Experimental animals
Five-week-old female SKH-1 hairless mice with the mean weight (mean ± SD) 25 ± 4.2 g were purchased from Orient Bio Inc. (Seongnam, Republic of Korea) and used in carrying out the studies. The mice were obtained at the small unit of animal care and use department in Wonju College of Medicine, Yonsei University, Republic of Korea.
Housing and husbandry
Handling of mice was done in accordance with the use and care protocols of Institutional Animal Care and Committee (IACUC) at Wonju College of Medicine, Yonsei University, Republic of Korea. The mice were kept in spacious plastic cages (390 × 275 × 175 mm) with wood shaving bedding and identified by labeling with surgical skin markers marking at the tail. They were acclimatized for 7 days to the housing environment prior to treatment and were maintained in a controlled environment with atemperature of 22 ± 2 °C and 40-60% humidity under a 12:12-h light-dark cycle. Standard rodent chow food (5 L79, PMI Nutrition®, LAND O’LAKES, INC, Minnesota, USA) and primary filtered water were supplied free to access until the end of the experiment. At the start of the experiment, 50 mice were randomized into five groups, five mice each cage (n = 10 respectively) as follows: Normal control group (NC), Negative control group (NeC) treated with DNCB only + DW bathing, Positive control group (PC) treated with DNCB +0.1% tacrolimus ointment + DW bathing, 100% pure high concentration mineral water (PHMW) group treated with DNCB+ PHMW bathing, and 10% diluted high concentration mineral water (DHMW) group treated with DNCB + DHMW bathing. The study protocol of the experiment was approved by the Institutional Animal Care and Use Committee (IACUC) at Wonju campus, (Ethical approval no: YWC-160513-1) Yonsei University, Gangwon, Wonju, and Republic of Korea. All the experiments were conducted between 7 a.m. and 6 p.m. to minimize the effects of environmental changes.
Preparation of experimental water
A colorless clear solution of natural high mineral spring water (HMW) was supplied from Tae chang Co.Ltd. (Gyeokpo, Buan-gun, Republic of Korea). Mineral compositions of HMW were analyzed by a Thermo Electron x Series Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) and a Thermo Scientific iCAP 6500 duo Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES) in Korea Institute of Geosciences and Mineral Resources (Daejeon, Republic of Korea) and the result was as Table 1. All the experimental water were stored in the big plastic container covered with a lid at 4 °C to protect from light and humidity until use. DHMW was prepared by 10% dilution of PHMW. For the treatment of NeC and PC groups, distilled water (DW) was used.
Induction of allergic dermatitis with DNCB in skh-1 hairless mice
AD-like inflammatory skin lesions were induced in skh-1 hairless mice by sensitization with 200 μL/mouse/day 1% DNCB (dissolved in a 3:1 mixture of acetone and olive oil) for 1 week, and boosted with 150 μL/mouse of 0.5% DNCB every alternate day for 3 weeks according to previous established methods [20]. The 3 weeks of boosting and bathing was followed by one more week of bathing with sample waters only. DNCB solutions were topically applied to dorsal skin (approximately 4 cm2) on each mouse except NC mice. In intact PC mice, tacrolimus ointment (0.1% ProtopicCo.Ltd. Osaka, Japan) was topically applied on the dorsal skin, seven times a week for 4 weeks (day 8-35). After a total of 4 weeks treatment, mice were anesthetized with isoflurane (Hana Pharm. Co., Hwaseong, Republic of Korea) in the mixture of 70% N2O and 28.5% O2 to minimize suffering and distress and blood samples of all the mice were collected from retro-orbital veins in EDTA vacutainer tubes and kept in ice packs. Immediately after blood collection, mice were sacrificed by cervical dislocation. The collected blood sample was centrifuged for 5 min at 14000 rpm and the separated serum was stored in −80 °C before use. A time line diagram for this experiment is shown in (Fig. 1).
