Clinical data

The skin and serum were collected from 20 patients with CSU (CSU group) diagnosed and treated in our hospital. Besides, normal skin and serum collected from 20 plastic surgery patients were set as controls (Normal group). The inclusion criteria of patients in CSU group were shown as follows: (1) age ≥ 18 years but ≤ 75 years; (2) the patients were in the active stage of the disease without antihistamine treatment; (3) the course of the disease was more than 8 weeks; (4) the patients consented to participate in this study. The inclusion criteria of patients in normal group were listed as follows: (1) without skin diseases and age paralleled with the CSU group; (2) undergoing plastic surgery in our hospital; (3) willing to participate in this study. The exclusion criteria applied to both the two groups were as follows: (1) with disease of vital organs such as lungs, kidneys, cardiovascular and cerebrovascular diseases; (2) with disease of the immune system; (3) lactating women or pregnant women; (4) treated with corticosteroids and immunosuppressants, and diagnosed with chronic inducible urticaria or urticarial vasculitis; (5) suffering from active atopic diseases, such as allergic rhinitis, atopic dermatitis or asthma. All procedures followed the ethical principles of medical research and all patients signed an informed consent. This study was approved by the Ethics Committee of The First Affiliated Hospital of Heilongjiang University of Chinese Medicine (HZYLLKY202009027).

Construction and grouping of CSU animal models

Thirty-six healthy specific pathogen free (SPF) Sprague Dawley rats (6–8 weeks old, 180–200 g) were purchased from Hunan SJA Laboratory Animal Co.,Ltd. All animal experiments were approved by the Ethics Committee of The First Affiliated Hospital of Heilongjiang University of Chinese Medicine (HZYDWLLKY202003417) and performed in accordance with ethical guidelines. The rats were housed in an SPF environment with a stable temperature and humidity within their living environment as well as a consistent circadian rhythm of 12-hour light/dark cycle. After one week of adaptive feeding, the rats were subjected to the experiments. Furthermore, rats had ad libitum access to food and water.

The CSU rat model was constructed according to the previous method [17]. Aluminum hydroxide (Yuanye Bio-Technology, China) was dissolved in saline to make a solution with a final concentration of 10 mg/ml. Ovalbumin (1 mg, Sigma-Aldrich, St Louis, MO, USA) was added to aluminum hydroxide solution (1 ml) to prepare a suspension. Rats were intraperitoneally injected with 1 ml of suspension containing ovalbumin (1 mg). After 5 days, 1 ml of suspension containing ovalbumin (1 mg) and aluminum hydroxide was injected again. The number and maximum duration of pruritus in rats were observed each day, when they were significantly higher than those of normal control rats, it indicated that the CSU model was constructed successfully.

All animals were divided into 6 groups (n = 6 rats/group): (1) normal group: rats were intraperitoneally injected with the same amount of saline, and then intraperitoneally injected again with the same amount of saline after 5 days; (2) CSU group: no other treatment was given to rats after successful CSU modeling; (3) NC group: rats were injected with negative inhibitor after successful CSU modeling; (4) in-miR-155 group: CSU rats were injected with miR-155 inhibitor; (5) Tyr705 group: CSU rats were injected with JAK/STAT signaling pathway agonist Tyr705 (Absin, China) [4]; (6) in-miR-155 + Tyr705 group: CSU rats were injected with miR-155 inhibitor and Tyr705. Negative inhibitor and miR-155 inhibitor were both designed and constructed by BGI Genomics. Thirty min after the injection, the number and maximum duration of pruritus within one day were measured. The behaviors of the rats were defined as pruritus, such as rubbing their paws against the skin of their back, nose, or ears and then placing the paws back on the floor [18]. Upon completion of the measurements, the rats were euthanized by intraperitoneal injection of pentobarbital sodium (100 mg/kg), followed by collection of blood and back skin tissues with wheal or rash.

RT-qPCR

Total RNA was extracted from skin tissues of patients and rats using miRNeasy Mini Kit (217,004, Qiagen, Düsseldorf, Germany). The concentration and purity of RNA were detected by NanoDrop. RNA was reversely transcribed to cDNA in accordance with the instructions of the Reverse Transcription Kit (TaKaRa, Japan). The target gene was synthesized using the Thermal Cycler Dice® Real Time System according to the instructions of the SYBR GREEN Kit (TaKaRa, Japan). U6 was used as an internal control. The relative expression of the target gene was calculated by the 2-ΔΔCt method. The primer sequences were shown in Table 1.

