Eight weeks old BALB/c female inbred mouse strains were used in the experiments, which were purchased from Southern Medical University Experimental Animal Center and maintained in a pathogen-free animal house (approval numbers, CUMS-2018-0062-03). Mice were kept on environment with 12 hour light/dark cycles, water and food were taken randomly.
2.2 IMQ induced psoriasis
Mice were divided into 4 groups (control group, model group and two treatment groups viz., GNY-2.5 g/kg and GNY-5 g/kg groups) with 8 mice in each group. Each mouse from all the groups except the control group received a daily topical dose of 30 mg of imiquimod (5% aldara cream, Mingxinlidi Laboratory, China) on the shaved area in the back for 4 days to induce psoriasis lesions. All the mice applied with imiquimod developed skin inflammation.
2.3 Preparation of GNY decoction
The ingredients of GNY decoction are as follows: Licorice (Glycyrrhiza uralensis Fisch, gan cao), Changbai mountain Ginseng (Panax ginseng C. A. Mey, ren shen), monk fruits (Siraitia grosvenorii, Swingle and C. Jeffrey ex Lu et Z. Y. Zhang, luo han guo), dandelion (Taraxacum mongolicum Hand.-Mazz., pu gong ying), Solomon’s seal (Polygonatum odoratum Mill. Druce, yu zhu), ginger (Zingiber officinale Rosc., gan jiang), and jujube (Ziziphus jujuba Mill., da zao) with a ratio of 12 : 9 : 9 : 12 : 6 : 9 : 6 : 18. The raw herbal medicines were boiled twice for 1 h and then diluted using ddH2O to get the concentrations of 2.5 g/kg and 5 g/kg.
2.4 Treatment and scoring protocols
Mice in the control group and model groups were given double distilled water (ddH2O), while mice in the GNY-2.5 g/kg and GNY-5 g/kg groups received different concentrations of GNY decoction in the drinking water. On day 10, when the manifestations of psoriasis-like lesions in all the groups were subsided, we applied IMQ for the second time and the treatment was continued for 3 more consecutive days (Fig. 1A). We used PASI (Psoriasis area and severity index) scored to monitor psoriasis symptoms as follows: erythema (0–4), scales (0–4) and thickness (0–4) were scored. 0, no symptoms; 1, mild; 2, moderate; 3, severe; 4, very severe. Skin thickness was measured in a specified area using Vernier calipers (Neill-Lavielle, Kentucky, USA).
At the ended of psoriasis period (day 14), mice were put into anesthesia by intramuscularly injection of Zoletil 50 (Virbac, France, 20 mg/kg) and sacrificed by neck broken method. The skin samples were collected for histopathological analysis. For paraffin embedding, tissues were fixed in 4% methanol at room temperature overnight. Before performing standard dehydration and paraffin embedding procedures, tissues were washed in PBS for three times. Skin sample was prepared for paraffin sections (Leica, Solmas, Germany) and tissues were cut (8 µm) using a paraffin slicer. The sections were methanol-fixed and processed for H&E staining by following the steps: deparaffinization, dehydration and staining with hematoxylin solution (Phygene, Shanghai, China) or 0.2% eosin. Images were acquired using an eclipse upright optical microscope digital camera (Nikon Ci-E, Tokyo, Japan). Epidermal thickness was measured by selecting 10 random regions from skin sections of each mouse using 10 × magnification. We used baker’s scores to judge pathological manifestations of psoriasis: Munro's small abscess was found in the stratum corneum, 2.0 point; hyperkeratosis, 0.5 point; hypokeratosis, 1.0 point; granular layer thinning or disappearing in the epidermis 1.0 point; the spinous layer thickening, 1.0 point; skin protuberances and undulations, 0.5. 1.0 and 1.5 points according to the severity; infiltration of mononuclear or multinucleated cells in the dermis according to the severity, 0.5, 1.0 and 1.5 points; 0.5 point for the upper mastoid, and 0.5 point for telangiectasia.