Bathing method for treatment after induction of AD-like inflammation
Five mice were freely bathed in a plastic cage (390 × 275 × 175 mm) containing 4 cm depth of PHMW, DHMW and DW respectively for 15 min/day for 4 weeks. The mice of both PHMW and DHMW groups were bathed in PHMW and DHMW, and PC and DNCB control group were bathed in DW to provide the same bathing condition. All the experimental water were warmed around 37 °C before bathing.
Evaluation of the skin severity
The dermatitis severity was assessed by using skin scoring procedure, the frequency of scratching and skin test after triggering AD via DNCB. The dermatitis skin scoring procedure assessed eczema area and a severity index scoring system applied as follows: 0, no symptoms; 1, mild symptom; 2, moderate symptom; 3, severe symptom. The overall dermatitis score was defined as the sum of scores for erythema, edema, excoriation and scaling/dryness. The skin scoring was assessed once a week during the 4 weeks of treatment. Simultaneously, scratching actions such as rubbing their dorsal skin with their hind paws, their nose and ears were counted on 2nd and 4th week within 15 min in triplicate observation.
Measurement of total IgE
Blood samples were collected from the retro-orbital plexus of mice at the end of the experiment. Serum was obtained by centrifugation at 14000 rpm for 5 min and stored in −80 °C until use. The serum total IgE levels were determined by using the mouse IgE ELISA kit (BD Biosciences, San Diego, CA, USA) according to manufacturer’s manual instructions. The reaction product was measured calorimetrically at 450 nm with a microplate reader (BioTekInstrument, Winooski, VT, U.S.A).
Measurement of cytokine concentration
Inflammatory cytokines such as interleukin (IL)-1β, IL-13 and tumor necrosis factor-alpha (TNF-α) were measured in serum by using multiplex array kit (Bio-Rad, San Diego, CA, U.S.A.) and run on Luminex technology (Bio-Plex Multiplex Bead array system TM, Bio-Rad Hercules, CA, U.S.A.) according to manufacturer’s instruction. Raw fluorescence data were analyzed by software using a 5-parameter logistic method.
Determination of total ROS
The level of total ROS production in serum was assessed by measuring the oxidation of 2-4-dichlorodihydrofluorescein diacetate (DCFH-DA) (Abcam, Cambridge, MA, U.S.A) by following manufacturer’s manual instructions. In brief, 50 μL of samples were prepared in the 96-well plate. One hundred μL of 10 μM DCFH-DA was added into each well and the plate was incubated for 30 min in the dark. Fluorescence at 488 nm excitation/525 nm emission was analyzed by using DTX-880 multimode microplate reader (Beckman Coulter Inc., Fullerton, CA, U.S.A).
Measurement of MDA
The level of MDA, a marker of oxidative stressin serum was measured using thiobarbituric acid reactive substances (TBARS) assay kit (Cell Biolabs, Inc., San Diego, CA, U.S.A). The assay was performed according to manufacturer’s instructions. The reaction product was measured calorimetrically at 532 nm with a microplate reader (Biotek instruments, Winooski, VT, U.S.A).
Measurement of GPx
GPx activity in serum was measured for H2O2 scavenging capacity by modified Cayman’s GPx assay kit (Cayman Chemical Co., Ann Arbor, MI U.S.A) according to the instruction of the manufacturer. The oxidation of NADPH to NAD+ was measured at the absorbance at 340 nm at least 3 times using automated micro plate reader (Beckman Coulter, Inc., Fullerton, CA, U.S.A) at one-minute interval.
Experimental outcomes
This study provides the in vivo bathing effect of HMW on immunomodulation and redox balance in DNCB-induced AD- like inflammation in hairless mice.
Statistical analysis
Data values were expressed as the mean ± standard deviation (S.D). The mean values among groups were analyzed and compared using one-way ANOVA followed by subsequent multiple comparison tests (Tukey) with Prism version 5.0 software packages (Graph Pad Software Inc., U.S.A). Significant differences were considered statistically at *p < 0.05, ** p < 0.01 and *** p < 0.001.