Molecular mechanism of luteolin against in�ammation based on integration of network pharmacology, transcriptomics and proteomics

Background: Luteolin (3', 4', 5,7-tetrahydroxyavone), a natural �avonoid exists in various medicinal plants, has strong anti-inammatory effect. However, anti-inammatory mechanism of luteolin has not been fully explored. Hence, we aimed to systematically investigate druggability and anti-inammatory mechanism of luteolin based on network pharmacology. Methods: The absorption, distribution, metabolism, excretion, and toxicity of luteolin were evaluated by TCMSP server. Targets associated with luteolin and in�ammation were collected from public databases, and the overlapping targets between luteolin and in�ammation were analyzed by Draw Venn diagram. Then the protein-protein interaction network of luteolin against in�ammation was constructed to get core genes. Further, gene function and pathway enrichment analysis were performed. Finally, in vitro experiment was carried out to estimate the accuracy of predicted target genes. Results: ADME results indicated that luteolin has great potential to be developed into a drug. 226 overlapping targets (targets of luteolin against in�ammation) were screened by matching 280 targets of luteolin with 9015 targets of in�ammation. 9 core targets of luteolin against in�ammation were identi�ed, including MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1. Gene function were mainly involved in metabolism, energy pathways and signal transduction. Pathway enrichment results suggested that metabolic pathways, pathways in cancer, PI3K-AKT signaling pathway, Ras signaling pathway and so on might be the critical pathways of luteolin against in�ammation. RT-qPCR and ELISA results indicated that luteolin decreased the expression of most of core genes at protein and mRNA levels (MMP9, MAPK1, HSP90AA1, EGFR, SRC and HRAS). Conclusions: The anti-inammatory mechanism of luteolin were systematically investigated based on network pharmacology, RT-qPCR and ELISA. Luteolin is expounded to have great potential to be developed into a drug and target various genes and pathways to perform systematic anti-inammatory effect


Background
In ammation, a defense responses to stimulation from infection and tissue damage, is related to the pathogenesis of a lot of diseases, such as sepsis, arthritis, atherosclerosis, asthma and so on 1,2 .Thus the effective anti-in ammatory treatment can reduce risk and prevent in ammation-related diseases and is becoming more clinically used and attracting the attention of basic researchers.However, long-term use of steroids or NSAIDs to treat in ammatory diseases can bring multiple systemic side effects.Therefore, there is an urgent need to develop new strategies for in ammatory disease.
The development of new anti-in ammatory drugs from Traditional Chinese Medicine has gradually become a research hotspot for its abundant resources and few side effects 3 .Chrysanthemi Flos with identi ed quality markers including luteolin is proved to inhibit the activation of MAPK and NF-κB in RAW 264.7 macrophages, which are involved in the progress of type 2 diabetes, atherosclerosis and cancer 4,5 .
A clinical trial shows that Reseda luteola L. containing about 40% luteolin decreases UVB-induced erythema, which is a in ammatory skin condition 6,7 .It is con rmed that os lonicerae avonoids isolated from Lonicerae Japonicae Flos containing luteolin decrease the expression of TNF-α, IL-1β and CRP to perform protective effect on ulcerative colitis in vivo, and HPLC-PDA assay is conducted to reveal that luteolin is one of the classi ed markers of Lonicerae Japonicae Flos 8, 9 .The above researches suggest that anti-in ammatory activity of avonoids from natural plants, especially luteolin, have been received widespread attention.
Luteolin (the structure was shown in Fig. 1) has anti-in ammatory, anti-oxidant, neuroprotective, cardioprotective, anti-diabetic, anti-microbial and anti-allergic effect as previous study 10 .It's worth noting that the anti-in ammatory effect of luteolin is one of its important pharmacological effect, which has been con rmed to be related to a variety of pharmacological activities.Luteolin exerts renal protection through inhibiting in ammation by activating Nrf2/ARE pathway in rats 11 .Luteolin can reduce in ammation to play a therapeutic role in rats with Non-alcoholic steatohepatitis 12 .Luteolin also can inhibit NF-κB and MAPKs activation to decrease in ammation, and thus improve insulin sensitivity in vitro 13 .Theoharides et al. conduct preliminary study on clinical trial on children with Autism Spectrum Disorder, and nd that treatment with luteolin signi cantly improves gastrointestinal and allergic symptoms in about 75% of children, and improves attention in 50% of children through performing antiin ammatory effect 14 .As luteolin exhibits strong anti-in ammatory activity at micromolar concentrations, luteolin and medical plants containing luteolin have become potential anti-in ammatory drugs 15 .However, the anti-in ammatory mechanism of luteolin has not been systematically elucidated.
Network pharmacology combines the concepts and methods of systems biology, bioinformatics and pharmacology to explore the systematic action of Traditional Chinese Medicine or compounds, and develop new drugs 16 .This study intended to estimate the potential of luteolin to develop into a drug and systematically obtain the anti-in ammatory targets and mechanism of luteolin by using network pharmacology and in vitro experiment.The owchart was shown in Fig. 2.