2.6 Immunohistochemistry and Immunofluorescence
Skin samples were harvested at the end of psoriasis period (day 14) and 5 mice from each group were used. Tissues were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek Inc, California, USA), snap-frozen overnight at -80℃. Before embedding, skin samples were dehydrated with buffer containing 30% sucrose in PBS overnight and with 30% sucrose-PBS:OCT in a ratio of 1:1 gradually for 4 h. The tissues were sectioned (7 µm) using a cryostat and frozen at -20℃ until used. For immunohistochemistry, the frozen sections were dehydrated with different concentrations of ethanol and fixed in acetone for 10 min and, 3% hydrogen peroxide and 5% goat serum were used as blocking agents. Inflammatory cells were stained with biotin-anti-mouse Ly6C/6G, CD11c from Biolegend (California, USA). Streptavidin-HRP or goat anti rabbit IgG (Yeasen, Shanghai, China) was used as secondary reagent. Sections were developed with DAB (Vector Laboratories, California, USA) solution and counter stained with hematoxylin before visualization under the microscope (Leica, Solmas, Germany).
For immunofluorescence, skin samples were embeded and sliced similarly as described above. Then the sections were fixed with acetone on ice for 10 min and immersed in PBS for 15 min. PBST containing 2% BSA (Biofroxx, Karlsruhe, Germany) was used for blocking for 45 min. Slides were incubated with rabbit mAb to TGF-β (Affinity Biosciences, Jiangsu, China) or RoR γt (Abcam, Cambridge, UK) for 60 min at room temperature. Then fluorescent labelled anti-rabbit secondary antibodies were added and incubated for 60 min at room temperature (Biotime, Nanjing, China). DAPI (Biotime) solution was used for staining the nuclei. After each step, slides were washed with PBST for 3 times with an incubation period of 5 min in the solution every time. Pictures were taken using a confocal microscope (Cannon, Tokyo, Japan).
2.7 Flow cytometry
To explore the levels of Th17 and Treg cells in mice from each group, spleen cells were harvested at the end of the experimental period. Single cells were prepared by grinding and incubated with antibodies against specific cell surface markers on ice for 30 min in dark. The following antibodies were used: Anti-mouse CD4+-APC, CD4+-PE, CD25+-APC, Foxp3+-BV570, IL-17A+-PE (BD Biosciences, New Jersey, USA). Acuri C6 or LSR II (BD Biosciences) was used to acquire data and analysis was done using Flow Jo software version 7.6 (Tree Star, San Carlos, USA).
2.8 RNA isolation and RT-qPCR
Total RNA was isolated from the skin using Trizol (Invitrogen, California, USA) extraction procedure, and dissolved in RNAse-free RQ-DNAse water (Promega, Madison, WI). The mRNA samples were reverse-transcribed with the PrimeScript RT reagent Kit with gDNA Eraser (Thermo Scientific, Cambridge, UK) by following manufacturer’s instructions. Each qRT-PCR reaction was performed using SYBR Premix Ex Taq II Tli RNaseH Plus (Takara biotech, Osaka, Japan) using a LightCycler 96 (Roche, Basel, Switzerland) thermocycler. β-actin was used for quantitative control. Transcript levels were calculated relative to control and the relative fold inductions were calculated using the 2– ΔΔCt algorithm. The primer sequences for different genes used are given below:
β-actin (ACCGTGAAAAGATGACCCAG, GTACGACCAGAGGCATACAG)
TNF-α (ACGCTCTTCTGTCTACTGAACT, ATCTGAGTGTGAGGGTCTGG)
IL-10 (CTGCTAACCGACTCCTTAATGC, GCTCCACTGCCTTGCTCTTAT)
IL-17F (GTCAGGAAGACAGCACCA,AGCCAACTTTTAGGAGCA) ,
IL-23-P19 (AGCAACTTCACACCTCCCTAC,ACTGCTGACTAGAACTCAGGC )
The experiments were repeated three times to confirm the results.
2.9 Statistical analysis
The data were analyzed using GraphPad Prism 8 software and the results are presented as mean ± SEM. Two-tailed unpaired Student’s t test was used for comparisons between 2 groups, and one-way analysis of variance with Bonferroni or Newman–Keuls correction was used for multiple comparisons. Probability values ≤ 0.05 were considered as significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001 and NS, not significant. Error bars depict SEM with a 95% confidence interval.