was adjusted to 5.5 using triethanolamine (#90279, Sigma-Aldrich, US). This process resulted in the preparation of 5%, 10%, and 15% FA-embedded carbomer hydrogels.
Characterization of FA embedded carbomer hydrogel
The particle size analysis was conducted as follows: the FA gel sample (1 mL) was placed in a 5 mL centrifuge tube and mixed with 2 mL of ethanol. The samples were dispersed by ultrasonication for 5 min, and then 1 mL of the upper layer solution was transferred to a sample cell for particle size measurement using the Marvin laser particle size analyzer (ZS90). Transmission electron microscopy (TEM) analysis was performed as follows: The FA gel sample was dispersed in ethanol, following the same procedure as mentioned above. The resulting sample solution was placed onto an ultra-thin carbon film and air-dried. TEM imaging was carried out using the FEI Tecnai G2F30 microscope operating at an acceleration voltage of 200 kV. Rheological properties were measured using a rotational rheometer (#MCR92, Anton Paar, Austria) following a previously described protocol [26]. An oscillatory strain amplitude sweep was performed with a strain range from 0.1 to 1000% at a frequency of 1 Hz and a temperature of 25 °C. Subsequently, a shear strain of 0.2% was applied, and an oscillatory frequency sweep ranging from 0.1 to 100 rad/s was conducted at 25 °C. The model of the test rotor was a parallel plate of 50 mm, with a gap setting of 1 mm.
The swelling detection of FA hydrogels was performed as described below. The dry FA hydrogels (W1) were immersed in phosphate buffered saline (PBS), and removed at 2, 4, 6, 8 h. After removing water with filter paper, the weights of FA hydrogels were recorded (W2). The swelling ratio= [(W2-W1)/W1]×100%.
In vitro antioxidant ability of of FA hydrogels
The scavenging abilities of 2,2’-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and ROS production by FA hydrogels were investigated as described previously [27]. To evaluate the hydrogel’s ability to scavenge DPPH radicals, FA hydrogels (200 µL) were prepared in a 24-well plate and immersed in 1 mL of anhydrous ethanol. DPPH solution dissolved with 0.5 mM ethanol was added. After incubation for 1 h in the darkness, OD value at 517 nm was measured (ODFA). PBS was added instead of FA hydrogels for control (ODCon). The scavenging efficiency of DPPH radicals = [(ODCon – ODFA)/ODPositive]×100%.
The scavenging ability of ABTS radicals was measured as described below. The FA hydrogels were incubated with ABTS working solutiong for 1 h in the dark. The OD value at 734 nm was measured. The scavenging efficiency of ABTS radicals = [(ODCon – ODFA)/ODPositive]×100%.
The HaCaT cells were treated with 100 µM hydrogen peroxide (H2O2) for 4 h to establish cell damage model. FA hydrogel extraction solutions were obtained by immersing hydrogel in DMEM/F12 for 24 h at 37 °C. Then, medium containing H2O2 was replaced with FA hydrogel extraction solutions, or DMEM/F12 medium. After incubation for 24 h, DCFH-DA probe was added. After incubation for 30 min in the dark, cells were washed with PBS twice, and fluorescence intensity was observed by fluorescence microscopy.
Animal model establishment of radiation-induced skin injury
A 60Co γ radiation source (Beijing Institute of Radiation Medicine, Beijing, China) was utilized to induce skin injuries. The animal experiments were performed in accordance with national and institutional guidelines. The experimental protocol was approved by the Ethics Committee of Animal Experiments of the Beijing Institute of Radiation Medicine (Approval number: IACUC-DWZX-2020-773). Seventy-two female Wistar rats (190–210 g, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd, China) were randomly divided into six groups, including control, 40 Gy irradiation, 40 Gy irradiation + Aloe gel, 40 Gy irradiation + 5% FA gel, 40 Gy irradiation + 10% FA gel, and 40 Gy irradiation + 15% FA gel, with 12 rats in each group. The rats were housed under controlled conditions at a temperature of 22 ± 2 °C, a humidity of 50% ± 5%, and provided with sterilized feed and purified water ad libitum. After 1 week of acclimatization, the rats were used for the experiments. Anesthesia was induced using 1% pentobarbital sodium at a dosage of 40 mg/kg. The fur on the buttocks and back of the rats was shaved, and the rats were immobilized on an irradiation board. The buttocks and back of the rats in the experimental groups were exposed to a single dose of 40 Gy irradiation (266.24 cGy/min), while the rest of their bodies were shielded with lead bricks (Supplementary Fig. 1). After irradiation, the rats in the group 40 Gy irradiation, 40 Gy irradiation + Aloe gel, 40 Gy irradiation + 5% FA gel, 40 Gy irradiation + 10% FA gel, and 40 Gy irradiation + 15% FA gel were topically treated with carbomer hydrogel without FA, Aloe gel, 5% FA gel, 10% FA gel, and 15% FA gel, respectively, twice a day for 10 consecutive days. The weights of the animals were recorded on days 0, 7, 14, 21, and 28 after irradiation. The skin changes in the irradiated areas were observed daily after irradiation, and parameters such as peeling, redness, damage, and exudation were recorded. The degree of radiation-induced skin damage was analyzed using the Douglas and Fowler scores (Supplementary Table 1). On the 14th, 21st, and 28th days after radiation, skin tissues from the buttocks and back were collected for further analysis based on previous reports [27, 28]. Pentobarbital sodium (150 mg/kg) intraperitoneal injection was used to euthanize rats.
