Mass Spectrometry Analysis of Gleditsiae Spina
Production of Freeze-dried Powder of the Gleditsiae Spina
Gleditsiae Spina, mainly used as medicine, is obtained from the dry spines of the leguminous Gleditsia sinensis Lam. In this study, Gleditsiae Spina was provided by Beijing Xinglin Pharmaceutical Co. (Beijing, China), and identified by Professor Mao Kechen according to the Chinese Pharmacopoeia standards.
Gleditsiae Spina pieces (200 g) were placed in a vessel and cooked twice for 1 h each time. The two resulting decoctions were mixed to obtained a volume of 450 mL which was transferred to a vacuum freeze dryer (Epsilon 2-4LSC; Martin Christ Gefriertrocknungsanlagen, Harz, Germany) to obtain 18.72 g of freeze-dried powder, with a production rate of 8.28%. The powder was placed into a closed container containing silica gel to keep it dry. The powder was dissolved in deionized water to prepare a 100 mg/mL solution and stored at −20 °C for subsequent experiments.
Identification of the Gleditsiae Spina Components Using High Performance Liquid Chromatography and Mass Spectrometry
Gleditsiae Spina freeze-dried powder (100 mg) was dissolved in 50% methanol and sonicated for 10 min. The solution was centrifuged at 3,000 r/min for 5 min (Beckman Coulter, Brea, CA, USA), the supernatant was collected and filtered through a 0.22 μm filter (Millipore, Burlington, MA, USA). The filtered solution was subjected to mass spectrometry analysis using a Q-Exactive Orbitrap quadrupole-electrostatic field orbitrap mass spectrometer equipped with a thermal spray ion source and a Vanquish ultra-high performance liquid chromatography system (Thermo Fisher Scientific, Waltham, MA, USA).
Target Prediction
Prediction of Active Components in Gleditsiae Spina
Based on the mass spectrometry results of Gleditsiae Spina, the active ingredients were initially selected. The molecular structure of the active pharmaceutical ingredients and 3Dsdf were obtained on the Pubchem platform. The 3Dsdf files of the active ingredients were uploaded to the SwissADME platform, and the pharmacokinetics and drug similarity were screened. The compound ingredients were required to have good gastrointestinal absorption, and the drug similarity received more than 3 positive evaluations. The potential targets of the ingredients were predicted on the PharmMapper platform (19).We required the norm fit value to be greater than 0.75。
Construction of the Active Ingredient-Target Network of the Gleditsiae Spina
The “active ingredient-target” network of the Gleditsiae Spina was constructed and analyzed by Cytoscape 3.7.2 software (20). "node" was used to indicate the component or target, and "edge" was used to indicate the relationship among them. The “Network Analyzer” analyzing tool built in Cytoscape 3.7.2 software was used to analyze the network characteristics, including Degree, Betweeness, and Closeness, to study important components and target relationships of Gleditsiae Spina.
Ovarian Cancer-Related Targets
The differentially expressed genes of patients with ovarian cancer were obtained from GEO database (Series: GSE54388 and GSE14407, Samples: normal tissue GSM1314222-GSM1314227,GSM360039-360049, GSM359984 and tumor tissue GSM1314228-GSM1314243, GSM359972-359983). Differential Genes with an adj. P-value < 0.05 and |log2(fold change)|>1 were considered to be of significantly differential expression and ovarian cancer-related targets.
Construction of protein interaction network and screening of key targets
The PPI were constructed by BisoGenet3.0 (21). The targets related to the active ingredients of Gleditsiae Spina, and the targets of disease were introduced into BisoGenet, each generated a PPI network. The intersection network of the two PPI networks was extracted through the Merge function in Cytoscape, and CytoNCA2.1 (22) was used to analyse the nodes of the intersection network. The targets were mapped and visualized by Cytoscape 3.7.2 and the protein-protein internetwork (PPI) of the shared genes were constructed through the String APP in Cytoscape.
Pathway enrichment analysis
The Metascape platform (23) was used to perform pathway enrichment analysis on the target. The platform integrates many authoritative functional databases such as GO, KEGG, etc., and supports batch genes or annotates, enriches and analyses proteins and builds PPI networks. The platform is updated once a month to ensure data is reliable. Imported potential ovarian cancer targets were inserted into the Cytoscape platform for GO and KEGG analysis, the results were saved and visualized with R software3.6.1.
Molecular Docking and Molecular Dynamics Simulation
In order to further determine the credibility of the relationship between the ovarian cancer target and the core components of Gleditsiae Spina, the top two compounds of traditional Chinese medicine-compound-target were selected as ligands genistein and luteolin, and four important targets were selected to analyse molecularly docking.