ADME-Related properties of luteolin
Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP:http://lsp.nwu.edu.cn/tcmsp.php)comprehensively provides the active compounds of all TCM recordeed in the "Pharmacopoeia of the People's Republic of China".TCMSP also contains information of absorption, distribution, metabolism, and excretion (ADME) characteristics for each compound, and druglikeness (DL) and oral bioavailability (OB) are the two most important indicators for ADME characteristics estimation of compounds 17 .The criteria of OB ≥ 30% and DL ≥ 0.18 are important for drug development from natural plant sources 18 .

Collection Of Luteolin And In ammatory Targets
Targets of luteolin were captured by using PharmMapper (http://www.lilab-ecust.cn/pharmmapper/submit le.html), which is a reverse docking server for targets shing.All the targets in PharmMapper are extracted from TargetBank, DrugBank, BindingDB and PDTD and it contains more than 7000 receptor-based pharmacophore models.PharmMapper can harveste targets of compounds through pharmacophore localization and provide the rst 300 targets based on the t score for each compound 19 .An sdf le for luteolin was acquired from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and input to PharmMapper with default values."In ammation" and "anti-in ammation" performing keywords were input to Comparative Toxicogenomics Database (CTD: http://ctdbase.org/)and Genecards (https://www.genecards.org/)database to obtain targets for in ammation 20 .All targets were input to UniProt (http://www.uniprot.org/)to get corresponding gene symbols.

Construction of Protein-protein interaction (PPI) network of luteolin against in ammation
The targets of in ammation and luteolin were analyzed by using Draw Venn Diagram (http://bioinformatics.psb.ugent.be/Webtools/Venn/) to get the overlapping genes, which represented the targets of luteolin against in ammation.Then STRING 11.0 database (https://string-db.org/)was used to construct PPI network of luteolin against in ammation.Protein interactions with a scoring value > 0.4 and "Homo sapiens" were selected as the high con dence basis.Cytoscape 3.7.1 software was used to visualize the PPI network of luteolin against in ammation.

Core Genes Analysis
Core genes of PPI network of luteolin against in ammation were mined by Cytohubba and MCODE plugins of Cytoscape 3.7.1 software.The parameters of CytoHubba were set as: core genes = top 10 nodes ranked by degree, Maximum Neighborhood Component (MNC), and Maximal Clique Centrality (MCC) 21 .The parameters of MCODE were set as follows: degree cutoff = 2, node score cutoff = 0.2, Kscore = 2, Max deapth = 100 22 .Then the results of Cytohubba and MCODE analysis were analyzed by using Draw Venn Diagram to get overlapping genes, which represented the core genes.
Gene Ontology (go) Enrichment Analysis GO, a functional system, is designed to expound gene functions and properties of gene products 23 .GO are mainly involved in biological process (BP), cellular component (CC) and molecular function (MF).The target genes of luteolin against in ammation were imported into FunRich software to carry out GO analysis.P value 0.05 were considered to be statistically signi cant for GO enrichment 24 .
Kyoto Encyclopedia Of Genes And Genomes (kegg) Pathway Analysis KEGG pathway analysis designed to systematically analyze gene function links gene lists to higher-order functional information to receive signi cantly enriched biological pathways 25 .All targets of luteolin against in ammation were subjected to KEGG pathway analysis by using the DAVID Bioinformatics Resources 6.8 database (https://david.ncifcrf.gov/).All the results of KEGG pathway analysis with p value 0.05 were selected and visualized by OmicShare tools (https://www.omicshare.com/tools/index.php/)and the luteolin-target-pathway network was constructed by Cytoscape 3.7.1 26 .

