Materials andequipment
Low (0.035 g/g,batchNo.20200506), medium (0.07 g/g, batchNo.200401), and high (0.14 g/g, batchNo.20200507) concentrations of CTGO (NEW CICON Pharmaceutical Co., Ltd), Gallic acid (China Institute for Food and Drug Control, content: 91.5%, Batch No. 110831-201906), Gallic acid methyl ester (Chengdu Pufei De Biotech Co., Ltd., content: 98.0%, Batch No. 17092604), Ethyl gallate (Chengdu Pufei De Biotech Co., Ltd., content: 98.0%, Batch No. 19042902), Ellagic acid (China Institute for Food and Drug Control, content: 88.8%, Batch No. 111959-201903), β,β-Dimethylacrylalkannin (China Institute for Food and Drug Control, content: 98.0%, Batch No. 111689-201805), L-shikonin (China Institute for the Control of Pharmaceutical and Biological Products, Batch No. 110769-200506), Methanol and acetonitrile of HPLC grade were purchased from Sigma-Aldrich Company. All other reagents were of analytical grade and supplied by Tianjin Beilian Fine Chemicals Development Co., Ltd. Compound dexamethasone acetate cream (China Resources Sanjiu Medical and Pharmaceutical Co., Ltd., 20201024X), 2, 4-dinitrochlorobenzene (DNCB) (Shanghai Jiudin Chemical Technology Co., Ltd., 97-00-7), IL-4, INF-γ test box (Shanghai Enzyme Linked Biotechnology Co., Ltd., 05/2020, 06/2020), PCR kit (Beijing Quanshijin, AQ601-01), Microscope (Leica, Germany, EQ2016-037), Thermo Fisher, Multiskan FC, and an electronic balance (Germany Sartorius, CPA124S) were obtained.
Experimental animals
SixtySprague-Dawley (SD) rats (180–220 g body weight and aged 5–7 weeks) were purchased from the Laboratory Animal Center ofXinjiang Medical University(License number:SCXK (new) 2018-0002).Allof therats were fed in an environment free of specific pathogens: temperature 20–26°C, humidity 40%–70%, artificial light, 12-h light-dark cycle, and free access to water and food.
Database and analysis software
The following databases were used: TCMSP (http://tcmspw.com/index.php/), CNKI (https://www.cnki.net/),CTD(http://ctdbase.org/),ETCM(http://www.tcmip.cn/ETCM/index.php/Home/Index/),TCMID(http://47.100.169.139:8000/tcmid/),BATMAN-TCM (http://bionet.ncpsb.org.cn/batman-tcm/), GEO (https://www.ncbi.nlm.nih.gov/geo/), DrugBank (https://go.drugbank.com/), STRING (https://string-db.org/),PubChem (https://pubchem.ncbi.nlm.nih.gov/), and PDB (http://www1.rcsb.org/). The softwareCytoscape 3.7.2,R3.6.1 language, Chem3D, PyMOL, AutoDock Vina-1.5.6,and BIOVIA Discovery Studio 2020 were used.
Untargeted UHPLC-Q-Orbitrap MS analysis
Chromatographic separation was performed on aQ-Exactive-Plus (Thermo Fisher Scientific Inc., Waltham, MA, USA)using aKromasil Classic 100-5-C18 column (100 ×4.6 mmi.d., 5 μm) at30°C with a flow rate of 1.0mL/min and awavelength of 300 nm.The mobile phase consisted of water containing 0.1% formic acid (solvent system A) and acetonitrile (solvent system B), andgradient elutionwas conductedas follows: 0–5 min,5% B, 5–45 min, 5%–12% B, 45–50 min, 12% B, 50–80 min, 12–20% B, 80–95 min, 20%–25% B, 95–105 min,25%–67% B, 105–115 min, 67%–70% B, 115–125 min,70%–75% B, 125–135 min, 75% B. Ten microlitresof the sample solution was injected for analysis.
