Chemicals and reagents
Raw Moutan Cortex pieces were bought from Guangzhou Zhixin Chinese Sliced Herbal Medicine Co. Ltd. (Guangzhou, China) and identified by associate professor Jizhu Liu (School of Traditional Chinese Medicine, Guangdong Pharmaceutical University). Voucher specimens were deposited at the Herbarium Centre, Guangdong Pharmaceutical University. The reference substances, including gallic acid, 5-hydroxymethylfurfural (5-HMF), oxypaeoniflorin, paeoniflorin, benzoic acid, methyl paraben, quercetin, paeonol, 3,8-dihydroxy-2-methylchromone, 3-hydroxy-4-methoxyacetophenone (IS) with purity ≥ 98%, were obtained from Chroma Biotechnology Co. Ltd. (Chengdu, China). Their chemical structures are shown in Fig. 2. Methanol and acetonitrile were HPLC grade (Oceanpak). Purified water was Watsons water. All other reagents used in this study were analytical grade. Nitrogen was purchased from Guangzhou Junduo Gas Company (purity > 99.9%) (Guangzhou, China). ELSA kit: thromboxane B2 (A002774), 6-keto-prostaglandin F1α (A0021984), Human coagulation factor II (FII)(A011227), Human coagulation factor Ⅶ (FⅦ) (A08571), Human coagulation factor X (FX)(A036278), all purchased from Shanghai Fusheng Industrial. RNAex Pro Reagent (AG11728, ACCURATE BIOTECHNOLOGY, HUNAN), EvoM-MLV RT Kit with gDNA Clean for qPCR (AG11728, ACCURATEBIOTECHNOLOGY, HUNAN).
Preparation of Moutan Cortex extract
The RMC pieces were soaked with 95% v/v ethanol at a solid-liquid ratio of 1:8 for 2 h, then reflux extracted for 2 h. After filtering, the residue was extracted again under the same conditions. The twice filtrates were collected and combined, and vacuum concentrated into an alcohol-free extractum. Last, the extractum was dissolved in water (equal to 6 g/ml of RMC) and stored in seal at 4°C.
Pharmacokinetic study
Twenty-four male Sprague-Dawley rats (weighing 300 ± 20 g) were purchased from Animal Experiment Center, Guangdong Academy of Medical Sciences (Certificate no. SCXK2013-0002). The rats were kept in an environmentally controlled breeding room at a temperature of 22 ± 2°C and a relative humidity of 55 ± 10% for 1 week before starting the experiments. They were fed with standard laboratory food and water. Both the animal’s care and the study protocol were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and followed the guidelines of the Ethics Committee of Guangdong Pharmaceutical University.
The rats were randomly divided into following four groups: normal groups (Normal groups), BHS model groups (BHS groups), normal group administrated with RMC water extraction (N-RMC group), BHS group administrated with RMC water extraction (BHS-RMC group), respectively. N-RMC group and BHS-RMC group rats were respectively orally administrated RMC water extraction (equal to 6 g/ml of RMC) for continuous 7 days at a dose of 10 ml/kg, once a day, one hour after the sixth day of administration. The rats in BHS groups and BHS-RMC group were subcutaneously injected with 10% aqueous suspension of yeast (10 ml/kg) and then subcutaneously injected with 0.1 mg/ml adrenaline hydrochloride solution twice (at a dose of 0.8 ml/kg, 4 hours apart) while body temperature remained relatively steady high. Those rats were soaked in ice-water for 5 min keeping their heads outside surface between two subcutaneous injections of adrenaline hydrochloride solution[36, 37]. At the same time, the normal groups and N-RMC group rats were injected with the same dose of saline (10ml/kg) subcutaneously. All rats were fasted for 12 h but free for water before taking the blood. On the seventh day, 0.3 ml blood samples were collected from fossa orbitalis vein at the time point of 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h after oral administration. All blood samples were immediately centrifuged at 12000 r/min for 10 min at 4°C and the serum samples were obtained and stored at -80°C until analysis.
Preparation of serum sample
Firstly, 100 µl serum was spiked with 10µL IS working solution to mix evenly, then 1ml methanol was added to protein precipitation, and centrifuged at 4000 r/min for 10 min at 4°C. Secondly, the supernatant was collected with a clean tube carefully and filtered through 0.22 µm microporous filter head. Thirdly, the filtrate was evaporated to dryness under a stream of nitrogen at 40°C. Finally, the residue was dissolved in 100 µl methanol and 10 µl of aliquot was injected into UHPLC -DAD system for analysis.
