Preparation of FA embedded carbomer hydrogel
The three concentrations (5%, 10%, and 15%) of FA embedded carbomer hydrogels have been ready as follows. Firstly, 0.75 g of carbomer 940 (#C832684, Shanghai Macklin Biochemical Know-how Co., Ltd, Shanghai, China) was dispersed in 85 mL, 80 mL, and 75 mL of distilled water, respectively, and allowed to swell for twenty-four h. Glycerol (5 g) and ethyl p-hydroxybenzoate (0.001 g) have been added to the carbomer 940 gels at completely different concentrations. After thorough stirring and mixing, 5 g, 10 g, and 15 g of FA (#F809521, C10H10O4, purity > 99.99%, Shanghai Macklin Biochemical Know-how Co., Ltd, Shanghai, China) have been added to the carbomer 940 gels of various concentrations, respectively. The pH worth was adjusted to five.5 utilizing triethanolamine (#90279, Sigma-Aldrich, US). This course of resulted within the preparation of 5%, 10%, and 15% FA-embedded carbomer hydrogels.
Characterization of FA embedded carbomer hydrogel
The particle dimension evaluation was performed as follows: the FA gel pattern (1 mL) was positioned in a 5 mL centrifuge tube and blended with 2 mL of ethanol. The samples have been dispersed by ultrasonication for five min, after which 1 mL of the higher layer answer was transferred to a pattern cell for particle dimension measurement utilizing the Marvin laser particle dimension analyzer (ZS90). Transmission electron microscopy (TEM) evaluation was carried out as follows: The FA gel pattern was dispersed in ethanol, following the identical process as talked about above. The ensuing pattern answer was positioned onto an ultra-thin carbon movie and air-dried. TEM imaging was carried out utilizing the FEI Tecnai G2F30 microscope working at an acceleration voltage of 200 kV. Rheological properties have been measured utilizing a rotational rheometer (#MCR92, Anton Paar, Austria) following a beforehand described protocol [26]. An oscillatory pressure amplitude sweep was carried out with a pressure vary from 0.1 to 1000% at a frequency of 1 Hz and a temperature of 25 °C. Subsequently, a shear pressure of 0.2% was utilized, and an oscillatory frequency sweep starting from 0.1 to 100 rad/s was performed at 25 °C. The mannequin of the take a look at rotor was a parallel plate of fifty mm, with a niche setting of 1 mm.
The swelling detection of FA hydrogels was carried out as described under. The dry FA hydrogels (W1) have been immersed in phosphate buffered saline (PBS), and eliminated at 2, 4, 6, 8 h. After eradicating water with filter paper, the weights of FA hydrogels have been recorded (W2). The swelling ratio= [(W2-W1)/W1]×100%.
In vitro antioxidant capability of of FA hydrogels
The scavenging talents of two,2’-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and ROS manufacturing by FA hydrogels have been investigated as described beforehand [27]. To guage the hydrogel’s capability to scavenge DPPH radicals, FA hydrogels (200 µL) have been ready in a 24-well plate and immersed in 1 mL of anhydrous ethanol. DPPH answer dissolved with 0.5 mM ethanol was added. After incubation for 1 h within the darkness, OD worth at 517 nm was measured (ODFA). PBS was added as an alternative of FA hydrogels for management (ODCon). The scavenging effectivity of DPPH radicals = [(ODCon – ODFA)/ODPositive]×100%.
The scavenging capability of ABTS radicals was measured as described under. The FA hydrogels have been incubated with ABTS working solutiong for 1 h at midnight. The OD worth at 734 nm was measured. The scavenging effectivity of ABTS radicals = [(ODCon – ODFA)/ODPositive]×100%.
The HaCaT cells have been handled with 100 µM hydrogen peroxide (H2O2) for 4 h to determine cell injury mannequin. FA hydrogel extraction options have been obtained by immersing hydrogel in DMEM/F12 for twenty-four h at 37 °C. Then, medium containing H2O2 was changed with FA hydrogel extraction options, or DMEM/F12 medium. After incubation for twenty-four h, DCFH-DA probe was added. After incubation for 30 min at midnight, cells have been washed with PBS twice, and fluorescence depth was noticed by fluorescence microscopy.
