Anti-inflammatory Mechanism of Rhein in Treating Asthma Based on Network Pharmacology

Background: Network pharmacological methods were used to predict the anti-inflammatory targets and related pathways of rhein in the treatment of asthma and to elucidate its mechanism of action. In addition, we validated the anti-inflammatory effects of rhein in vitro. Methods: The corresponding targets of rhein were obtained from the TCMSP database, and molecular docking was performed. A network of predicted rhein targets was established and analysed with Cytoscape 3.7.1. The anti-inflammatory targets in the TTD database were searched to build a PPI network, which was merged with the ingredient-target network to screen anti-inflammatory targets associated with rhein. A network of anti-inflammatory rhein targets during the in vivo treatment of asthma was constructed to screen the anti-inflammatory targets related to asthma. KEGG enrichment analysis was performed with the Enrichr database and Cytoscape 3.7.1. The expression levels of proteins in the MAPK/NF-κB signalling pathway were assessed by Western blot analysis. Results: Altogether, 17 targets were obtained. Epidermal active growth factor receptor (EGFR), E-selectin (SELE), macrophage migration inhibitory factor (MIF), and mitogen-activated protein kinase 14 (MAPK14) might be important anti-inflammatory targets of rhein during asthma treatment. We selected the MAPK signalling pathway to determine the anti-inflammatory effects of rhein. Conclusion: The anti-inflammatory mechanism of the treatment of asthma with rhein may be related to MAPK14, EGFR, SELE, and MIF, as well as their signalling pathways. To prevent the exacerbation of asthma, instead of targeting a single pathway or a single target, all these targets and their signalling pathways should be controlled holistically. Rhein may reduce inflammation by inhibiting the MAPK/NF-κB pathway.


Background
Asthma is an abbreviation of bronchial asthma. Asthma is a kind of chronic inflammatory disorder of the airways involving cells and corresponding components, and it is characterized by inflammation, hyperresponsiveness, stenosis and airway remodelling. Chronic inflammation is considered the hypostasis of asthma [1,2].
Rhein is an effective monomer component, and it is separated and purified from traditional Chinese 3 medicines, such as rhubarb. Rhein is a monanthraquinone 1,8-dihydroxyl anthraquinone derivative.
Rhein has anti-inflammatory, antibacterial, antitumour and other effects [3,4,5,6]. However, the efficacy of the anti-inflammatory effects of rhein in the treatment of asthma has not been reported.
Network pharmacology has become an emerging research method in recent years. Network pharmacology is considered a new model for the next generation of drug research. The construction of biomolecule networks, such as the drug-target-pathway, is the basis of network pharmacology [7,8].
Network pharmacology can explore the efficacy of drugs from the component-target pathway, and the pathogenesis of diseases can also be reversed through drugs with known efficacy [9,10]. Hence, we used network pharmacology to explore the anti-inflammatory mechanism of rhein in the treatment of asthma and to provide new ideas for treating asthma.

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 antiinflammatory target protein PPI network.

Screening of the anti-inflammatory targets of the rhein effect and construction of an antiinflammatory 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).

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.

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.

Rhein and its predicted targets
Seventeen predicted targets of rhein were searched in STITCH, DrugBank databases, and a drugtarget interaction network of rhein and its potential targets were constructed by using Cytoscape 3.7.1 (Fig. 1).

Rhein-target network results
The rhein-target protein interaction networks were constructed by using String Database (Fig. 2).

The reverse docking scores of rhein and its related targets.
The PDB-IDs were imported into systemsDock for molecular docking studies. The docking score was above 5, verified the reliability of the predicted targets ( Table 1).

Network of rhein anti-inflammatory targets during treatment of asthma
The Cytoscape 3.7.1 merge function was used to combine the rhein-predicted target network with the anti-inflammatory target PPI network in the TTD, considering the overlapping results and a high confidence interval above 0.7, rhein anti-inflammatory targets, including mitogen-activated protein kinase (MAPK14), receptor tyrosine-protein kinase erbB-2 (ERBB2), tumour necrosis factor receptor superfamily member 1A (TNFRSF1A), and epidermal growth factor receptor (EGFR) were identified (Fig. 3).

KEGG pathway enrichment of the target genes
According to the KEGG pathway enrichment of the target genes, the anti-inflammatory target proteins 7 of rhein associated with asthma were involved in 87 signalling pathways. The Enrichr analysis results were sorted in descending order of the combined score. The top ten pathways included the MAPK signalling pathway, hepatitis C signalling pathway, epithelial cell signalling during Helicobacter pylori infection, immune signalling pathway, and proteoglycans in cancer signalling pathway and et al (Fig.   4).

The cytotoxicity of rhein on HBE cells
To examine the cytotoxicity of rhein on HBE cells, the cells were treated with various concentrations of rhein (0.1 to 1μM) in RPMI medium and fetal bovine serum (FBS) for 24 h. There was no observed cytotoxicity of rhein in the HBE cells when the final concentration of rhein was less than 40μM (Fig.5).