Hematoxylin and eosin (HE) staining
Skin tissue samples measuring 1 cm × 1 cm were collected from the buttocks and back areas and fixed in 4% paraformaldehyde. The tissues were then embedded in paraffin. The sections were dewaxed using the following steps: incubation in xylene for 15 min, anhydrous ethanol for 5 min, 85% alcohol for 5 min, and 75% alcohol for 5 min, in that order. Subsequently, the sections were stained with hematoxylin for 5 min and eosin for 30 s. After staining, the slices were immersed in anhydrous ethanol for 5 min, followed by incubation in xylene for 5 min, and finally sealed with transparent neutral gum.
Masson staining
The sections were stained with hematoxylin for 5 min. After washing with tap water, the tissues were differentiated with 1% hydrochloric acid alcohol for 3 s and rinsed with running water for 3 min. Acid fuchsin solution was used to stain the sections for 5 min, and phosphomolybdic-phosphotungstic acid was used to incubate the slides for 5 min. After incubating with aniline blue solution for 5 min, the sections were differentiated with 1% glacial acetic acid for 1 min. After dehydration, the slides were sealed with neutral gum and observed using an inverted microscope (Olympus, Japan). Pathological sections of three animals in each group were selected for Masson staining. Three random fields of each section were used to calculate the collage deposition ratio with Image J software. Collagen deposition = (collagen tissue area/whole tissue area)×100%.
Immunofluorescence staining
The sections were placed in EDTA solution (Servicebio, Wuhan, China) and heated for 8 min in a microwave for antigen repair. Afterward, the sections were washed with PBS and incubated with bovine serum albumin for 30 min to block nonspecific binding. Following the removal of bovine serum albumin, the sections were incubated with primary antibodies overnight at 4 °C and washed with PBS three times (5 min each time). The sections were then incubated with secondary antibodies for 1 h. After washing with PBS, the sections were incubated with DAPI solution (Servicebio, Wuhan, China) for 1 min. Finally, the sections were observed using a fluorescence microscope (Nikon, Japan). The antibodies used in this research are listed in supplementary Table 2.
Detection of skin blood flow using laser doppler blood flow monitor
Laser Doppler technology can be employed to monitor the blood perfusion of capillaries, venules, arterioles, and anastomotic branches in the skin, thereby reflecting changes in blood flow and the degree of skin damage. Rats were first anesthetized, and the skin blood flow was recorded using a laser Doppler blood flow monitor (Moor Instruments, UK) on the 14th, 21st, and 28th days after radiation.
Evaluation of radiation-induced skin injury based on sweep source-OCT
Sweep-source optical coherence tomography (OCT) systems can be used to observe radiation-induced skin injury in real-time. This method enables non-invasive and radiation-free imaging [29]. By calculating and visualizing the optical attenuation coefficient, both qualitative and quantitative analysis of radiation-induced skin injury can be achieved. Wistar rats were anesthetized with 1% pentobarbital sodium on the 14th, 21st, and 28th days after irradiation, and the lesion site was scanned and analyzed. A sweep-source OCT system (Thorlabs, US) was used for three-dimensional reconstruction and quantitative analysis of skin damage.
Detection of inflammatory factors in the serum
The whole blood from the abdominal aorta were collected with a sterile injection syringe. After natural coagulation of blood at room temperature for 15–20 min, the blood samples were centrifuged at 4000 rpm/min at 4 °C for 20 min. The serum was extracted for the detection of inflammatory factors using the ELISA method according to the instructions. The Caspase-1, NLRP3, IL-1β, and IL-18 ELISA kits were purchased from Jiangsu Meimian Industrial Co., Ltd (Nanjing, Jiangsu province, China).