Animal mannequin institution of radiation-induced pores and skin damage
A 60Co γ radiation supply (Beijing Institute of Radiation Drugs, Beijing, China) was utilized to induce pores and skin accidents. The animal experiments have been carried out in accordance with nationwide and institutional tips. The experimental protocol was authorized by the Ethics Committee of Animal Experiments of the Beijing Institute of Radiation Drugs (Approval quantity: IACUC-DWZX-2020-773). Seventy-two feminine Wistar rats (190–210 g, Beijing Weitong Lihua Experimental Animal Know-how Co., Ltd, China) have been randomly divided into six teams, together with management, 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 every group. The rats have been housed underneath managed circumstances at a temperature of twenty-two ± 2 °C, a humidity of fifty% ± 5%, and supplied with sterilized feed and purified water advert libitum. After 1 week of acclimatization, the rats have been used for the experiments. Anesthesia was induced utilizing 1% pentobarbital sodium at a dosage of 40 mg/kg. The fur on the buttocks and again of the rats was shaved, and the rats have been immobilized on an irradiation board. The buttocks and again of the rats within the experimental teams have been uncovered to a single dose of 40 Gy irradiation (266.24 cGy/min), whereas the remainder of their our bodies have been shielded with lead bricks (Supplementary Fig. 1). After irradiation, the rats within 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 have been topically handled with carbomer hydrogel with out 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 have been recorded on days 0, 7, 14, 21, and 28 after irradiation. The pores and skin adjustments within the irradiated areas have been noticed each day after irradiation, and parameters resembling peeling, redness, injury, and exudation have been recorded. The diploma of radiation-induced pores and skin injury was analyzed utilizing the Douglas and Fowler scores (Supplementary Desk 1). On the 14th, twenty first, and twenty eighth days after radiation, pores and skin tissues from the buttocks and again have been collected for additional evaluation based mostly on earlier studies [27, 28]. Pentobarbital sodium (150 mg/kg) intraperitoneal injection was used to euthanize rats.
Hematoxylin and eosin (HE) staining
Pores and skin tissue samples measuring 1 cm × 1 cm have been collected from the buttocks and again areas and stuck in 4% paraformaldehyde. The tissues have been then embedded in paraffin. The sections have been dewaxed utilizing the next steps: incubation in xylene for 15 min, anhydrous ethanol for five min, 85% alcohol for five min, and 75% alcohol for five min, in that order. Subsequently, the sections have been stained with hematoxylin for five min and eosin for 30 s. After staining, the slices have been immersed in anhydrous ethanol for five min, adopted by incubation in xylene for five min, and eventually sealed with clear impartial gum.
Masson staining
The sections have been stained with hematoxylin for five min. After washing with faucet water, the tissues have been differentiated with 1% hydrochloric acid alcohol for 3 s and rinsed with working water for 3 min. Acid fuchsin answer was used to stain the sections for five min, and phosphomolybdic-phosphotungstic acid was used to incubate the slides for five min. After incubating with aniline blue answer for five min, the sections have been differentiated with 1% glacial acetic acid for 1 min. After dehydration, the slides have been sealed with impartial gum and noticed utilizing an inverted microscope (Olympus, Japan). Pathological sections of three animals in every group have been chosen for Masson staining. Three random fields of every part have been used to calculate the collage deposition ratio with Picture J software program. Collagen deposition = (collagen tissue space/complete tissue space)×100%.
Immunofluorescence staining
The sections have been positioned in EDTA answer (Servicebio, Wuhan, China) and heated for 8 min in a microwave for antigen restore. Afterward, the sections have been washed with PBS and incubated with bovine serum albumin for 30 min to dam nonspecific binding. Following the removing of bovine serum albumin, the sections have been incubated with main antibodies in a single day at 4 °C and washed with PBS 3 times (5 min every time). The sections have been then incubated with secondary antibodies for 1 h. After washing with PBS, the sections have been incubated with DAPI answer (Servicebio, Wuhan, China) for 1 min. Lastly, the sections have been noticed utilizing a fluorescence microscope (Nikon, Japan). The antibodies used on this analysis are listed in supplementary Desk 2.
Detection of pores and skin blood circulate utilizing laser doppler blood circulate monitor
Laser Doppler know-how will be employed to watch the blood perfusion of capillaries, venules, arterioles, and anastomotic branches within the pores and skin, thereby reflecting adjustments in blood circulate and the diploma of pores and skin injury. Rats have been first anesthetized, and the pores and skin blood circulate was recorded utilizing a laser Doppler blood circulate monitor (Moor Devices, UK) on the 14th, twenty first, and twenty eighth days after radiation.
Analysis of radiation-induced pores and skin damage based mostly on sweep source-OCT
Sweep-source optical coherence tomography (OCT) programs can be utilized to look at radiation-induced pores and skin damage in real-time. This technique permits non-invasive and radiation-free imaging [29]. By calculating and visualizing the optical attenuation coefficient, each qualitative and quantitative evaluation of radiation-induced pores and skin damage will be achieved. Wistar rats have been anesthetized with 1% pentobarbital sodium on the 14th, twenty first, and twenty eighth days after irradiation, and the lesion web site was scanned and analyzed. A sweep-source OCT system (Thorlabs, US) was used for three-dimensional reconstruction and quantitative evaluation of pores and skin injury.