Inflammation prevention effects of rhein on HBE cells via MAPK/NF-κB pathway
To observe the inflammation prevention effects of rhein on inflammation induced HBE cells,we treated cell samples with rhein (0.1 to 1μM/mL) and induced inflammation by applying LPS (1μg/mL)+OVA (0.1mg/ml). MAPK/NF-κB activation, which plays a vital role in inflammatory responses, was analyzed by western blotting analysis to evaluate the effects of rhein in treating on HBE cells. The results showed that rhein treatment dose dependently inhibited phosphorylation of MAPKp38 and significantly reduced the expression of NF-κB of p65 in the HBE-cell activated MAPK/NF-κB pathway by LPS+OVA recombinant. Rhein treatment is associated with the MAPK/NF-κB pathway;thereby rhein would prevent inflammation (Fig.6).

Discussion
It has been reported that rhein has anti-inflammatory activities [12,13]. The results of this study show the anti-inflammatory effects of rhein. Youdong Xu et al. explained the anti-inflammatory effects of rhein based on its molecular mechanism [14]. MAPK14, EGFR, EERB2, TNFRSF1A, etc. are the main targets of the anti-inflammatory effects of rhein. Rhein can directly act on EGFR, MAPK14, EERB2, and TNFRSF1A and can also indirectly act on other targets to exert its anti-inflammatory effects [15].
EGFR is an epidermal growth factor receptor that is widely distributed in epithelial tissues and plays an important regulatory role in the development of respiratory inflammation [16]. GFR inhibitors can effectively inhibit acute inflammation of the rat respiratory tract caused by exogenous zinc ions [17]. 8 EGFR inhibitors reduce the symptoms of allergic asthma caused by dust mites by reducing the production of pro-inflammatory factors, such as IL-6 and IL-8 [18]. Thus, it is speculated that rhein interacts with EGFR, blocking the binding of EGFR to pro-inflammatory cytokines, to exert an antiinflammatory effect.
MAPK14 plays an important role in the cellular cascade triggered by pro-inflammatory cytokines or extracellular stimuli and is a key signalling molecule in the lung inflammation induced by S. pneumonia [19,20]. Inflammatory factors, such as IL-1β, IL-6 and TNF-α, can positively regulate signalling pathways, such as ERK and nuclear factor-κB (NF-κB), by activating the p38 MAPK signalling pathway and cascading to amplify the inflammatory response [21,22]. The transcriptional cascade regulated by P38 MAPKs leads to the production of pro-inflammatory factors, such as TNF-α and IL-1β, which in turn leads to the activation of enzymes involved in inflammation [23]. It is speculated that rhein exerts its anti-inflammatory effect by inhibiting the release of pro-inflammatory factors, such as TNF-α and IL-1β, by inhibiting MAPK14.
TNFRSF1A is a type 1 TNF receptor that mediates inflammatory responses mainly by activating NF-κB, p38, and ERK1/2 to induce IL-6 and IL-8 synthesis and apoptosis [24,25]. It is thus concluded that rhein interacts with TNFRSF1A to reduce the secretion of anti-inflammatory factors, such as IL-6, and exert an anti-inflammatory effect.
EERB2, also called HER2, NEU, and CD340, is a 185-kDa cell membrane receptor encoded by the proto-oncogene erbB-2 and is a member of the EGFR family. EERB2 can induce IL-6 autocrine activity, which in turn affects the JAK-STAT pathway-or NF-κB pathway-mediated inflammatory responses [26].
It is speculated that rhein interacts with EERB2 and may exert anti-inflammatory effects by regulating the secretion of IL-6 [27,28,29].
In this study, the anti-inflammatory mechanism of rhein was confirmed by network pharmacology.
The main targets of the anti-inflammatory effect of rhein and the related signalling pathways were predicted. Rhein exerted an anti-inflammatory effect by acting on multiple targets. However, network pharmacology research is based on network modelling, database resource development and software application. The network model has certain differences from the in vivo environment. 9 We have demonstrated that rhein can reduce the inflammatory responses induced by LPS + OVA via the MAPK/NF-κB pathway in vitro. In the future, the anti-inflammatory effects of rhein need to be verified by further animal experiments.

Conclusion
In summary, as far as we know, it is the first time to investigate the mechanism of rhein in treating asthma by its anti-inflammatory effect. Network pharmacologic analysis indicated that rhein may not only has the anti-inflammatory function by regulating MAPK14 EGFR EERB2 TNFRSF1A, but also has the effect of treating asthma by regulating the MAPK/NF-κB signaling pathway. Additionally, the expression of MAPK/NF-κB pathway proteins were validated in HBE cell lines by western blotting. All

Conflict of interest statement
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, "Anti-inflammatory Mechanism of Rhein in Treating Asthma Based on Network Pharmacology".
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request Supplementary Table SII .

Authors' contributions
All authors read and approved the final manuscript.

Ethics approval and consent to participate
The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee. Written informed consent was obtained from individual or guardian participants.  Rhein and its predicted targets Seventeen predicted targets of rhein were found in the STICH,Drugbank databases.