Animals and bacteria

Six-week-old C57/BL6 female mice (from the Experimental Animal Center of Yangzhou University, Yangzhou, China) were used in this experiment. They were fed solid food (without antibiotics) and given free access to water. The animals were acclimated for a week at 24 ± 1 °C and a 12-h normal light-and-dark cycle before the experiment. The animal experiments were approved by the Ethics Committee for Experimental Animal Research of Yangzhou University. Additional file 1 is the ethics approval document.

H. pylori SS1 (gift from Nanjing University) was cultured on Colombia Blood Agar Base (Thermo Scientific, Waltham, USA) containing 5 μg/mL trimethoprim, 2 μg/mL amphotericin B, 10 μg/mL vancomycin, and 3.8 μg/mL polymyxin. For mouse infection, H. pylori were grown under shaking in brain heart infusion (BD, Franklin Lakes, USA). Then, the cultures were incubated in an anaerobic incubator (Hua Yue, Guangzhou, China) at 37 °C with 5% O2, 10% CO2, and 85% N2 for 4 days.

H. pylori infection model

After fasting overnight, the mice were gavaged once per day for five consecutive days with 300 μL phosphate-buffered solution (PBS) containing 1 × 109 CFU/mL H. pylori. The control mice were inoculated with saline solution.

AD-like skin lesions mouse model

Several concentrations of DNCB (Sigma-Aldrich, St. Louis, MO, USA) were used to induce AD-like lesions of the skin after 6 weeks of infection with H. pylori. In the first stage of inflammation induction, the control group (HP−AD−) was treated only with a carrier (acetone: olive oil = 4:1), whereas in the H. pylori-negative AD group (HP−AD+) and the H. pylori-positive AD group (HP+AD+), 200 μL of 1% DNCB solution was applied to the same back area two times over the course of one week. Following the sensitization, 100 μL of 0.5% DNCB solution was repeatedly applied on the back skin twice a week for 14 days.

Measurement of dermatitis score

The severity of AD-like skin lesions was measured by scoring as described previously [18]. Each of the four symptoms (erythema/hemorrhage, edema, excoriation/erosion, and scaling/dryness) was graded as follows: 0 (none), 1 (mild), 2 (moderate), or 3 (severe). The sum of all individual scores for the four symptoms of AD-like changes was calculated (maximum score: 12).

Scratching behavior of mice

On the 21st day, after stimulation with DNCB, the mice were placed separately, and we counted how many times in a 60-min period the mice scratched their backs or brushed against the cage. Continuous scratching was counted as one time, and the number of scratches was counted.

Histological examination

Tissue slides were made as previously reported [15]. After hematoxylin and eosin (H&E) and toluidine blue staining, anti-filaggrin (Signal, College Park, USA) and anti-loricrin (Abcam, Cambridge, UK) antibodies were used for immunohistochemistry. The Ultravision Quanto detection system (Thermo Fisher Scientific, MA, USA) was employed for the staining. Following the staining process, the tissue was dehydrated and sealed in a fixed culture medium (Sinopharm Group; Shanghai, China), and it was examined under an optical microscope (Olympus, Tokyo, Japan).

Quantification of total serum inflammatory cytokines and immunoglobulin E (IgE)

Blood samples were collected from the mice and centrifuged at 4 °C for 30 min. Enzyme-linked immunosorbent assay (ELISA) kits (ELISA LAB, Wuhan, China) were employed for the measurement of serum IgE, IL-4, and IL-31 levels, following the guidelines provided by the manufacturer. We measured the absorbance at 450 nm using a microplate reader (BioTek Epoch, Vermont, USA).

Western blot analysis

After homogenizing the back tissue in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and supplementing it with protease and phosphatase inhibitor cocktails (Beyotime, Shanghai, China), a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) was used to determine the levels of protein in the lysates. Subsequently, equivalent protein quantities were loaded and separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by transfer onto polyvinylidene fluoride membranes (Vazyme, Nanjing, China). The membranes were treated with primary antibodies against anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT3, and anti-STAT3 (Cell Signaling Technology, MA, USA) as well as anti-actin (Santa Cruz Biotechnology, CA, USA) at 4 °C overnight after being blocked with QuickBlockTM blocking buffer (Beyotime, Shanghai, China). Following the washing step, the membranes were subjected to incubation with secondary antibodies (anti-mouse or anti-rabbit from Cell Signaling Technology) conjugated to horseradish peroxidase (HRP). Finally, using enhanced chemiluminescence tools (ECL, NCM Biotech, Suzhou, China), immunoreactive signals were detected.

Statistical analysis

Data were analyzed and plotted through GraphPad Prism 5 software. Continuous variables were represented by the mean ± standard error of mean (SEM), while counts and percentage values were used to represent discrete variables. Student t-tests or one-way analysis of variance (ANOVA) were performed to compare values between groups, as appropriate. Post hoc tests were performed using Tukey’s honestly significant difference (HSD) method to identify significant differences among multiple groups. P < 0.05 was considered statistically significant.