Herb materials

Coptidis Rhizoma (C. chinensis Franch.), Phellodendri Chinensis Cortex (P. chinense Schneid.), Angelicae Sinensis Radix (A. sinensis (Oliv.) Diels), Rehmanniae Radix (R. glutinosa Libosch.), Curcumae Longae Rhizoma (C. longa L.), and sesame oil (Sesamum indicum L.) were purchased from Jilin Xiancao Medicine Co., Ltd. Identification and authentication of the herb materials were carried out at Changchun University of Chinese Medicine, and voucher specimens were deposited at the Jilin Ginseng Academy, Changchun University of Chinese Medicine, Jilin, China.

Chemicals and reagents

Rabbit anti-ERK1 + ERK2 antibody (bs-0022R), rabbit anti-phospho-ERK1/2 antibody (bs-3016R), rabbit anti-p38 MAPK antibody (bs-0637R), rabbit anti-Phospho-p38 MAPK (Thr180 + Tyr182) antibody (bs-0636R), rabbit anti-JNK1 + JNK2 + JNK3 (bs-2592R) and rabbit anti-phospho-JNK1 + JNK2 + JNK3 (T183 + T183 + T221) (bs-4163R) antibody were obtained from Beijing Biosynthesis Biotechnology Co., Ltd. Compound dexamethasone acetate cream was obtained from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd. Curcumin, sesamin, berberine, coptisine hydrochloride, phellodendrine, and ferulic acid standards with a purity greater than 98% were obtained from the China National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Assay kits for IgE, IL-4, tumor necrosis factor (TNF)-α, IL-6, IL-2, and interferon (INF)-γ were purchased from RD, USA.

Animals

BALB/c female mice (18–22 g) were purchased from the Hongda Biotechnology Co., Ltd, Changchun, Jilin, China (Certificate: No. SCXK (Liao) 2015–0001) and acclimatized for 7 days. They were kept under controlled ambient temperature (24 ± 2 °C) and humidity (60 ± 10%) with a 12-h light–dark cycle. Water was available ad libitum. The animal protocol was approved by the Animal Care Ethics Committee of Changchun University of Chinese Medicine (Changchun, Jilin, China, Approval No. 20181011).

Preparation of CTCMDO and detection solution

The prescription of CTCMDO was composed of five herbs including Coptidis Rhizoma (4.5 g), Phellodendri Chinensis Cortex (4.5 g), Angelicae Sinensis Radix (7.5 g), Rehmanniae Radix (15 g), and Curcumae Longae Rhizoma (4.5 g). The above five herbs were ground to pieces and mixed together. The mixture of these herbal medicines was extracted with sesame oil (180 g) for 45 min at 120 °C, and removed the herb residue via hot filtration. The hot oil extract mixed with beeswax (Apis cerana Fabricius) according to the weight ratio of 3:1, stirred to completely dissolved, and then cooled down at room temperature to obtain the CTCMDO.

12 g of CTCMDO were accurately weighed and placed into a Soxhlet extractor. Petroleum ether was added and refluxed until the solution was colorless. Then replaced petroleum ether with equal amount of methanol, refluxed until methanol was colorless, then collected methanol extract. The methanol extract was dried under Rotavapor R-100 (BUCHI, Switzerland), then dissolved extract in 2 mL methanol (HPLC grade) and filtered through a 0.22 mm-membrane before detection.

UHPLC‑MS/MS for the identification of ingredients in CTCMDO

Chromatographic analysis was performed using an UltiMate 3000 RS (Thermo Scientific, Sunnyvale, CA, USA) system equipped with a quaternary pump, vacuum degasser, auto sampler, and diode array detector. The mobile phase was 1% chromatographic formic acid aqueous solution (A) and 0.1% formic acid acetonitrile (B). The detailed mobile phase procedure is shown in Table 1. The chromatographic column was a C18 column (150 × 2.1 mm 1.8 µm) with a temperature of 35 °C, the injection volume was 10 μL, and the flow rate was 0.3 mL/min.

Table 1 Gradient elution program table

The Q-Exactive high-resolution mass spectrometer (Thermo Scientific, Sunnyvale, CA, USA) 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. The automatic MS2 mode of the mass spectrometer was selected to analyze the sample. The following operating parameters were used: spray voltage: 3.8 kV (positive), capillary temperature: 300 °C, collision gas: high-purity argon (purity ≥ 99.999%), sheath gas: nitrogen (purity ≥ 99.999%), 40 Arb, auxiliary gas (Aux gus heater temp): nitrogen (purity ≥ 99.999%), 350 °C, data collection time: 30.0 min, scan range: 150.0–2000.0 m/z. The recorded data were processed using the applied Thermo Xcalibur software system.

Establishment of the CTCMDO fingerprint

High-performance liquid chromatography (HPLC) analysis of the CTCMDO sample solution was performed on an Agilent 1260 Infinity HPLC system (Agilent, Santa Clara, CA, USA) equipped with a UV detector. Chromatographic separation was conducted on a Sepax Bio-C18 column (4.6 × 250 mm, 5 μm, from Sepax Technologies, Delaware, USA). The solvent system was composed of solvent A (acetonitrile) and solvent B (0.1% phosphoric acid in water) (Table 2). The operating conditions were as follows: detection wavelength, 278 nm, flow rate, 1.0 mL/min, column temperature, 30 °C, injection volume, 20 μL. Ten batches of CTCMDO were prepared and measured separately to identify the phytochemical constituents.