Mass detection was performed using an UHPLC-Q-Orbitrap HRMS equipped with a
Dual ESI source operating in both the positive and negative modes with the following operating parameters: ESI spray voltage, –2.8 kV (positive ion, 3.2 kV), sheath gas, 40 arb, auxiliary gas, 10 arb, air curtain gas (CUR), 35, ion source temperature (heat temp), 350°C, capillary temperature, 300°C, –70 V, focusing voltage (FP), –350 V, and DP2, –10 V. The data dependent acquisition(DDA)mode was adopted for sample analysis: the collection range of q-orbitrap was 100–1500 m/z, and the fragment ion scanning range was 50–1500 m/z. Collision gas (NCE): 20, 40, 60. The mass spectrometry (MS) resolution was 70,000 full width at half maximum (FWHM) (200 m/z), and the MS2 resolution was 17600 FWHM (200 m/z). The Xcalibur 4.0 software package was used for data acquisition and analysis (Thermo Fisher Scientific Inc., Waltham, MA, USA).
To prepare the sample, we first weighed 0.05 gofTurkish galls beforeplacingintoa conical flask. Next, 25 mL of 80% methanol was added before a 1-h reflux in a water bath at 75°C. Then,a pipette was used to transfer 1 mL of the supernatant into a 5-mL volumetric flask. In parallel, 0.1 g of Comfrey was weighed precisely and placed into another conical flask, before adding 50 mLof chloroform and subjecting it toa 1-h reflux in a water bath at 75°C. Next, the sample was filtered, and the solvent was evaporatedinthewater bath. Theresidue was transferred intothe abovementioned 5-mLvolumetric flask with about 4 mL acetonitriletobring the total volume to 5 mL.
Network Analysis
“Turkish galls”and“comfrey”weresearched for inthe TCMSP, CTD, TCMID, ETCM, and BATMAN-TCMdatabases andtheliterature to obtain the components of Turkish galls and comfrey. These agentswere combined with the compounds identified by UHPLC-Q-Orbitrap-HRMS to buildthe active ingredient databaseof CTGO.Targetscorresponding to the componentswere obtainedthrough the TCMSP and BATMAN-TCM databases, and a target database of CTGO was constructed. “Eczema” was searched inGEO and DrugBank databasesto obtain eczema targets. R3.6.1 language software was used to obtain the intersection targets of drugs and diseases.
The intersection targets and corresponding componentsand the top 20 pathways of significance were inputto Cytoscape 3.7.2 software to construct a “component-target-pathway” network, and the size of degree value was reflected by the size of the nodes.All intersection targets were imported into the String data platformto builda protein-protein interaction(PPI)network. The PPI networkwasimportedthe Cytoscape 3.7.2 softwareto build with PPI topology analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the intersection targetsbyR3.6.1 language software, and a threshold q< 0.05 was set. Biological processes and target pathways with significant differences were screened.
Molecular docking
The three core components of gallic acid, ellagic acid, and shikonin in the component-target network were docked with the four core targets of TNF, IL-1β, TLR4, and albumin (Alb) in the PPI network. First, the composition and target files were downloaded from PubChem and PDB, respectively.The componentfile’s energy minimization was completed by Chem3D, and the targetfile’s dewatering and impurity removal were completed by PyMOL.Next, the file was converted to PDBQT format using AutoDock Vina. The active pocket of the target was determined, molecular docking was performed, and binding energy values were obtained.The best docking model diagram was drawn by BIOVIA Discovery Studio.Finally, the binding ability was evaluated by the affinity of the component and the target.An affinity < 0 means that the ligand and the receptor can bind spontaneously, and the lower the affinity, the stronger thebinding effect. An affinity < –5.0 kcal/mol indicatesgood bindingactivity.