Table 1 Primer Sequences for RT-qPCRHistamine release assay

Histamine release rate was determined according to the previous literature [4]. Serum from patients and rats was mixed with mast cells after incubation at 37 °C for 20 min. After centrifugation (3000 rpm, 15 min), the supernatant was collected. The histamine content in the supernatant was detected by Histamine ELISA Kit (COIBO, Shanghai, China), and the histamine release rate was calculated.

Hematoxylin and eosin (H&E) staining

After the rats were euthanized, their skin tissues were isolated and washed with saline (4 °C) to remove surface bloodstains. Next, the tissues were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin and then cut into 5 μm serial sections. Later, the tissue sections were deparaffinized with xylene, followed by a gradient ethanol to remove xylene. Subsequently, the sections were stained with hematoxylin (Solarbio, China) for 10 min, differentiated in 10% hydrochloric acid in ethanol for 10 s, and immersed in 1% ammonia for 30 s. The sections were subsequently stained with eosin (Solarbio, China) for 3 min. Finally, the tissue morphology was observed under a light microscope (Olympus, Tokyo, Japan), the photographs were collected, and eosinophil counts were performed.

Determination of immunoglobulins

The levels of immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) in rat serum were detected by immunoturbidimetry according to the instructions of the immunoglobulin detection kits (Jining Shiye, Shanghai, China).

Detection of inflammatory cytokines and immunoglobulin E

Rat serum was collected, then the levels of interleukin (IL)-18, IL-6, IL-2, interferon-gamma (IFN-γ) and immunoglobulin E (IgE) in rat serum were detected based on the instructions of ELISA kit (Keshun, Shanghai, China). The samples and antibodies were sequentially added into the ELISA plate and incubated at room temperature for 1 h. Then, the diluted horseradish peroxidase-labeled conjugated antibody was added into each well for another incubation at room temperature for 0.5 h. After that, the supernatant was discarded, and 100 µL of colorimetric substrate 3’-3-5’-5-tetramethylbenzidine (TMB) was added into each well and incubated in a dark box at room temperature for 30 min. Next, 100 µL of stop solution was added to each well, and the absorbance value at 450 nm was measured by a Microplate Reader (Decca, China). Finally, the corresponding cytokine levels in each sample were calculated according to the standard curve.

Flow cytometry

The peripheral blood ( 100 µL) of rats was gathered, supplemented with 10 µL of antibody, and then incubated for 20 min at room temperature away from light. Later, erythrocytes were lysed with Red Blood Cell Lysis Buffer (Thermo Fisher, USA) for 10 min, centrifuged for 5 min, washed with 2 ml of phosphate-buffered saline and then resuspended. Lastly, flow cytometry (BD FACSCanto II, BD Biosciences, Franklin lakes, NJ, USA) was used to detect the proportion of CD3+, CD4+, CD8 + T, CD4 + CD25 + Treg.

Western blot

Total proteins were extracted from rat skin tissues on ice using RIPA Lysis Buffer. Then, the protein concentration was determined by a BCA kit (Beyotime, China). Next, the total protein was added to 5 × sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) Protein Sample Buffer (Solarbio, China) and boiled for 5 min. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Rio-Bad, USA), followed by blocking with 5% skimmed milk powder for 2 h. Subsequently, the PVDF membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies were purchased from Abcam (Cambridge, MA, USA), including JAK2 (ab108596, 1 : 5000), p-JAK2 (ab32101, 1 : 10,000), STAT3 (ab68153, 1 : 2000), p-STAT3 (ab267373, 1 : 1000), IRF-9 ( ab271043, 1 : 1000), and GAPDH (ab8245, 1 : 10,000). The PVDF membranes were rinsed with Tris-buffered saline containing Tween 20 (TBST, Solarbio, China) and incubated with secondary antibody (Abcam, USA) at room temperature for 2 h. Next, BCL luminescent solution (Solarbio, China) was uniformly added to the membranes, which were subsequently scanned and photographed with FluorchemHD2 imaging system. Gray scale values of protein bands were analyzed by Image J. GAPDH was used as an internal reference protein to calculate the relative expression of the target proteins.

Statistical analysis

The experimental data were analyzed statistically by SPSS 24.0 software. The differences between two groups were analyzed by t test, and the differences among multiple groups were compared by one-way analysis of variance. The results were expressed as mean ± standard deviation (SD), and P < 0.05 was used as the criterion for a significant difference.