DMAG, 3,3'-Di-O-methylellagic acid 4'-glucoside (CAS:51803-68-0, purity ≥ 98%) was obtained from Chengdu Push Bio-technology Co., Ltd (Chengdu, China).
HEL cells were purchased from American Type Culture Collection (Rockville, MA, USA). The cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37◦C in a humidified atmosphere with 5% CO2.
4.0 × 104 HEL cells were seeded in 6-well plates and were treated with DMAG (10, 20 and 40 μM) for 6 days. The cells were harvested and washed with PBS for twice. Then cells were fixed with fixing solution (methanol: glacial acetic acid = 3:1 (v/v)), and stained with Giemsa solution (Solarbio, Beijing, China) for 5 min. The stained cells were finally photographed under electron microscope (10Í).
After treatment for 6 days, cells were harvested for F-actin staining by using phalloidin (Solarbio, Beijing, China) according to the manufacturer’s instructions. In brief, the cells were fixed with 4% paraformaldehyde for 15min and transfused with 0.05% Triton X-100 for 10min at the room temperature. After that, cells were washed twice with PBS and TRITC Phalloidin (Solarbio, Beijing, China) was performed for 30min in dark at room temperature, then add DAPI to counter-stain the nucleus for 5 minutes. Finally, the representative images were captured using the inverted fluorescence microscope (Nikon Ts2R/FL, Japan).
Flow cytometry analysis for megakaryocyte differentiation
After treating with DMAG (10, 20 and 40 μM) for 6 days, HEL cells were harvested and washed with PBS for twice, then labeled with FITC-anti-CD41 (4A Biotech, Beijing, China) and PE-anti-CD61 (BioLegend, USA) antibody on ice in the dark for 30 min. The samples were resuspended in 400 μL PBS for analysis by flow cytometry (BD Biosciences, San Jose, CA, USA).
Megakaryocytes ploidy assay
HEL cells were treated with DMAG (10, 20 and 40 μM) for 6 days and then harvested for DNA ploidy analysis using CycleTEST™ PLUS DNA Reagent Kit (Cycletest Plus DNA Reagent, BD) according to the manufacturer’s instructions. Then, we used the cell cycle analysis module of high-content screening (HCS) to detect the megakaryocytes ploidy again. Briefly, HEL cells were collected and washed twice with PBS, then cells were transferred to a 96-well plate at a density of 2 × 105cells/well. DAPI (100 nM, Solarbio, Beijing, China) was added and incubated at room temperature in the dark for 10 minutes. Lastly, the sample was detected by the ImageXpress Micro4 (Molecular Devices, USA).
Establishment the thrombocytopenia mice model and treatment with DMAG
Specifc-pathogen-free Kunming mice (KM), 8- to 10-week-old, were purchased from Da-shuo Bio-technology Limited (Chengdu, Sichuan, China). The mice were maintained under standard condition (22 ± 2°C, 55 ± 5 % humidity and 12 hours light / dark cycle). All experimental procedures were approved by the laboratory animal ethics committee of the Southwest Medical University (Luzhou, China). Except for the control group, the other mice were irradiated by X-ray (4 Gy) to establish thrombocytopenia mice model. According to the level of peripheral blood, the mice were randomly divided into 4 groups (6 male mice and 6 female mice in each group): control group, model group, TPO positive group, DMAG group. The mice in control group and model group were intraperitoneally administered with normal saline per day. The mice in TPO positive group and DMAG group were intraperitoneally administered with TPO (3000 U/kg) or DMAG (5 mg/kg) per day. On day 0, 3, 7, 10, and 14, the blood were collected from eyes’ venous plexus for hematologic parameters analysis by Hematology Analyzer (SYSMEX XT-1800Iv, Kobe, Japan). On day 10, the femurs were collected and fixed in 10% formaldehyde for 24 hours. After decalcification for a month, the femurs were embedded in paraffin and cut into 5µm thick sections. Then the samples were stained with hematoxylin and eosin (H&E). Images were captured using Olympus BX51microscope (Olympus Optical).
Acquisition of candidate targets of DMAG against thrombocytopenia
PharmMapper (https://lilab.ecust.edu.cn/pharmmapper/index.php) and Swiss database (http://www.swisstargetprediction.ch/index.php) were used to identify targets of DMAG. The GeneCards database (https://www.genecards.org/) and DisGeNET database (http://www.disgenet.org/) were used to retrieve targets related to thrombocytopenia. The common targets of DMAG and thrombocytopenia were considered as potential targets of DAMG against thrombocytopenia. Venn diagram was drawn on Jvenn website (http://jvenn.toulouse.inra.fr/app/example.html) to obtain the overlapped targets of DMAG and thrombocytopenia. The component-target-disease network was constructed by cytoscape_v3.7.1 software.
Construction of protein-protein interaction (PPI) network and identification of core targets of DMAG against thrombocytopenia
PPI network was constructed by STRING database (http://string-db.org) and visualized by Cytoscape_v3.7.1 software. The screening condition of core targets of DMAG was as follows: Degree was greater than or equal to twice the median, Betweenness Centrality (BC) and Closeness Centrality (CC) were greater than or equal to the median.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of core targets
Database for Annotation, Visualization and Integrated Discovery database (DAVID, https://david.ncifcrf.gov/) was used to obtain Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Visualization of GO and KEGG pathway analyses by using GraphPad Prism v126.96.36.199 software and OmicShare website (https://www.omicshare.com/tools/).
Molecular docking simulation and molecular dynamics simulation
Molecular docking simulation was used to explore the binding ability between DMAG and its core targets. The crystal structures of core targets were obtained from the RCSB Protein Data Bank (https://bivi.co/visualisation/rcsb-protein-data-bank). Sybyl-X 2.0 software was used for structural modification of these structures, including residue modification and repair, hydrogenation and charging. The 3D structure of DMAG was constructed based on the PubChem database (https://pubchem.ncbi.nlm.nih.gov), its partial atomic charges were calculated by the Gasteiger Hückel method, energy minimizations were performed using the Tripos force field and the Powell conjugate gradient algorithm convergence criterion of 0.01 kcal/mol Å. After binding pocket was generated using the Protomol generation technique of SYBYL, the molecular docking between DMAG and core proteins were simulated by Sybyl-X 2.0(Ragunathan et al. 2018). Molecular docking simulation was visualized utilizing Pymol and Ligplus software. Molecular dynamics simulation is a widely used tool to explore the dynamic binding of compounds to proteins. We use the DMAG-protein complex obtained by molecular docking to establish a molecular dynamics model using AMBER18 software. Then, the biological macromolecule system was optimized, and the conditions were set as follows: the DMAG-protein complex was dissolved in the TIP3P water model containing H2O, Na+ ions and Cl+ ions, and the temperature was heated to 300 K. After all optimizations are completed, a continuous simulation of 25ns will be performed.
HEL cells were collected after treated with DMAG (10, 20 and 40 μM) for 4 days. Total protein was extracted by RIPA lysis buffer (CST, MA, UAS) supplemented with protease inhibitors (Sigma, St Louis, MO). Protein was quantified with the Quick Start™ Bradford 1 × Dye Protein Assay Reagent (Bio-Rad, CA, USA). An equal amount of protein (30 μg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk powder in phosphate-buffered saline (PBS) for 60 min, the membrane was incubated with primary antibodies overnight at 4 ◦C followed by the HRP-bound secondary antibody for 60 min at 37 ◦C. The protein bands were visualized with ECL Western Blotting detection reagent (4A Biotech Co., Ltd., Beijing, China) and detected by the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The proteins were quantified with ImageJ software. Primary antibodies were as follows: β-actin (CST, MA, USA), ITGB3 (Proteintech, USA), ITGA2B (Proteintech, USA), PLEK (Proteintech, USA), VWF (Proteintech, USA), BCL2 (CST, MA, UAS), BAX (CST, MA, UAS) and TNFα (CST, MA, UAS). Secondary antibodies were as follows: Mouse Anti-rabbit IgG (CST, MA, USA) and Anti-mouse IgG (CST, MA, USA). A β-actin antibody was used as a control.