Instrument and Chromatographic conditions
The chromatography experiment was carried out using UPLC system (Thermo Fisher Scientific, USA). Chromatographic conditions: column: Acquity UPLC® BEH Shield RP18 Column (2.1mm × 100 mm, 1.7µm, Waters, USA). The column temperature was maintained at 30°C. mobile phase: Acetonitrile (A) and 0.4% (v/v) formic acid in water (B). The gradient conditions: 0–10 min, 5–15% A; 10–20 min, 15–35% A; 20–30 min, 35–95% A; 30–32 min, 95% A. The samples were kept at 4°C in the auto-sampler. flow rate: 0.2 mL/min; sample size: 2 µL; column temperature: 30°C. detection wavelength 254 nm.
Preparation of standards and quality control solutions
Primary stock solution of standards was prepared in methanol separately at concentrations of 2.075 mg/ml (gallic acid), 3.690 mg/ml (5-HMF), 0.640 mg/ml (oxypaeoniflorin), 1.125 mg/ml (3,8-dihydroxy-2-methylchromone), 2.235 mg/ml (paeoniflorin), 3.250 mg/ml (benzoic acid), 0.260 mg/ml (Methyl paraben), 2.385 mg/ml (quercetin), 0.482 mg/ml (paeonol) and 0.460 µg/ml (3-hydroxy-4-methoxyacetophenone, IS), respectively. Working standard solutions were prepared daily. All standard stock and working solutions were stored at 4°C.
Quality control (QC) samples were prepared by the same procedure to attain low, medium and high concentration levels. The concentrations were 186.75, 93.38 and 20.75 µg/ml for gallic acid; 405.90, 110.70 and 18.45 µg/ml for 5-HMF; 6.40, 3.20 and 0.96 µg/ml for oxypaeoniflorin; 129.38, 73.13 and 16.88 µg/ml for 3,8-dihydroxy-2-methylchromone; 145.28, 67.05 and 16.76 µg/ml for paeoniflorin; 243.75, 97.50 and 40.63 µg/ml for benzoic acid; 6.50, 3.90 and 1.30 µg/ml for methyl paraben; 715.50, 357.75 and 107.33 µg/ml for quercetin; 12.05, 7.23 and 2.41 µg/ml for paeonol.
Method validation
The selectivity of the method was evaluated by comparing the chromatograms of blank serum, blank serum spiked with the nine analytes and IS, serum samples of N-RMC and BHS-RMC rats administrated with RMC. Calibration standards of the mixture of nine components were prepared by spiking the standard mixture working solutions into 100 µl blank serum and 100 µl IS. The linearity of calibration curve was constructed by plotting the peak area ratios versus the concentration of nine components. The QC serum samples at three concentration levels were respectively analyzed in five replicates on the same day and on three consecutive validation days to evaluate the intra-day and inter-day precision and accuracy. The accuracy was expressed by the percentage difference between amount spiked and determined while the precision was presented with the relative standard deviation (RSD%). The stability of nine components was evaluated to determine the changes in three different concentrations at low, medium and high levels of QC samples subjected to three different storage conditions. Short-term stability was evaluated by assessing QC samples at the room temperature (25°C) for 24 h. The freeze-thaw stability was inspected by implementing three freeze (-80°C) – thaw (room temperature, 25°C) cycles on consecutive days. Long-term stability was determined by storing at -80°C for 30 days. Extraction recovery was determined by calculating the peak area ratio of QC samples to corresponding post-extracted samples spiked with the analytes and internal standard at three concentrations of low, medium and high levels. The matrix effect was evaluated by comparison of the peak areas of nine components/IS in the post‐extracted spiked samples and standard working solutions at the same concentration. The QC serum samples at three concentration levels were analyzed in five replicates, respectively.
The index of TXB2 and 6-Keto-PGF1 detect
It takes appropriate amount of serum and the levels of TXB2 and 6-Keto-PGFlα in the serum of rats in Normal group, BHS groups and BHS-RMC groups were detected by ELISA kit.
Statistical analysis
The concentrations of nine components of all samples were processed by PKSolver pharmacokinetics software. Statistical comparisons among groups were performed with GraphPad Prism 8 using a One-Way ANOVA analysis. Data expressed as mean ± SD and a P value ≤ 0.05 was considered to be statistically significant.
Network Analysis
Targets for nine compounds: the targets of nine compounds were predicted by
PharmMapper (http://lilab.ecust.edu.cn/pharmmapper/) limited to “Homo sapiens”; also including targets predicted from TCMSP (http://lsp.nwu.edu.cn/tcmspsearch.php). Subsequently, top 300 targets were predicted from the PharmMapper. Among the targets, those with z’-score > 0 was selected as the potential targets for the correlative ingredients. The PharmMapper online tool is a web server for potential drug target identification by reversed pharmacophore matching the query compound against anin-house pharmacophore model database[38].
Disease-related targets: By reviewing the literature on target proteins of traditional Chinese medicine for activating blood and cooling blood, it combined with RMC medicinal properties (bitter, acrid, slightly cold, homeward liver, kidney meridian), screen anti-inflammatory, improve hemodynamics, anti-coagulation, anti-platelet aggregation from TTD database equal to disease-related target protein. Finally, it locked the target proteins that may be related to activating blood and cooling blood[39–47].
