2.1 Experimental animals
8-week-old BALB/c mice, female, weighing between 20g and 22g, were purchased from Beijing Hua fukang Biotechnology Co. (Beijing, China) and maintained in microisolator cages in a specific pathogen-free facility at the Xi'an Medical University Laboratory Animal Center. All mice were fed with standard chow diet and tap water. All experimental protocols were approved by the Institutional Ethics Committee on Animal Use of Xi'an Medical University. All methods were carried out in accordance with the guidelines and regulations of Institutional Ethics Committee on Animal Use of Xi'an Medical University.
After 1 week of adaptation, Mice were divided into 4 groups. Eight mice were included each group. Normal: normal control, with no sensitization and vehicle application; Model: negative control, with DNCB sensitization and vehicle application; DEX: positive control, with DNCB sensitization and dexamethasone application; HCC: with DNCB sensitization and Hydrogenated coral calcium application. Administration is by gavage.
2.2 Reagents
2,4-dinitrochlorobenzene (DNCB) was purchased from Sigma Aldrich (St.Louis, USA). Sodium dodecyl sulfate (SDS) and Sodium carboxymethyl cellulose were purchased from Solarbio Life sciences (Beiing, China). Acetone olive oil and acetone was purchased from Thermo Fisher Scientific (Shanghai, China). Dexamethasone was purchased from Sigma Aldrich (St.Louis, USA). Calcium hydrogenated coral solution was provided from Rizhao Life Valley Biotechnology Development Co., Ltd (Shandong, China).
(1) 0.5% Sodium carboxymethyl cellulose: take 1g of sodium carboxymethyl cellulose dissolved in 150ml of hot water and placed in a hot magnetic stirrer, dissolve at high temperature until all the solids are dissolved and then fix the volume to 200ml.
(2) Acetone olive oil mixed solution: Acetone and olive oil volume at a ratio of 3: 1 evenly mixed.
(3) 1% DNCB solution: weigh 1.2g DNCB solid dissolved in 12ml acetone plus olive oil (acetone 9ml + olive oil 3ml) mixed solution, mix well.
(4) 0.4% DNCB solution: Weigh 48mg DNCB solid dissolved in 12ml acetone plus olive oil (acetone 9ml + olive oil 3ml) mixed solution, mix thoroughly.
(5) Calcium hydrogenated coral solution: effective concentration 5mg/ml, dissolved with 0.5% sodium carboxymethyl cellulose.
2.3 Measurement of Dermatitis Score
Mice were scored double-blind according to the severity of skin manifestations on the back of mice, including erythema or hemorrhage, scratches or vesicles, skin edema, and scaling or mossy lesions[11]. The scores of erythema or hemorrhage, scratches or erosions, and scaling or mossy changes were as follows: 0–4, with 4 indicating that the symptom involved > 75% of the skin area on the back of the mice; 3 indicating that the symptom involved 40%-75%; 2 indicating that the symptom involved 10%-40%; 1 indicating that the symptom involved < 10%; and 0 indicating that the symptom was absent. Skin edema was also scored on a scale of 0 to 4. A score of 4 indicated that the skin was significantly higher than the skin surface and dark red; a score of 3 indicated that the skin was less swollen, light red and dry; a score of 2 indicated that the skin was uneven and darker than normal mice; a score of 1 indicated that the skin was smooth and close to normal mice, with almost no detectable signs of edema; and a score of 0 indicated none of the above. Finally, the total score of the four manifestations was taken as the score of the mouse.
2.4 Measurement of Scratching Behavior
The previous day of sacrifice, all mice were set in a separated plastic cage for stabilizing for 1 h after sensitization with 200 ml of 0.4% DNCB. Scratching behavior of each group was recorded for 10 min. The number of scratching was counted by acknowledging the scratches only the back side of the body. The scratching episode of each group was counted by a blind test. The number of scratching of each mouse was averaged from the counts of three researchers.
2.5 Histological Analysis for H&E Staining
Skin tissues from the upper back of mice were fixed in 4% formalin for 24h, dehydrated. Histological analysis of the skin was performed by staining 5‑mm sections of paraffin‑embedded tissues with hematoxylin and eosin. Inflammatory cells were counted by examining 10 different visual fields under a microscope at magnification, x400 in a blinded manner.
2.6 Cytokine assay
The left blood was subsequently collected from the orbital sinus, and plasma was obtained by centrifugation and stored at − 20°C until further use. Cytokine production was assessed using IL-17 and IgE ELISA Kit (MultiSciences (Lianke) Biotechnology Corporate Limited, China), according to the manufacturer’s instructions.
2.7 Statistical analysis
GraphPad Prism software version 8 (Cricket Software, USA) was used for data analyses. Specifically, the data were analyzed using one-way ANOVA or two-way ANOVA with SNK-q multiple comparisons test and column statistics. The results are reported as the means ± SEMs. P values < 0.05 were considered to indicate statistical significance.