Materials and methods

Ethics statement

BALB/c mice were provided by the Department of Experimental Animals of the Army Medical University, and all were female, weighing about 25 g. The mice were housed according to a 12 h circadian rhythm with a relative humidity of 50% and an ambient temperature of 25 °C. Animal experiments were approved by the Ethics Committee of Southwest Hospital, and all operations were performed in accordance with relevant ethical regulations.

Cell lines and cell culture

Human keratinocyte cell HaCaT was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured using DMEM culture medium (HyClone, USA) supplemented with 10% fetal bovine serum (ABW, Uruguay) and 1% penicillin/streptomycin (Beyotime, China). All cells were cultured at 37 °C in a 5% CO2 incubator.

Cell viability and apoptosis assay

NaCl was added to the DMEM culture medium at a concentration of 40 mM to achieve an osmolality of 380 mOsm/kg H2O. Similarly, mannitol, sodium gluconate (Na-Gluconate), and choline chloride (Choline-Cl) were prepared to reach the same osmolality of 380 mOsm/kg H2O, ensuring isotonic conditions. After 24 h of treatment with the prepared isotonic media, 10 μL of cell counting kit-8 (ShenEr, China) was added, and the optical density was measured at 450 nm to evaluate cell viability. Additionally, cells were collected after treatment and stained using the Annexin V-FITC/PI apoptosis detection kit (Beyotime, China). Apoptosis levels were then immediately detected using flow cytometry.

The AD mouse model

Six-week-old female BALB/c mice weighing 20–25 g were procured from the Experimental Animal Centre of the Army Medical University (Third Military Medical University, Chongqing, 400,038, China). All the animals were housed in polypropylene cages and were given unlimited access to sterilized fodder and water. All experimental procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (AMUWEC20223374). Animals were acclimatized to feeding conditions for 1 week before experimentation. At specific time points, the mice were sacrificed by cervical dislocation. After acclimation to the laboratory conditions, the back skin hair of all the mice was shaved with an electric razor, and the bare skin was treated with hair removal cream. The application of hair removal cream was repeated every three days to keep the skin clean. One hundred microliters of DNFB or solvent was uniformly smeared onto the shaved back of the mice three times a week. One hundred microliters of ovalbumin or saline was given twice a week, once for application to the shaved back skin, and once for intraperitoneal injection. Animals were sacrificed 24 h after the last exposure to reagents for the procurement of blood samples and skin tissue. All reagents were softly and evenly applied to the mice. Cages were sanitized and replenished with chow and water weekly.

Enzyme-linked immunosorbent assay (ELISA)

Skin tissue samples were weighed and homogenized on ice. The homogenates were centrifuged at 12,000 rpm at 4 °C for 15 min, and the supernatants were taken for cytokine assay. The concentrations of IL-4, IL-13, IL-25, IL-33, TSLP, and IgE (R&D Systems, USA) in the tissue homogenates were quantified using ELISA kits according to the manufacturer's instructions. HaCaT cells were inoculated in six-well culture plates. After stimulation with reagents, cell homogenates were taken, and a cytokine assay was performed according to the manufacturer's instructions. The concentrations of IL-25, IL-33, and TSLP in HaCaT cell homogenates were measured by ELISA kits according to the reagent instructions.

Western blotting

Cells were collected and lysed with RIPA buffer (Beyotime, China) containing protease inhibitors on ice for 30 min. Total proteins were quantified by the BCA Kit (Beyotime, China) according to the reagent instructions. Proteins were separated by gel electrophoresis and transferred to PVDF membranes. PVDF membranes were blocked in 5% bovine serum albumin for 2 h and incubated with primary antibody (1:1000) at 4 °C overnight. The membranes were then incubated with the secondary antibody for 1 h at room temperature and developed.

The primary antibodies used were against the following antigens: FLG (SCBT, USA), LOR (Thermofisher, USA), GAPDH (ShenEr, China), β-actin (Proteintech, China), NFAT5 (Affinity, USA), p38 MAPK (Zenbio, China), 4EBP1 (Proteintech, Chin), phospho-4EBP1 (Zenbio, China), 70S6K (CST, USA), phospho-70S6K (CST, USA), RPS6 (BIOSS, China), Phospho-RPS6 (BIOSS, China) (phospho-p38 MAPK (Zenbio, China), phospho-SGK-1 (Affinity, USA), phospho-SGK-1 (BIOSS, China), NFκB p65 (BIOSS, China), NFκB p50 (BIOSS, China), STAT3 (BIOSS, China), and phospho-STAT3 (BIOSS, China).

Immunofluorescence

Cells were cultured in 12-well slides. Cells were fixed with 100% acetone at 4 °C for 10 min and then permeabilized with 0.1% Triton X-100 at room temperature for 10 min. Fixed cells were treated with 1% bovine serum albumin. Incubated with phosphate-buffered saline (PBS) for 1 h and with FLG and LOR antibodies for 1 h. The cells were then incubated with FITC-labeled antibody for 1 h. Images were captured using fluorescence microscopy after DAPI staining.

Hematoxylin–eosin staining

Mouse skin tissues were fixed in 4% paraformaldehyde for 24 h, rinsed, dehydrated, and paraffin-embedded. The tissues were sectioned. Sections of 4–5 μm thickness were stained with hematoxylin–eosin staining kit (Solarbio, China) and then observed under a microscope.

Skin lesion assessment

The lesion scores were recorded for each group of mice except the blank group, and the severity of lesions was scored with reference to the five main components: erythema, desquamation, dryness/scarring, edema, and exudation. Each component has four levels: 0 (none), 1 (mild), 2 (moderate), or 3 (severe). The total score for dermatitis was calculated based on the total score of these individuals. The scores for each project were evaluated by two independent observers. Skin damage assessment was conducted every four days and every 8 day at the end of the experiment. The water content of the stratum corneum, trans-epidermal water loss (TEWL), and sebum content (measured in μg/cm2) were measured using a non-invasive skin tester. The same area was selected for each measurement, and a total of three measurements were taken and averaged.

Statistical analysis

GraphPad Prism software was used for statistical analysis, and the results of the statistical analysis were expressed as mean ± standard deviation. Student's t-test was used for comparison between two groups. One-way ANOVA and LSD t-test were used for analysis of differences between groups. P-value < 0.05 indicates statistical significance.