PPI analysis
Screen for common targets at the crossover of compounds and diseases and import into the STRING database (https://string-db.org/, version 11.0), selecting "Homo sapiens" as the species source. The protein-protein interactions (PPIs) of proteins common to compounds and diseases targets were analyzed by String at the high confidence. The purpose of PPI was to get relevant targets, and to remove targets with low confidence.
Gene pathway analysis
Based on previous steps, compound-related targets and disease targets were prepared, and the drug-disease crossover genes were screened. Then, the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/home.jsp, ver. 6.8) was used to identify the signal pathways and biological functions related to compound-disease target genes, which were analyzed by KEGG pathway enrichment (p < 0.05) and GO enrichment (p < 0.05).
Network construction
The compounds-target (C-T) and compounds-target -pathway network was built by network analysis software (Cytoscape 3.3.0).
Molecular docking
The molecular structures of the compounds were downloaded from PUBMED
(https://www.ncbi.nlm.nih.gov/) and then imported into Oppen Babel (2.4.1) software to transform the three-dimensional (3D) structure to mol2 file format. Several key targets are extracted from the major hub network, including F2, F7, F10, PLAU, MAPK14, MAPK10, AKT1 and NOS3. The 3D structures of Prothrombin(F2, pdb = 3tu7), Coagulation factor VII (F7, pdb = 5paw), Coagulation factor FX (F10, pdb = 3q3k), Urokinase-type plasminogen activator (PLAU, pdb = 1ejn), Mitogen-activated protein kinase 14 (MAPK14, pdb = 1a9u), Mitogen-activated protein kinase 10 (MAPK10, pdb = 3cgf), threonine-protein kinase(AKT1, pdb = 3ocb) and Nitric oxide synthase( NOS3, pdb = 1maj) were downloaded from the PDB database (https://www.rcsb.org/) (source: Homo sapiens; method: X-ray; resolution:> 1.5; number of ligands:> 1) using Sybyl-7.3 for processing. After removing the water molecules residues, hydrogenation, use the Surflex-Dock program Docking with its own ligand, the score obtained is used as the threshold value of the target. The compounds were docked with the target protein obtained by screening and scored. The target with a score greater than the target threshold (> 5) regarded as the master targets for RMC[48]. Then docking sites were analyzed using Discovery Studio 4.5 software to observe the Interaction between compound and target.
Cell Culture and Treatments
TNF-α was used to induce endothelial factor damage in Human Umbilical Vein Endothelial Cells (HUVEC) leading to inflammation and coagulation dysfunction. HUVECs were maintained in ECM complete growth medium (ECM, 5% FBS, 1%P/S and 1%EGCS). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. After three or four passages, the HUVECs were digested with 0.25% trypsin and seeded in a 6-well plate with a density of 1 × 105 cells/ml for ELISA analysis and RT-qPCR. First, we determined the effects of different doses of drug on the viability of HUVECs cells using the MTT methods(MTT results are shown in Supplementary Fig. 1).The cells used for experiment were divided into the following groups: control group (giving complete growth medium), model group (giving 25 ng/ml TNF-α), paeonol (50 ug/ml + 25 ng/ml TNF-α), paeoniflorin (500 ug/ml + 25ng/ml TNF-α), quercetin (1 ug/ml + 25 ng/ml TNF-α) and oxypaeoniflorin (500 ug/ml + 25 ng/ml TNF-α), for 8 h, respectively. Afterwards, take the supernatant and measure according to the ELISA kit, and detect the OD value at the wavelength of 450. Meanwhile, Total RNAs were extracted according to the manufacturer’s instructions, which were used to generate cDNA using EvoM-MLV RT Kit with gDNA Clean for qPCR. ChamQTMUniversalSYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Q711-02) was performed on a Real-time PCR System. GAPDH was used as an internal control to normalize RNA expression. The primers information used in RT-qPCR were shown in Table 1.
Table 1
The primers used in RT-qPCR were shown in Table 1.
Primer | sequence (5' to 3') |
GAPDH | F : ACGGATTTGGTCGTATTGG |
R : TCCCGTTCTCAGCCTTG |
MAPK14 | F : TTTGCTGGCTCTTGGAAC |
R : CGATCTCCCTGCACCTT |
MAPK10 | F : AGCAATAATCAGGCTTCCC |
R : TCATCAACCATCCACTTCC |
AKT1 | F : TGTGAAGGAGGGTTGGCT |
R : GCGCCACAGAGAAGTTGTT |
F10 | F : CGGCTACGACACCAAGC |
R : ACTTGAGGAAGGCGGTGA |
F2 | F : GCAAGCACCAGGACTTCA |
R : GAGGTCGCAGTACCCAAAG |
F7 | F: CTGAAGGCGGTTGTTTAGC |
R: GGAAGCAGGTGGGGAATA |