Eight-week-old female C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea). The mice were adapted for one week before the start of experiments. During the experiment, animals were maintained under specific pathogen-free conditions in the animal facilities at Dongguk University School of Medicine. The animal care and use committee of the research institute at Dongguk University Hospital approved all studies used in this investigation.
Chemicals and reagents
Primary antibodies against p38 MAPK, p-p38 MAPK, p-IκBα, p-NF-κB p65, and β-actin and horseradish peroxidase-linked anti-rabbit IgG secondary antibody were supplied by Cell Signaling Technology (Danvers, MA, USA). IMQ cream (5%) (Aldara; 3M Health Care, UK) was purchased from Dong-A Pharmaceutical Co. (Seoul, Korea).
Preparation of herbal formula SC-E1
All herbal medicines of SC-E1 were purchased as dried herbs from Omniherb (Daegu, Korea) and prepared according to our previous study . Briefly, A mixture of Gypsum, Gardenia jasminoides, Glycyrrhiza uralensis, Pueraria lobata, and Platycodon grandiflorum at 16:6:2:6:3 ratios was macerated with 800 ml of 70% ethanol, stirred for 24 h at room temperature (RT), and filtered twice using 8 μm pore size Whatman filter paper. After rotary evaporation at 40 ~ 45 °C, the concentrate was lyophilized, yielding 15.9 g of dried power (yield ratio 15.9%). All constituents of SC-E1 have been recognized as standard products by the Korea Food and Drug Administration (KFDA).
Induction of psoriasis and the administration with SC-E1
IMQ cream was used to induce psoriasis-like skin symptoms in mice as previously described . Briefly, hair on the backs of the C57BL/6 mice was shaved using an electric shaver, after which they were treated with a skin-hair-remover (Niclean, Ildong, Korea). The experimental scheme is shown in Fig. 1. The mice were then randomly divided into five groups (n=5/group): (1) Normal group (vehicle cream; Petrolatum), (2) IMQ/distilled water (DW) control group, (3) IMQ/SC-E1 (250 mg/kg), (4) IMQ/SC-E1 (500 mg/kg), and (5) IMQ/SC-E1 (1000 mg/kg). On day 0, SC-E1 (250 mg/kg, 500 mg/kg, and 1000 mg/kg) was administered to the mice orally once a day by oral gavage needle for 5 days before and together with IMQ application for another 7 days (totally 12 days of treatment). For the control IMQ group, distilled water (DW) was administered instead of SC-E1. The normal group was treated with only a vehicle cream (Petrolatum).
Scoring severity of dermatitis
The extent of: (1) erythema, (2) scaling, and (3) thickening was scored as 0 (none), 1 (slight), 2 (moderate), and 3 (marked). The cumulative dermatitis score (erythema plus scaling plus thickening) was used to indicate the severity of dermatitis of the back skin (scale 0-9).
Assay of cytokines production
To measure cytokine levels, mice serum was collected at 24 h after the final administration and stored at -70℃ until analysis. To measure the cytokine levels in skin tissue, the dorsal skin of mice was removed and stored at -80℃. For analysis, skin was homogenized using a Bullet Blender TM Blue (Next Advance, Averill Park, NY) at 4℃, after which the supernatants were at -30℃. The concentration of TNF-a, IFN-g, IL-17A, and IL-23 in the mouse serum and skin tissue was measured using the Quantikine mouse IL-17A (R&D system, Minneapolis, MN, USA), TNF-a, IFN-g, and IL-23 (eBioscience, San Diego, CA, USA). ELISA (enzyme-linked immunosorbent assay) was performed in accordance with the manufacturer’s instructions.
Paraformaldehyde-fixed and paraffin-embedded back skin samples from the mice were sliced and then stained with hematoxylin and eosin (H&E). Based on the histological finding, the several representative symptoms of dermatitis were assessed in a blind manner on the epidermis or dermis (epidermal thickening, stratum corneum, and inflammatory cell infiltration). The inflammatory cells were counted per 5 high-power fields (X400) for each section under the microscope.
Spleen weight and size
The spleen of each mouse was removed, weighed and sized at the time of sacrifice.
Western blotting analysis
Total proteins of skin lesion tissues were extracted by homogenization in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Briefly, equal amounts of protein (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). The membranes were blocked in PBST containing 5% skim milk (BD Diagnostic Systems, Sparks, MD, USA) for 1 h and incubated with primary antibodies against p38 MAPK (1:1000), p-p38 MAPK (1:1000), p-IκBα (1:1000), anti-p-NF-κB p65 (1:1000), and β-actin (1:2000) overnight at 4°C. After three washes with PBST, membranes were incubated with horseradish peroxidase-linked anti-rabbit IgG secondary antibody (1:3000) for 1 h at room temperature. After washing three times with PBST, detection was performed using an enhanced chemiluminescence solution (Amersham™ ECL™ Prime Western Blotting Detection Reagent; GE Healthcare, Buckinghamshire, UK). Bands were visualized using a Fusion Solo 2M chemiluminescence imaging system (Vilber Lourmat, France) and the intensity of each band was analyzed using ImageJ 1.48v software (NIH, Bethesda, MD, USA).
All groups were compared by one-way analysis of (ANOVA), followed by the Duncan test. The results were expressed as the means ±S.D. A p < 0.05 was considered significant.