Cell culture and radiation-induced cell injury
The HaCaT cell line, a type of human keratinocyte, was used in this study. The cells were cultured using DMEM/F12 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37 °C with 5% CO2. The cells were treated with radiation at a total dose of 18 Gy (100.68 cGy/min). FA solutions (200 µg/mL) were prepared using DMSO and diluted with DMEM/F12 to concentrations of 200, 100, 50, and 25 µg/mL. After radiation, the cell medium was replaced with different concentrations of FA solution or vitamin C solution (25 µg/mL, #A4403, Sigma-Aldrich, US). After 24 h of incubation, cells were used for detection.
ROS detection
The DCFH-DA probe (#D399, Thermo Fisher, US) was diluted with DMEM/F12 medium to a final concentration of 10 µmol/L. The irradiated cells were washed three times with PBS (#10010023, Thermo Fisher, US). Then, the DCFH-DA probe was added, and cells were incubated for 20 min in a 37 °C cell incubator. The supernatant was discarded, and cells were washed three times with DMEM/F12 medium. Flow cytometry was used to measure ROS intensity.
SOD detection
After removing the cell medium, the cells were scraped off with a cell scraper. The cells were then centrifuged at 2000 g for 10 min at 5 °C, and the supernatant was removed. The cells were washed twice with PBS. Ultrasound treatment of cells was performed on an ice bath. The cells were then centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant was transferred to a new test tube. The prepared sample solution was used for SOD detection according to the instructions of the kit (Beiren Chemical Technology Co., Ltd, Japan).
CCK8 assay
To each well, 10 µL of CCK8 reagent (Beiren Chemical Technology Co., Ltd, Japan) was added. After incubation for 4 h at 37 °C in a 5% CO2 incubator, the absorbance value at 450 nm was measured. The formula for cell viability is [(ODFA – ODBlank) / (ODCon – ODBlank)]×100%.
Western blotting
Proteins from the skin tissues and cells were isolated using lysate containing RIPA lysate, protease inhibitor, and phosphatase inhibitor. The protein concentration was determined using the bicinchoninic acid (BCA) assay. Approximately 30 µg of proteins were loaded and separated through SDS-PAGE. The proteins were then transferred to a Polyvinylidene fluoride (PVDF) membrane (Millipore) at 100 mA current for 150 min. The membrane was blocked with tris buffered saline with tween (TBST) containing 5% skim milk. After washing with TBST, the membrane was incubated with primary antibodies overnight at 4 °C. Secondary antibodies were used to incubate the membranes at room temperature for 2 h, followed by washing with TBST. The proteins were detected using the Image Quant Las 500. The antibodies used in this research are listed in supplementary Table 2.
Transcriptome sequencing
The assessment of RNA purity and integrity was conducted utilizing a NanoPhotometer spectrophotometer and an Agilent 2100 bioanalyzer, respectively. For the construction of libraries, the NEBNext® Ultra™ RNA Library Prep Kit for Illumina was utilized. Quantification was initially undertaken with a Qubit 2.0 Fluorometer, and the insert size was subsequently evaluated using the Agilent 2100 bioanalyzer. The determination of the library’s effective concentration was achieved through qRT-PCR. The library amplification process involved the integration of fluorescently-labeled dNTPs, DNA polymerase, and adapter primers. During the extension phase of each sequencing cluster, fluorescently-labeled dNTPs were incorporated, triggering the emission of corresponding fluorescence signals. The calculation of FPKM (Fragments per kilo base per million mapped reads) values for individual genes was based on gene length and the number of mapped reads. The analysis of differential expression across comparison groups was performed employing DESeq2 software, with the adjustment of P-values according to the Benjamini & Hochberg method [30]. Criteria for significant differential expression were established by adjusted P-values and the magnitude of |log2foldchange|. Differentially expressed genes were analyzed functionally using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) with the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. The thresholds for Padj < 0.05 and FDR < 0.05 were applied.
Statistical analysis
All experimental data were obtained by independently repeating the experiments at least three times. The experimental data were presented as mean ± SD and analyzed using GraphPad 8 software. A t-test was conducted to compare data between two groups, and ANOVA was used to compare data among multiple groups, with P < 0.05 indicating significant statistical differences.
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