Tools used and data sources
The TCMSP, PubChem, TTD, STITCH, DrugBank databases were used to search for the chemical composition of the drug and its targets. Genes related to diseases, gene-to-protein interactions, and protein-protein interaction information can be found in the GenBank and String databases. We looked for signalling pathways related to biomolecules in the Enrichr and KEGG databases. Cytoscape 3.7.1 and systemsDock were used for network construction and molecular docking respectively.
Prediction of the targets of rhein
The data about the three-dimensional chemical structure of rhein was searched and exported from the TSCM and PubChem databases. Then, the data was imported into the Swiss target prediction database, and reverse molecular docking was performed. Predictive targets were obtained by setting the target set to Homo sapiens. The results were used in further studies.
Molecular docking studies of rhein and its target proteins
The PDB-ID of the target proteins was queried in the PDB database. The data was imported into systemsDock for molecular docking studies. The docking score was used to determine the matching degree between rhein and the target. The score ranged from 0-10, and the greater the value was, the stronger the interaction was [11].
Construction of the rhein-target network
A drug-target interaction network of rhein and its potential targets were constructed by using Cytoscape 3.7.1.
Construction of the rhein-target protein interaction network
By using String Database, the rhein-target protein interaction network was constructed by setting the protein type to Homo sapiens, setting the minimum interaction threshold to medium confidence, and keeping the remaining parameters silent.
Anti-inflammatory target protein screening
In the TTD database, information on the anti-inflammatory target proteins was searched using the keyword anti-inflammation, and this information was put into Cytoscape 3.7.1 to construct an anti-inflammatory target protein PPI network.
Screening of the anti-inflammatory targets of the rhein effect and construction of an anti-inflammatory target network
The rhein-target protein network was combined with the anti-inflammatory protein PPI network, and the results, which were called the anti-inflammatory targets of the rhein effect, were imported into the String database to construct the network. Screening values above 0.7 indicated a high confidence of protein interactions.
Search for asthma-related genes in humans
In the NCBI Gene Database http://www.ncbi.nim.nih.gov, asthma and Homo sapiens are searchable keywords, and the asthma-related genes of a human being can be acquired.
Network of the anti-inflammatory targets of rhein in the treatment of asthma
The anti-inflammatory target genes of rhein and human asthma-related genes were imported into the String database to construct the response network of the anti-inflammatory targets of rhein during the in vivo treatment of asthma and to screen the anti-inflammatory targets related to the incidence of asthma.
KEGG pathway enrichment of the target genes
The Enrichr was used to analyse the KEGG biological pathway enrichment of the target genes to predict the anti-inflammatory targets of rhein.
Exploration of safe doses of rhein in HBE cells
Cell viability was evaluated by the CCK-8 assay. The drug dose was selected within a range that is nontoxic to cells, and this dose ensured comprehensive accurate results of the subsequent experiments.
Culture and Treatment of HBE cells
The HBE cell lines were previously obtained from the American Collection of Cell Culture (ATCC, USA). The cells were cultured in appropriate bottles with sterile RPMI-1640 medium (Sigma-Aldrich) supplemented with 1% antibiotic+amphiphilic solution (Sigma-Aldrich) and 10% foetal bovine serum (FBS, Sigma-Aldrich) and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% atmospheric air.
Cell viability assay
Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay. HBE cells (2×105 cells in 200 μl of RPMI medium+2% FBS per well in a 96-well plate) were treated with rhein at concentrations of 0.1, 0.5 and 1.0µM. After 24 hours, the supernatant was removed, and 100 μl of the medium was added with CCK-8 solution to form crystals of formazan in the viable cells. The plate was incubated for 4 hours. The wells were immediately analysed in a spectrophotometer at a wavelength of 450 nm.
Western blotting analysis
Cells were lysed with RIPA lysis buffer including protease inhibitor (Solarbio,Beijing,China).Total protein concentration of obtained extract was quantified with a BCATM protein assay kit (Pierce,Appleton,WI,USA).After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),the protein were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore,Temecula,CA,USA).Afterwards,the membrane was blocked with 5% milk for 1 h,and then incubated with primary antibodies against NF-kBp65(A2547)、p38MAPK(A1401)、p-p38MAPK(AP0297)、β-Tubulin(AS014) (all purchased from ABclonal) overnight at 4℃.The primary antibodies were diluted with 5% milk at a dilution of 1:1000.Subsequntly,the primary antibodies were probed with the secondary antibody HRP Goat Anti-Rabbit IgG(H+L)(AS014)(1:3000;purchased from ABclonal) for 1 h at room temperature.Subsequently,the signals of protein bands were captured with Image LabTM Software (Bip-Rad).
Statistical analyses
Descriptive statistics were performed using Prism 7.0 software. The measurement data are expressed as the mean ± S.E.M. Comparisons among groups were performed by one-way ANOVA. Comparisons of two groups were performed by t-test. P < 0.05 was considered statistically significant.