The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform 2.3 (TCMSP), PubChem 1.2, Therapeutic Target Database 2020 (TTD), STITCH 5.0, DrugBank databases 5.1.7 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 12.21 and String Databases 11.0. We searched signalling pathways related to biomolecules in the Enrichr [11] and Kyoto Encyclopedia of Genes and Genomes (KEGG) . Cytoscape 3.7.1 and systemsdock 4.2.6 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 2.3 and PubChem 1.2. Then the data was imported into the Swiss target prediction database 2019, 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 Protein Data Bank-ID (PDB-ID) of the rhein-target proteins was queried in the PDB database 1.1. 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 [12].
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 ofthe 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 2020, information on the anti-inflammatory target proteins was searched by using the keyword anti-inflammatory, nine anti-inflammatory target proteins of them were selected for subsequent use [13], and these target proteins were put into Cytoscape 3.7.1 to construct an anti-inflammatory target protein protein-protein interaction network (PPI).
Screening of the anti-inflammatory targets of the rhein effect and construction of an anti-inflammatory target network
By choosing Multiple proteins,setting organism to Homo sapiens,the rhein-target proteins and the anti-inflammatory proteins 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 National Center for Biotechnology Information (NCBI), 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.
Drug and Reagents
Rhein (purity ≥ 99%) was provided by MedChemExpress (Shanghai China) Co.,Ltd. The human bronchial epithelial (HBE) cell lines were obtained from the American Collection of Cell Culture (ATCC, USA). Cell culture reagents including fetal bovine serum (FBS) , Roswell Park Memorial Institute-1640 (RPMI-1640) medium, and antibiotic+amphiphilic solution, dimethyl sulfoxide (DMSO),ovalbumin (OVA, grade V) were supplied by Sigma-Aldrich Co. LLC. Lipopolysaccharide (LPS) was purchased from Solarbio Co.,Ltd. NF-kBp65(A2547), p38MAPK(A1401), p-p38MAPK(AP0297), β-Tubulin(AS014) were provided by ABclonal Tech (Wuhan China) Co.,Ltd.
Exploration of safe doses of rhein in HBE cells
Cell viability was evaluated by the Cell Counting Kit-8 (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 for 24 hours. The culture medium was changed every 2 days. Cells were grown to 80% confluence prior to treatment. Rhein was dissolved in culture grade DMSO (final concentration < 0.1%) in serum-free media. To investigate the protective effect of rhein, primary cultured HBE cells were treated with 0.1, 0.5, and 1.0 μM of rhein for 2 hours and then treated with 100μg/ml LPS+ 1μg/ml OVA for 24 hours while the OVA+LPS group received 100μg/ml LPS + 1μg/ml OVA treatment alone. The inflammation model refers to the reference [14].
Cell Viability Assays
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. Cell viability (%) was expressed as a percentage relative to the untreated control cells.
Western Blot Analysis
To observe the inflammation prevention effects of rhein on inflammation induced HBE cells, we treated cell samples with rhein (0.1 to 1μM) and induced inflammation by applying LPS (1μg/mL)+OVA (0.1mg/ml). MAPK/NF-κB activation, which plays an important role in inflammatory responses, was analysed by western blot analysis to evaluate the effects of rhein in treating on HBE cells. Cells were lysed with Radioimmunoprecipitation (RIPA) lysis buffer including protease inhibitor (Solarbio, Beijing, China). Total protein concentration of obtained extract was quantified with a The bicinchoninic acid (BCA) protein assay kit (Pierce, Appleton, WI, USA). After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Temecula, CA, USA). Afterwards, the membrane was blocked with 5% milk for 1 hour, 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. Subsequently, the primary antibodies were probed with the secondary antibody Goat HRP Goat Anti-Rabbit IgG (H+L) (AS014) (1:3000; purchased from ABclonal Tec.) for 1 hour at room temperature. Subsequently, the signals of protein bands were captured with Image LabTM Software (Bip-Rad).
Statistical analyses
Descriptive statistics were performed using Graphpad Prism 7.0. The measurement data is expressed as the mean ± SD. Comparisons among groups were performed by one-way Analysis of variance (ANOVA). Comparisons of two groups were performed by T-test. P < 0.05 was considered statistically significant.