Detection of inflammatory components within the serum
The entire blood from the stomach aorta have been collected with a sterile injection syringe. After pure coagulation of blood at room temperature for 15–20 min, the blood samples have been centrifuged at 4000 rpm/min at 4 °C for 20 min. The serum was extracted for the detection of inflammatory components utilizing the ELISA technique based on the directions. The Caspase-1, NLRP3, IL-1β, and IL-18 ELISA kits have been bought from Jiangsu Meimian Industrial Co., Ltd (Nanjing, Jiangsu province, China).
Cell tradition and radiation-induced cell damage
The HaCaT cell line, a sort of human keratinocyte, was used on this research. The cells have been cultured utilizing DMEM/F12 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37 °C with 5% CO2. The cells have been handled with radiation at a complete dose of 18 Gy (100.68 cGy/min). FA options (200 µg/mL) have been ready utilizing DMSO and diluted with DMEM/F12 to concentrations of 200, 100, 50, and 25 µg/mL. After radiation, the cell medium was changed with completely different concentrations of FA answer or vitamin C answer (25 µg/mL, #A4403, Sigma-Aldrich, US). After 24 h of incubation, cells have been used for detection.
ROS detection
The DCFH-DA probe (#D399, Thermo Fisher, US) was diluted with DMEM/F12 medium to a ultimate focus of 10 µmol/L. The irradiated cells have been washed 3 times with PBS (#10010023, Thermo Fisher, US). Then, the DCFH-DA probe was added, and cells have been incubated for 20 min in a 37 °C cell incubator. The supernatant was discarded, and cells have been washed 3 times with DMEM/F12 medium. Movement cytometry was used to measure ROS depth.
SOD detection
After eradicating the cell medium, the cells have been scraped off with a cell scraper. The cells have been then centrifuged at 2000 g for 10 min at 5 °C, and the supernatant was eliminated. The cells have been washed twice with PBS. Ultrasound therapy of cells was carried out on an ice bathtub. The cells have been then centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant was transferred to a brand new take a look at tube. The ready pattern answer was used for SOD detection based on the directions of the package (Beiren Chemical Know-how Co., Ltd, Japan).
CCK8 assay
To every properly, 10 µL of CCK8 reagent (Beiren Chemical Know-how Co., Ltd, Japan) was added. After incubation for 4 h at 37 °C in a 5% CO2 incubator, the absorbance worth at 450 nm was measured. The formulation for cell viability is [(ODFA – ODBlank) / (ODCon – ODBlank)]×100%.
Western blotting
Proteins from the pores and skin tissues and cells have been remoted utilizing lysate containing RIPA lysate, protease inhibitor, and phosphatase inhibitor. The protein focus was decided utilizing the bicinchoninic acid (BCA) assay. Roughly 30 µg of proteins have been loaded and separated by means of SDS-PAGE. The proteins have been then transferred to a Polyvinylidene fluoride (PVDF) membrane (Millipore) at 100 mA present 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 main antibodies in a single day at 4 °C. Secondary antibodies have been used to incubate the membranes at room temperature for two h, adopted by washing with TBST. The proteins have been detected utilizing the Picture Quant Las 500. The antibodies used on this analysis are listed in supplementary Desk 2.
Transcriptome sequencing
The evaluation of RNA purity and integrity was performed using a NanoPhotometer spectrophotometer and an Agilent 2100 bioanalyzer, respectively. For the development of libraries, the NEBNext® Extremely™ RNA Library Prep Package for Illumina was utilized. Quantification was initially undertaken with a Qubit 2.0 Fluorometer, and the insert dimension was subsequently evaluated utilizing the Agilent 2100 bioanalyzer. The dedication of the library’s efficient focus was achieved by means of qRT-PCR. The library amplification course of concerned the combination of fluorescently-labeled dNTPs, DNA polymerase, and adapter primers. Throughout the extension section of every sequencing cluster, fluorescently-labeled dNTPs have been included, triggering the emission of corresponding fluorescence alerts. The calculation of FPKM (Fragments per kilo base per million mapped reads) values for particular person genes was based mostly on gene size and the variety of mapped reads. The evaluation of differential expression throughout comparability teams was carried out using DESeq2 software program, with the adjustment of P-values based on the Benjamini & Hochberg technique [30]. Standards for important differential expression have been established by adjusted P-values and the magnitude of |log2foldchange|. Differentially expressed genes have been analyzed functionally utilizing Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) with the Database for Annotation, Visualization and Built-in Discovery (DAVID) software. The thresholds for Padj < 0.05 and FDR < 0.05 have been utilized.
Statistical evaluation
All experimental information have been obtained by independently repeating the experiments not less than 3 times. The experimental information have been offered as imply ± SD and analyzed utilizing GraphPad 8 software program. A t-test was performed to match information between two teams, and ANOVA was used to match information amongst a number of teams, with P < 0.05 indicating important statistical variations.