First, the crystal structure of the three proteins in pdb format from the RCSB database was downloaded, and the SDF from the PubChem database. We also downloaded the 3D chemical structure of the candidate compound and used Open Babel 2.4 to convert to the pdb format file. AutoDock Tools 1.5.6 was used to delete the water molecules in the ligand, separate the ligand from the receptor, add non-polar hydrogen, calculate the Gasteiger charge, and save the pdbqt format file. The selected potential core constituent ligands were subjected to energy minimization treatment, the ligand atom type was given, the charge was calculated, and stored in pdbqt format. Molecular docking operations were performed using Autodock Vina 1.1.2, and reflect the matching degree and docking activity between the target and the ligand through the docking score value, where we believe that a docking score > 4.25 means that the ligand and the target have binding activity, a score > 5.0 means good matching activity, and a score > 7.0 means strong docking activity (24).
The MD simulation of docked complexes were carried out using Desmond version 2020. Here, OPLS3e force field was used to initiate the MD simulation, and the system was solvated using TIP3 water model. The neutralization of the system was performed by adding counter ions. Energy minimization of the entire system was performed using OPLS3e, as it is an all-atom type force field. The geometry of water molecules, the bond lengths and the bond angles of heavy atoms was restrained using the SHAKE algorithm. Simulation of the continuous system was executed by applying periodic boundary conditions and long-range electrostatics was maintained by the particle mesh Ewald method. The equilibration of the system was done using NPT ensemble with temperature at 300 k and pressure at 1.0 bar. The coupling of temperature-pressure parameters was done using the Berendsen coupling algorithm. On post-preparation of the system, the production run was performed for 200 ns with a time step of 1.2 fs and trajectory recording was done for every 200 ps summing up to the recording of 10,00 frames. The calculation of the RMSD (Root mean square deviation) was done for the backbone atoms and was analyzed graphically to understand the nature of protein-ligand interactions. RMSF (Root Mean Square Fluctuation) for every residue was calculated to understand the major conformational changes in the residues in comparison between the initial state and dynamics state.
Cell Verification
Cells and cell proliferation
Human ovarian cancer cell line A2780 purchased from ATCC was used in this study. A2780 cells were cultured in RPMI-1640 (Gibco Company, USA) with 10% foetal bovine serum (Gibco Company, USA) and 1% penicillin-streptomycin (Gibco; 10000 units/mL Penicillin, 10000 μg/mL Streptomycin) and cultured at 37°C in a humidified atmosphere under 5% CO2. Cells used in this experiment were in the exponential growth phase.
For proliferation tests, The MTT was used to evaluate cell proliferation.3000 cells were seeded in each of the non-edge well of 96-well plates. Freeze-dried powder of the Gleditsiae Spina was added after the cells adhered to the wall. The freeze-dried powder of maximum freezing 20 mg/mL degree was diluted into the cells by multiple dilutions. After 24 hours, 20 𝜇L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) solution (5 mg/mL in phosphate-buffered saline (PBS)) was added to the culture medium in each well at a final concentration of 0.5 mg/mL and the cells were incubated at 37 °C for 4 hours. The supernatants were replaced with 150 𝜇L of dimethyl sulfoxide (Sigma, St. Louis, MO, USA). Then the 96-well plates were measured by Microplate reader at 490 nm.
Western blot
After exposure to the test compounds (1.25mg/mL, 2.5 mg/mL Freeze-dried powder of the Gleditsiae Spina) for 24 h, the A2780 cells were harvested and lysed with RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) containing Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA, USA). A2780 cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected. The protein concentration was determined using the Pierce BCA Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts (30 μg/lane) of total protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA (Amresco, Solon, OH, USA) at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-HPSE1(1:1000, CST,USA), anti-MMP9(1:1000, CST,USA), anti-β-Catenin(1:1000, CST, USA), anti-N-cad (1:1000 CST, USA), anti-E-cad(1:1000 CST, USA),anti-PI3K/p-PI3K (1:1000, CST,USA), anti-AKT/p-AKT (1:1000, CST,USA), anti- YAP/TAZ(1:1000, CST,USA)and β-actin (1:10000, CST,USA). After washing the membranes in Tris-buffered saline with 0.1% Tween-20 (TBST), the membranes were probed with secondary antibodies (1:10,000) for 1 h at room temperature. The signals were detected using an Odyssey Infrared Imaging System (Li-cor Biosciences, Lincoln, NE, USA). The relative density of the protein bands was measured by Odyssey version 3.0 software (LI-COR Biosciences). Each experiments were repeated three times. The ratios of the protein band intensities relative to that of β-Actin were calculated for each sample using Image J.