Animal experiment
Sixty rats were randomly divided into a normal group, a model group, a positive group (compound dexamethasone acetate cream), a CTGO high-dose group, a CTGO medium-dose group, and a CTGO low-dose group, with 10 rats in each group. Except for the normal group, the rats in the other groups were treated with 7% DNCB solution by rubbing the solution on the back to establish the eczema model. The hair on the neck (A: 2 cm × 2 cm) and back (B: 4 cm × 4 cm) of the animals was removed using an electric shaver. The rats were sensitized by applying 100 μL of 7% DNCB acetone solution with a pipetting gun on the skin of site A. Severe pruritus, frequent scratching, and rolling behaviors were observed in rats, lasting for about 2 h. After 1 week, 200 μL of 7% DNCB acetone solution was applied to site B for excitation. Theratswere stimulated once every 5 days, and severe pruritus could be seen in rats, with frequent scratching and rolling behavior in each stimulation. Erythema, papules, edema, scratches, and desquamation gradually appeared on the skin at site B. After each stimulation, the skin lesions were recorded and scored. After six sessions of stimulation, erythema, papules, scab, and exudation appeared in skin lesions on the back of each rat, at which point the stimulation was stopped, indicating that the model had been successfully established(Miao MS, et al., 2017).
Eachgroup was administered low (0.035 g/g), medium (0.07 g/g), or high (0.14 g/g) concentrations of CTGO, and the positive group was administered compound dexamethasone acetate cream (0.75 mg/g, 0.3 g each time, once a day). The normal and model groups were administered with saline.After smearing the experimental drug,2–3 layers ofmedical gauze were wrapped and fixed with a non-irritating adhesive tape. After 4 h, the covering was removed and washed with warm pure water for continuous administration for 14 days.
After the last administration, the rats were fasted with water for 12 h. The rats were mildly anesthetized with 1% sodium pentobarbital (4 mL/kg, i.p.). Blood samples were collected from the abdominal aorta, whichwere placed in a centrifuge tube for 30 minand centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was stored at –20°C. One part of the obtained skin tissue was immersed in 10% neutral formalin for fixation, and the other part was stored at −80°C.
On the 1st, 5th, 10th, 15th, 20th, 25th, 30th, and 35th days of modeling, and on the 0th, 9th, and 14th days of administration, the grades of erythema, edema, exudation, desquamation, liche-like change, desquamation, and other indexes were evaluatedfrom 1 to 4 grade. After 14 days of administration, Skin conditionwerephotographed, andnaked eye observation was performed.
Skin tissuewasremoved from10% neutral formalinsolution, dehydrated,permeated, paraffin impregnated, embedded, sectioned, stained, and sealed. Histomorphological observation was performed with a microscope, and radiographs were taken.
The levels of IFN-γ and IL-4 in the serum of rats were determined according to the instructions of the ELISA kit.
The mRNA expression levels of TLR4, NF-κB (p65), IL-1β, and TNF-α in skin tissue samples were detected by RT-qPCR. RNA was extracted, its purity was determined, and cDNA was obtained by reverse transcription. PCR upstream and downstream primers were added (2 µL each, refer (Table 1)for specific information) to the PCR reaction system of 20 µL according to the kit instructions. The reaction procedures were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 5s, and annealing at 60°C for 35s, detection was conducted after 45cycles. The 2-△△CT method was used to analyze the difference in target gene expression between the control group and test groups.
Table 1. PCR primer sequence
Gene
|
Sequence
|
β-actin
|
F-CCCATCTATGAGGGTTACGC
|
R-TTTAATGTCACGCACGATTTC
|
TLR4
|
F-TCCTTTCCTGCCTGAGACCA
|
R-TGTCTCAATTTCACACCTGGAT
|
NF-κB
|
F-TGTATTTCACGGGACCTGGC
|
R-CAGGCTAGGGTCAGCGTATG
|
IL-1β
|
F-AAATGCCTCGTGCTGTCTGA
|
R-TTGGGATCCACACTCTCCAG
|
TNF-α
|
F-GTCCCAACAAGGAGGAGAAGTT
|
R-CTCCGCTTGGTGGTTTGCTA
|