Table 2 Gradient elution schedule

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 ligand and receptor. 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. Four targets with the highest degree value were selected, and they were imported into the System Dock Web Site server and then molecular docked with the active ingredients of CTCMDO. The results were obtained and evaluated by analysis of the docking score [22, 23].

Preparation of CTCMDO in different administration groups

According to the previous description in ‘Preparation of CTCMDO and detection solution’, a half-dose prescription, a single-dose prescription and a double-dose prescription were taken into Sesame oil (180 g), respectively. Subsequently prepared ointment according to ‘Preparation of CTCMDO and detection solution’, CTCMDO-L (low-dose group, specifications of CTCMDO: 75 mg/g, crude drug/ointment), CTCMDO-M (medium-dose group, specifications of CTCMDO: 150 mg/g, crude drug/ointment), CTCMDO-H (high-dose group, specifications of CTCMDO: 300 mg/g, crude drug/ointment) were obtained, respectively. In the following animal experiments, the ointment was prepared on the basis of daily application dose for future use.

Animal’s models and treatment

The establishment of the DNCB-induced AD model mice was based on previous publication with minor modification [24]. Mice (n = 10) were divided into the following 6 groups: NOR group (the normal group, free access to food and water without other treatment), DNCB (DNCB-sensitized group as a negative control group), DEX group (Compound Dexamethasone Acetate Cream group as a positive control group), CTCMDO-L, -M and -H group. Except the NOR group, the back skin area (2 cm × 2 cm) of the mouse was shaved with a tool, and the residual hair was removed by applying depilatory cream. from day -7 to day -4, 100 μL of 1% DNCB solution (dissolved in a 4:1 mixture of acetone and olive oil) for sensitization (for 3 days), then the mice were observed without any further treatment (for 4 days), from day 0 to day 18, all administration groups were treated with back application therapy, separately. The application dose of DEX and CTCMDO group in each administration group was 0.1 g/cm2 (ointment/back skin area, the ointment completely covered the wound).

Determination of ear swelling

On the 17th day of administration treatment, the mice in each administration group were sensitized for the second time, 100 μL of 0.5% DNCB solution was applied to the ear skin. After 24 h, the mice were euthanized, the left and right ear biopsies were collected (about 8 mm) by a puncher and quickly weighed on an analytical balance, and the difference in weight between the ears was used as the swelling degree.

ELISA for detection of the levels of cytokines (INF-γ, TNF-α, IL-6, IL-2, IL-4, IgE)

The total serum samples were collected 24 h after DNCB application on day 18, and the serum levels of INF-γ, TNF-α, IL-6, IL-4, IgE, and IL-2 were detected using mouse ELISA kits in accordance with the manufacturer’s instruction. Quantitation was done with an ELISA reader using 450 nm filters. Cytokine concentrations were calculated using a linear regression equation obtained from the standard absorbance values.

Histological analysis

After the back skin was fixed in 10% formalin for 24 h, it was dehydrated and then embedded in paraffin and sliced (4-micron thickness). The sections of back skin were stained with hematoxylin and eosin (H&E) and observed under an optical microscope. The thickness of the epidermis and dermis and the number of inflammatory cells were measured using the Leica Application Suite. The magnification was × 200.

Immunohistochemistry

The skin sections for immunohistochemistry were processed in the same manner as those for histological analysis. After deparaffinization and rehydration, the skin sections were boiled in 10 mM sodium citrate buffer for antigen retrieval. The 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 nonspecific binding. After blocking, the sections were incubated with antibodies against SP overnight at 4 °C. After washing with PBS, the sections were incubated with secondary anti-goat antibody for 1 h at room temperature. The 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 magnification was × 200.

qRT-PCR analysis

After administration of CTCMDO for 17 days, the expression of p38 MAPK mRNA in mouse skin tissue was detected, and the total RNA of the mouse skin tissue was extracted by the TRIzol method for RT-PCR amplification. The p38 MAPK upstream primer was 5′-GATTGAGAT-GATTTTGGAG-3′ and downstream primer 5′-GTATGTTAAG-TATATGATTG-3′, and the amplified fragment was 482 bp. The PCR reaction conditions were as follows: 45 s at 94 °C, 50 s at 55 °C, 75 s at 72 °C, after 40 cycles to obtain the p38 MAPK mRNA cycle threshold (Ct), used ΔΔCt (ΔΔCt = Ct target gene − ΔCt internal reference gene) analysis and calculated the relative expression of the 2−ΔΔCt target gene.

Western blot analysis

The skin tissues of mice in each group were accurately weighed, and cut into small pieces with scissors, and extracted the protein from the tissues in accordance with the kit instructions to obtain the total protein of each group. A BCA kit was used to determine the protein concentration. Following 10% SDS-PAGE gel electrophoresis, the PVDF membrane was placed in 5% skimmed milk powder blocking solution for 1 h and washed three times with TBST, 5 min each time. Next, The primary antibodies (p65, p-p65, ERK, p-ERK, JNK, p-JNK, p38, and p-p38 primary antibodies diluted 1:1000) were diluted and incubated them at room temperature for 1 h. Then, washed the membrane by TBST for three times, 5 min each time, next, added diluted horseradish peroxidase-labeled secondary antibody for 1 h, washed the membrane three times, 5 min each time, followed by analysis using the Chemi Doc XRS system (Bio-Rad, Richmond, CA, USA).

Statistical analysis

All of the data are expressed as the mean and standard deviation and represent three independent experiments. Statistical analyses were performed using SPSS 13.0. Treatment effects were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. A value of P < 0.05 was used to indicate statistically significant differences.

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