Dinitrochlorobenzene

AD was induced in mice by repeated local exposure of Dermatophagoides farinae extract (DFE; house dust mite extract) and 2,4-dinitrochlorobenzene (DNCB) to the ears, as previously described [17 (link)]. A schematic of the experimental procedure is provided in Figure 1. For the induction of AD, the mice were divided into four groups (control, AD-only, EF-2001-only, and AD + EF-2001), and the surfaces of both earlobes were stripped five times with surgical tape (Nichiban, Tokyo, Japan). After stripping, 20 µL of 1% DNCB was painted onto each ear, followed by 20 µL of DFE (10 mg/mL) four days later. DFE and DNCB treatment was administered once a week for four weeks. Animals received EF-2001 (2 mg/kg orally administered) throughout the four weeks of AD induction.
The ear thickness was measured 24 h after DNCB or DFE application with a dial thickness gauge (Kori Seiki MFG, Co., Tokyo, Japan). At days 14 and 28, blood samples were collected by orbital puncture. Plasma samples were prepared from the cardiac puncture under ketamine/xylazine anesthesia and stored at −70 °C for further analysis. After blood collection, the ears were removed and used for histopathological analysis. The serum immunoglobulin (Ig)E, DFE specific IgE and IgG2a levels were measured at days 14 and 28 after the first induction using an IgE enzyme-linked immunoassay kit (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer’s instructions.

Wild-type male BALB/c mice were purchased from OrientBio Co. (Seongnam, Republic of Korea). The animals were housed at 2~3 mice per individually ventilated cage at a temperature of 22 °C ± 2 °C and a relative humidity of 55 ± 5% under a 12-h light-dark cycle in a pathogen-free animal facility at the World Institute of Kimchi. The mice were fed standard chow and had ad libitum access to water. To study the ameliorative effects of LAB administration on AD, AD-like lesions were induced by 2,4-dinitrochlorobenzene (Sigma-Aldrich, Saint Louis, MO) according to Kim, et al.23 (link). Mice were randomized into 4 groups (n = 5 per group): a non-induction group (naïve group, mice fed the vehicle), negative control group (NC group, mice sensitized with DNCB and fed the vehicle), positive control group (PC group, mice sensitized with DNCB and fed the anti-histamine agent kerotifen at 1 mg/kg), and WIKIM28 group (mice sensitized with DNCB and fed W. cibaria WIKIM28). Dorsal skin was shaved, and 200 μL of 1% DNCB in acetone/olive oil (3:1) was applied to the dorsal skin twice a week. Three weeks after the first induction, 0.2% DNCB was applied to the dorsal skin once a week. WIKIM28 or PBS (200 μL) were administered by intragastric gavage once daily. The vehicle and LAB were administered for 42 days. On day 43, the mice were sacrificed, after which the blood, dorsal skin, spleens, MLNs, and PLNs were removed for further analysis. All animal procedures were performed following the National Institutes of Health Guidelines for the Humane Treatment of Animals, with approval from the Institutional Animal Care and Use Committee of the World Institutes of Kimchi (WIKIM IACUC 201509). All sacrifices were performed under CO₂ condition, and all efforts were made to minimize suffering.

AD-like skin lesions were induced by DNCB (Sigma-Aldrich, St Louis, MO, USA) topical application in NC/Nga mice described in the methods of our previous study [20 (link)]. Briefly, after 1 week of acclimation, dorsal hair of NC/Nga mice was removed by using an electric shaver. After shaving hair, the mice were randomly divided into the following 6 groups, and 8 mice were allocated in each group (sample size was n = 8 per group): nontreated control group (Normal, naïve control group), DNCB-treated group (Control, negative control group), DNCB-treated + prednisolone 3 mg/kg (Sigma-Aldrich, St Louis, MO, USA) group (PD, positive control group), and DNCB-treated + F.NONI 250, 500, 1000 mg/kg group (F.NONI 250, F.NONI 500, F.NONI 1000). To induce AD-like skin lesions, 1% DNCB was dissolved in an acetone and ethanol mixture (2:3 v/v) and then was topically applied on the shaved dorsal area (200 µL) and right ear (100 µL) twice a week for sensitization. Following the sensitization, 0.4% DNCB dissolved in an acetone and olive oil mixture (3:1 v/v) was challenged on the dorsal skin (150 µL) and right ear (50 µL) repeatedly three times a week for 9 weeks. The mice in the normal and control groups were orally administered 0.5% carboxymethyl cellulose (0.5% CMC). Administration of PD (3 mg/kg prednisolone) and F.NONI (250, 500, 1000 mg/kg) was performed daily for 4 weeks. AD-like skin lesions were decided by dermatitis score, scratching behavior, and histological and immunological parameters.

BALB/c mice (male, 4 weeks old) were acclimated for 1 week upon arrival. Mice were randomly divided into five groups of five mice (only PBS, only DNCB, DNCB, 4 mg/mL Ca-EE, DNCB and 8 mg/mL Ca-EE, and DNCB and 8 mg/mL dexamethasone). DNCB, Ca-EE (200 μL/ear), and dexamethasone (200 μL/ear) were sequentially dissolved in PBS and applied to both ears of mice. Ca-EE and dexamethasone were treated at 200 μL/ear once a day for 28 days. On day 7, 200 μL of 1% DNCB was applied to each ear, and treatment with 200 μL of 0.5% DNCB was repeated every 3 days. On Day 28, all the mice were euthanized, and blood samples and ears were collected. The serum was isolated from the blood samplem and an IgE-ELISA assay was performed with an ELISA kit (BD Biosciences, Oxford, UK) according to the manufacturer’s instructions.

Six-week-old female BALB/c mice were obtained from Samtako (Osan, Korea). A total of 25 mice were divided into five groups (n = 5): the vehicle phosphate-buffered saline (PBS), DNCB vehicle (PBS), DNCB and VAE (100 and 300 mg/kg), and DNCB and dexamethasone (DX, 5 mg/kg) groups. For sensitization, 20 μL of DNCB was applied once each ear (face side and back side of ear, once) in first week. Then, both ears of BALB/c mouse were challenged with DNCB (1%, 20 μL/ear, once) for 3 weeks. VAE (100 and 300 mg/kg) or DX (5 mg/kg) was orally administered by gavage for five consecutive days per week at the time of the DNCB challenge. Mice were provided with ad libitum access to food and water. The room was maintained with a 12 h light-dark cycle (lights on at 07:00 and lights off at 19:00) and controlled temperature (21 ± 2 °C). Mice were handle in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the experimental protocols were approved by the Institutional Animal Care and Use Committee of Korea Research Institute of Bioscience and Biotechnology (permission number #KRIBB-AEC-21259).

The activity of glutathione transferase (GST) was measured as the rate of the produced dinitrochlorobenzene (DNCB)–glutathione (GSH) complex catalyzed by this enzyme [71 (link)]. The GST activity directly corelates with the increase in the sample absorbance. The absorbance was monitored spectrophotometrically at a wavelength of 340 nm every 30 s for 180 s at 25 °C, and activity was expressed as units per milligram of protein (U/mg protein).
The glutathione (GSH) content was determined following the protocol described by [72 (link)]. Briefly, the supernatant fraction of intestinal homogenates was deproteinized (in 10% sulfosalicylic acid). Ellman’s reagent (5,5-dithio-bis-(2-nitrobenzoic acid)) in Tris–Cl (pH 8.9) and reduced glutathione were used as standard. Absorbance was measured at 412 nm, and data are expressed as µmol/mg.