2.1 Identification of components of CTCMDO
2.1.1Sample processing
Precisely weigh 12g of CTCMDO, wrap it with filter paper, and place it in a Soxhlet extractor. Add appropriate amount of petroleum ether and heat until the petroleum ether is colorless. After discarding the petroleum ether, add an appropriate amount of methanol solution and reflux until the methanol is colorless. Pour out the extract, evaporate the solvent, add 2 ml of methanol to dissolve, filter through a 0.22 μm microporous membrane, and take the additional filtrate to obtain.
2.1.2 Chromatographic and Mass spectrometry conditions
Chromatographic analysis was performed in an UltiMate 3000 RS system equipped with a quaternary pump, va cuum degasser, autosampler and diode array detector. The mobile phase is 1% chromatographic formic acid aqueous solution (A)-0.1% formic acid acetonitrile (B). The detailed mobile phase procedure is shown in Table 1; the chromatographic column uses a C18 column (150×2.1mm 1.8um) with a temperature of 35℃; The injection volume is 10μl, and the flow rate is 0.3ml/min.
The Q Exactive high-resolution mass spectrometer equipped with an electrospray ionization source was used in the positive ion mode and the negative ion mode, controlled by the Thermo Fisher software system. Select the automatic MS2 mode of the mass spectrometer to analyze the sample. Use the following operating parameters: Spary Voltage: 3.8kV (Positive), Capillary Temperature: 300℃, Collision gas: High purity argon (purity ≥99.999%) Sheath gas: Nitrogen (purity≥99.999%), 40Arb, auxiliary gas (Aux gus heater temp): nitrogen (purity≥99.999%), 350℃ data collection time: 30.0min scan range: 150.0~2000.0m/z. The recorded data is processed by the applied Thermo Xcalibur software system.
2.1.3 Establishment of CTCMDO fingerprint
High performance liquid chromatography (HPLC) analysis for CTCMDO sample solution was performed on Agilent 1260 Infinity HPLC system equipped with a UV detector. Chromatographic separation was conducted on Sepax Bio-C18 column (4.6×250mm, 5μm, from Sepax Technologies, Delaware, USA). The solvent system composed of solvent A (acetonitrile) and solvent B (0.1% phosphoric acid in water) in the following gradient (Table 2). Operating conditions were as follows: detection wavelength, 278nm; flow rate, 1.0ml/min; column temperature, 30°C; injection volume, 20μl. 14 batches of CTCMDO were prepared and measured separately to identify phytochemical constituents.
2.2 Docking Procedure.
Molecular docking is a method of placing the ligand in the binding area of the receptor through computer simulation and calculating its physical and chemical parameters to predict the binding affinity and conformation of the two. The System Dock Web Site server (http://systemsdock.unit.oist.jp/iddp/home/index) evaluates the ligand-receptor binding potential through the molecular docking function of the Docking score. The four targets with the highest degree value were selected, and they were imported into the System Dock Web Site server, and molecular docked with the active ingredients of CTCMDO. The results were obtained and evaluated by analyzing the value of Docking Score (Xing Lvet al,.2020,Yanjiao Qi et al,2021).
2.3 Experimental validation
2.3.1 Preparation of CTCMDO
Take Coptischinensis , Phellodendron, Angelica, Rehmanniaglutinosa and Curcuma by prescription Put them in 360g sesame oil, fry at 120°C for 45min, Hot filtration, get medicated oil (The medicinal oil will be used for composition analysis), add beeswax at a ratio of 3:1 (medicinal: beeswax) and keep stirring until the beeswax is completely dissolved, After all the beeswax is dissolved, stop heating and keep stirring to room temperature and get CTCMDO.
2.3.2 Experimental procedures
Mice (n=10) were divided into 6 groups: NOR (blank group), DNCB (DNCB sensitized group as negative control group), and DEX (compound dexamethasone acetate treated group as positive control group), LOW (low dose group, Specifications of CTCMDO: 75mg/g, crude drug/ointment), MED (medium dose group, Specifications of CTCMDO: 150mg/g, crude drug/ointment), HIGH (high dose group, Specifications of CTCMDO: 75mg/g, crude drug/ointment).
The hair in the area of the back skin was shaved 1 day before the experiment, and the shaved back skin was treated with an application of 100μL 1% DNCB solution (dissolved in a 4:1 mixture of acetone and olive oil) for sensitization (for 3 days). The same volume of acetone/olive oil carrier was applied to the blank group. After the first induction, the mice were housed without any further treatment (for 4 days), in the secondary sensitization, 100μL of 0.5%DNCB solution was applied to the ear skin. The positive control group and the drug-administered group were treated with DEX and CTCMDO 4 hours before the second sensitization of DNCB, while the blank group and the model group were treated with blank matrix.
2.3.3 Determination of ear swelling
After stopping the drug, the mice were sacrificed, the left and right ears (about 8 mm) were removed with a puncher and quickly weighed on the analytical balance, and the difference in weight between the ears was used as the swelling degree.
2.3.4 ELISA detects the levels of cytokines (INF-γ, TNF-α, IL-6, IL-2, IL-4, IgE)
The total serum samples (n=5) were collected at 24h after DNCB application on day 19, and the level of INF-γ, TNF-α, IL-6, IL-4, IgE and IL-2 in serum were detected by the mouse Enzyme-Linked Immunosorbent Assay (ELISA) Kits. ELISA was performed according to the manufacturer’s instruction and quantitation was done with ELISA reader using 450nm filters. Cytokine concentrations were calculated using a linear regression equation obtained from standard absorbance values.
2.3.5 Histological analysis
After fixing the back skin in 10% formalin for 24 hours, it was dehydrated and then embedded in paraffin and sliced (4 microns thick). The sections of back skin were stained with Hematoxylin and Eosin (H&E) and observed by optical microscopy Thicknesses of the epidermis, dermis, and the number of inflammatory cells were measured using Leica Application Suite. The magnifification was×200.
2.3.6 Immunohistochemistry
The skin sections for immunohistochemistry were carried out in the same manner as histological analysis. After the deparaffifinization and rehydration, the skin sections were boiled in 10 mM sodium citrate buffer for antigen retrieval. Slides were treated with 3% H2O2 for 30 min to reduce endogenous peroxidase and with normal goat serum (in PBS with 5% NHS, 5% fetal bovine serum, 2% bovine serum albumin (BSA), 0.1% Triton X-100) to minimize nonspecifific binding. After blocking, the sections were incubated with antibodies against substance P overnight at 4℃. After washing with PBS, the sections were incubated with secondary anti-goat antibody for 1 h at RT. Sections were then stained using the Avidin/Biotinylated Enzyme Complex (ABC) Kit and the substrate chromogen mixture was prepared immediately before use. Immunoreactivity was viewed with an LAS. The magnifification was · 200.
2.3.7qRT-PCR method to detect the mRNA expression of p-p38 and p38 protein in mouse skin tissue
After administration for 11 days, the expression of p38MAPK mRNA in mouse skin tissue was detected, and the total RNA of mouse skin tissue was extracted by Trizol method for RT-PCR amplification, p38MAPK upstream primer 5'-GATTGAGAT-GATTTTGGAG-3', downstream primer 5'-GTATGTTAAG -TATATGATTG-3', the amplified fragment is 482bp; PCR reaction conditions: 45 s at 94 ℃, 50 s at 55 ℃, 75 s at 72 ℃, after 40 cycles to obtain p38MAPKmRNA cycle threshold (Ct), use ΔΔCt(ΔΔCt = Ct target gene-ΔCt internal reference gene) analysis, calculate the relative expression of 2-ΔΔCt target gene.
2.3.8 Western Blot analysis
Take the weighed skin tissues of each group of mice, cut them into small pieces with scissors, and extract the protein from the tissues according to the kit instructions to obtain the total protein of each group. Use the BCA kit to determine the protein concentration. Use 10% SDS-PAGE gel electrophoresis transfer membrane, put PVDF membrane into 5% skimmed milk powder blocking solution for 1 h, wash the membrane 3 times with TBST, 5 min each time; add diluted primary antibody (NF-κB, ERK1 /2, phospho-ERK1/2, JNK, phospho-JNK, p38, and phospho-p38 primary antibodies diluted 1:1000) Incubate at room temperature for 1h, wash the membrane with TBST 3 times, 5min each time; add diluted horseradish Peroxidase-labeled secondary antibody, react for 1 hour, wash the membrane 3 times, 5 min each time, and develop.
2.4 Statistical analyses
All data are expressed as mean-standard deviation and represent one of three independent experiments. Statistical analyses were performed using SPSS13.0. Treatment effects were analyzed using one-way analysis of variance followed by Dunnett’s test. A value of P<0.05 was used to indicate statistically significant differences.