Male BALB/c mice (6–8 weeks old, weighing 20 ± 2 g) were purchased from the Center of Laboratory Animals of Southern Medical University (Guangzhou, China). Mice were housed in a standard housing room under controlled conditions (maintained at 22 ± 2 oC, 45 to 55% relative humidity) and provided free access to food and water under a specific pathogen-free (SPF) environment. The animal protocols were approved by the Animal Experimental Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine.
Chemicals and reagents
Compound dexamethasone acetate cream (DXA) was purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd. (Shenzhen, China). Imiquimod cream was obtained from Sichuan Mingxin Pharmaceutical Co., LTD (Sichuan, China).
Preparation of FZHFZY and LC-ESI-MSn analysis
FZHFZY was provided by the Guangdong Provincial Hospital of Chinese Medicine and seven Chinese herbs involved were listed in Table 1. All these herbs had been cut into small pieces and soaked with water for 30 min, and then kept boiling for 1 h. After boiling twice, the water extracts was pooled together and then concentrated to the concentration of 0.5 g/mL by electric heating volatilization.
The main ingredients of FZHFZY were identified by LC-ESI-MSn analysis and the ESI-MS spectra of samples and reference compounds were acquired in both negative and positive ionization mode.
RAW 264.7 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). RAW 264.7 cells were cultured in DMEM medium containing 10% FBS, 100 μg/mL streptomycin and 100 IU/mL penicillin at 37 oC in a humidified 5% CO2 atmosphere.
RAW 264.7 cell proliferation assay in vitro
RAW 264.7 cell proliferation was measured using MTT assay. Briefly, Raw 264.7 cells in logarithmic growth were seeded into a 96-well microplate in triplicate. After 24-h incubation, various concentrations (150, 300, 600, 1,200, 2,400 and 4,800 µg/mL, respectively) of FZHFZY was added to each well. After further incubation for 24 h, 10 mL MTT (5 mg/mL) was added to each well. After 4-h incubation at 37 oC, the supernatant then was removed and 100 µL of DMSO was added into each well. Then the supernatants were collected and the absorbance (A value) value at 490 nm was measured using a Microplate Reader and the cell viability was indicated.
Administration of drugs
The BALB/c mice were randomly divided into the following 5 groups (n = 6): the control, vehicle, DXA (1 mg/kg/day) and FZHFZY (0.125 and 0.25 g/mL respectively). The control group was normal mice that were totally untreated. DXA and FZHFZY were topically administered to mice in the DXA and FZHFZY group respectively, while distilled water was given to the control and vehicle cream to induce psoriasis, respectively. Moreover, FZHFZY group was treated with CMF three days before using imiquimod cream and the topical cream treatment administration was applied for 7 consecutive days.
Imiquimod-induced psoriasis-like mouse model
In accordance with our preliminary study , mice were topically administrated with a dose of 62.5 mg of 5% imiquimod cream applied to a shaved area (3 cm × 2.5 cm) on their back for 7 consecutive days.
The Psoriasis Area and Severity Index (PASI) had three parameters, namely skin erythema, scaling and thickness , which served as a measure to assess the severity of the psoriasis-like lesion severity. Parameters were scored independently on a scale ranging from 0 to 4 as shown in Table 2.
Histological analysis and immunohistochemistry
The skin lesions samples of the mice were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm) were then made and stained with hematoxylin and eosin (H&E) for histological analysis. For immunohistochemical staining, antigen retrieval was conducted with citrate buffer (pH 6.0) followed by treatment with 3.0% H2O2 to quench endogenous peroxidase activity. The sections were incubated overnight at 4 °C with specific primary antibodies against F4/80. The sections were then incubated with biotinylated secondary antibodies for 1 h at room temperature followed by diaminobenzidine staining and hematoxylin counter staining.
Measurements of mRNA expression of inflammatory cytokines via RT-PCR
The mRNA levels of TNF-α, IL-6, IL-8, IL-17, IL-23 and IL-1β were determined by RT-PCR. Total mRNA was isolated from mouse skin tissue or cells using Trizol reagents and mRNA was then reversed transcription to cDNA. The primer sequences were shown in Table 3. The relative mRNA expression levels of cytokines versus GAPDH were measured using an ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, USA).
Western blotting analysis
Total protein from mouse skin samples was acquired with RIPA lysis buffer followed by centrifugation (12, 000 rpm and 15 min) at 4 °C. Equal amounts of proteins from each treatment group were subjected to fractionation by SDS-PAGE and electro-transferred to PVD membranes. The membranes were then blocked with 5% (w/v) skim milk in TBS-T containing 0.1% Tween-20 at room temperature for 2 h and subsequently incubated with primary antibody at 4 °C overnight. Then, the membranes were washed with TBS-T and blotted with the appropriate secondary antibody for 1 h. Finally, the protein bands were detected using the enhanced chemiluminescence (ECL). The band intensity was quantified using Image J software (NIH Image, Bethesda, MD, USA), and GAPDH was used as the loading control.
The data were statistically evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s test, and denoted as means ± standard deviation (SD). Statistically significant differences were identified as either P < 0.05 or P < 0.01. All analyses were carried out by GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA).