Cortex mori active ingredients screened
The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP http://lsp.nwu.edu.cn/) and Analysis Platform was searched using the keywords ‘cortex mori’ and related literature was reviewed for all chemical ingredients. The related compounds were identified through a screening of active compounds with oral bioavailability (OB)>30% and drug likeness (DL)>0.18 (Liu et al. 2013, Wang et al. 2017). OB is primarily a measure of how much the drug is absorbed into the body circulation, and the DL value is primarily used to determine how similar a compound is to known drugs, and these two sources provide a valuable reference for active ingredient analysis (Zhang et al. 2016, Xu et al. 2012). To improve accuracy and uniformity, target information and gene name standardization are compared in the UniProt database (https://www.uniprot.org/help/uniprotkb).
Acquisition of target sites of cortex mori
Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, http://tcmspw.com/tcmsp.php) and Analysis Platform was searched for the keywords ‘cortex mori’ to find all effect targets. For target gene selection, chose ‘Related Targets’ from the results obtained with Perl-5.32.0.1 software (https://www.perl.org/get.html).
Acquisition of disease-related genes
On the basis of the Online Mendelian Inheritance in Man (OMIM, https://www.omim.org) and Human Gene Database (GeneCards, https://www.genecaed.org), we identified a number of gene targets linked to lung cancer. A Venn diagram was drawn using the information obtained from UniProt (https://www.uniprot.org/help/uniprotkb), including their names, functions, symbols, and gene IDs (Wu et al. 2016, Wang et al. 2013). A comprehensive list of known gene targets was provided, facilitating further research and analysis of‘drug-disease’.
Construction of networks
Screened pharmaceutically active ingredients were introduced into the common target gene for ‘drug-disease’ interactions, and with Cytoscape 3.7.2 (http://www.cytoscape.org/), an interface was created for the visual composition network between active ingredient and cancer target. The nodes on the network diagram represent corti mori and gene proteins, and the edges represent the relationships between active ingredients and targets of action. In addition, the number of lines connecting target genes in a network determines the significance of node-to-node interactions.
Protein-Protein Interaction (PPI) network construction
The‘drug-disease’ target was delivered into the STRING biological database (https://string-db.org/) and at Organism, sorted the parameter‘homo sapiens’ for studying target protein interactions, saved the result file. The obtained files were entered into R×64 4.0.2 software for calculation, and the bar graph of the core targets of the protein interaction network was generated. PPI diagrams showed gene structures, proteins, and their relationships, with solid circles representing genes, and different colored lines connecting the circles. Lines represent biological processes involving proteins, including gene expression regulation, signal transduction, and cell migration.
GO and KEGG enrichment analysis
A version of R 4.0.2 was installed with the packages "colorspace", "stringi" and ‘ggplot2’, and the analysis of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was performed using a bioconductor package that includes ‘DOSE’ , ‘clusterProfiler’ , and ‘enrichplot’. Among them, it was considered significant when the P-value was less than 0.05, which indicated a strong correlation between cancer and the target protein. Biological functions of target proteins or related pathways may be regulated by cortex mori to induce anti-lung cancer effects. A bar graph was produced based on the analysis of the first 20 enrichment results.
Molecular docking stimulation
A search of the PDB database (https://www.rcsb.org) was conducted to find the target genes involved in the first 10 pathways of the KEGG enrichment results, of which by X crystal diffraction, 3D protein conformations with a crystal resolution lower than three were determined. Mol2 format files of the key active ingredients in GGQL were downloaded from the TCMSP platform. Proteins were processed using AutoDock Tools 1.5.6, which separated proteins, added nonpolar hydrogen, calculated Gasteiger charges, and assigned AD4 types, and turned all flexible bonds of small molecule ligands into rotatable bonds. Based on the original coordinates of the ligand and target protein, the docking box has been adjusted to include the target protein's structure. We selected the genetic algorithm for rigid docking and set the medium to the maximum number of evaluations when we docked the receptor protein rigidly. Autogrid4 and Autodock4 were used to obtain docking results, where binding energies were revealed. A partial docking diagram was created using PyMol software.
Cell culture and reagents
Jiajia Cui (Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China) kindly provided NCI-H1299 and A549 lung cancer cell lines. Cells were grown in 1640 medium containing 10% fetal bovine serum (BI) at 37℃ in an incubator with 5% CO2. Cyclomorusi was purchased from Chengdu must bio-technology company (Chengdu, China), its quality was conducted by Chengdu must bio-technology company and the quality of cyclomorusi meet the reporting standards (Supplementary 1). Shanghai Yuanye Bio-technology Company (Shanghai, China) supplied cortex mori and cis-platinum; Sigma-Aldrich (St. Louis, MO, USA) supplied dimethyl sulfoxide (DMSO). We dissolved cyclomorusin, cortex mori, and cis-platinum in DMSO. The DMSO concentration in the media was limited to 0.1% (v/v). DMSO treated cells were used as vehicle controls only.
Cell proliferation assay
Five thousand cells were plated into 96-well plates at a density of 5×103, and treated for 24 and 48 hours with various concentrations of cyclomorusin, cortex mori and platinum, or 0.1% DMSO (isotype control). By using the MTS assay (Promega) based on the manufacturer's instructions, cyclomorusin, cortex mori, and cis-platinum were evaluated on proliferation of A549 and NCI-H1299 cells. A solvent (only) used to dissolve cyclomorusin, cortex mori, and cis-platinum did not affect lung cancer cells' viability.
Apoptosis assays
Flow cytometry analysis was performed using Annexin V-FITC Apoptosis Detection Kit (KeyGen bio-technology company, Jiangsu, China) following treatment with cyclomorusin and cis-platinum for 24 hours.
Wound healing assay
A six-well plate was cultured with lung cancer cells (A549 and NCI-H1299 ). The supernatant was absorbed from the cells after they had reached nearly 100% confluence and the cells were scraped with a pipette tip sized for 10 liters. In the following step, the cells were washed in PBS to remove detached cells, and medium was added with cyclomorusin and cis-platinum. In order to determine the extent of metastasis of cells, a microscope was used at 0, 24 and 48 hours after the cells had been spread. During remodeling, the distance across the injured region diminished, normalized to the control at 0 h, and expressed as the extent of outgrowth. The solvent used to dissolve cyclomorusin and cis-platinum, lung cancer cells exhibited no differences in their migration properties.
Western blot analysis
The lysis buffer for the Radio Immunoprecipitation Assay (RIPA) was used to extract the cells at ice-cold temperatures (Beyotime, P0013B). We measured the protein concentration in lysates of tissues and cells using a BCA kit (Beyotime, P0010S). By electrophoresis on SDS-PAGE, 10L of proteins were separated, transferred to PVDF membranes and blocked for two hours with 5% skim milk powder, and then incubated with various primary antibodies at 4 °C overnight. TBST was used three times, each for 10 minutes, to wash primary antibodies before adding secondary antibodies. After shaking at room temperature for 2 hours, the secondary antibodies were removed and washed with TBST three times for 10 minutes each time, using the ECL system (BIO-RAD, Hercules, CA, USA) to show the protein bands. The primary antibodies for GADPH, p-PI3K (p85), PI3K (p85), p-AKT, AKT, p-mTOR, mTOR, Bcl-2, Bax, N-Cadherin and Vementin were purchased from Cell Signaling Technology.
Statistical analysis
Except in special circumstances, all data (n=3) are presented as mean + standard deviation. Student's test and one-way analysis of variance were used as statistical measures. P<0.05 was considered statistically significant.