Anti-in ammatory Effects Of Luteolin
The RAW 264.7 macrophages were purchased from Chinese Academy of Sciences (Shanghai, China) and cultured in dulbecco's modi ed eagle's medium (Gibico) supplemented with 10% fetal bovine serum (Gibico), 1% streptomycin and penicillin at 37℃ in a cell incubator containing 5% CO 2 .We treated RAW 264.7 macrophages with LPS (100 ng/mL, Sigma) for 24 h with/without the treatment of luteolin (5, 10 and 20 µM, Selleck) for 24 h, while cells in control group were treated with medium alone for 24 h 27 .
To determine the anti-in ammatory effect of luteolin, RT-qPCR analysis was performed to con rm the effect of luteolin on the expression of core genes at mRNA level.Total RNA was collected using Trizol reagent according to the kit's instructions.RNA reverse-transcribed to cDNA was carried out using the PrimeScript RT reagent Kit (TaKaRa) with gDNA Eraser.The SYBR green PCR Master Mix was used to detect mRNA expression and samples were normalized to β-actin.40 cycles of PCR were performed and Relative gene expression (fold change) was calculated by using 2 −ΔΔ CT method.Primers (Sangon Biotech) used in this study were lisetd as Table 1.Moreover, ELISA assay was performed to prove the effect of luteolin on the expression of core genes at protein level.The cell supernatants were collected to analyze by ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Table 1 The primers used in this study.

Statistical analysis
Statistical analysis was performed using the SPSS 22.0 software.All values were presented as Mean ± SD.One-way analysis of variance followed by least signi cant difference test were used for comparison.P < 0.05 was considered statistically signi cant.

ADME-related characteristics of luteolin
ADME characteristics of luteolin were studied in depth by TCMSP, such as OB, DL, Caco-2, BBB and Lipinski's rule of ve (including MW, AlogP, TPSA, Hdon and Hacc).DL and OB are the two most important indicators for ADME characteristics estimation of compounds.Notably, luteolin was satis ed with both DL ≥ 0.18 and OB ≥ 30% (Table 2).

PPI network construction and core genes of luteolin against in ammation veri cation
Nodes and edges represented targets and interactions of targets in PPI network, respectively.STRING database showed that PPI network had 226 nodes and 2180 edges.The circles represented the targets of luteolin against in ammation and the edges represented the interaction between targets (Fig. 4B).The results of CytoHubba analysis obtained 9 core genes, including MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1 (Fig. 5A).The results of MCODE obtained the top 2 most signi cant modules with scores of 15.647 and 10 (Fig. 5B).Convincingly, module 1 and module 2 comprised 9 core genes obtained from CytoHubba analysis, which further con rmed that the importance of MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1 (Fig. 5B).

Go Analysis For Target Genes Of Luteolin Against
In ammation GO analysis for target genes of luteolin against in ammation was performed by FunRich software.The results showed that 8 CC enrichment items comprised cytoplasm, cytosol, exosomes, lysosome, extracellular, extracellular space, extracellular region and caveola (Fig. 6A).9 items were enriched in MF, including transmembrane receptor protein tyrosine kinase activity, ligand-dependent nuclear receptor activity, protein-tyrosine kinase activity, protein serine/threonine kinase activity, catalytic activity, hydrolase activity, metallopeptidase activity, glutathione transferase activity and oxidoreductase activity (Fig. 6B).The study obtained 3 BP enrichmen items that contained metabolism, energy pathways and signal transduction (Fig. 6C).

Potential Molecular Pathways Of Luteolin Against In ammation
The results of KEGG pathway analysis revealed that 226 targets of luteolin against in ammation were mainly enriched in 99 signaling pathways (P < 0.05).Moreover, 31 signaling pathways visualized by Ominshare were directly involved in in ammation and might be the key mechanism of luteolin against in ammation, including FoxO signaling pathway, PI3K-Akt signaling pathway, Estrogen signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, MAPK signaling pathway, AMPK signaling pathway and so on (Fig. 7A).The luteolin-target-pathway network details of 31 signaling pathways and enriched genes visualized by Cytoscape 3.7.1 were showed in Fig. 7B.

Anti-in ammatory Effect Of Luteolin
Macrophages produce various pro-in ammatory mediators and chemokines when stimulated, so they are widely used to establish in ammatory cell models to study in ammatory diseases.RAW 264.7 macrophages, a mouse macrophage cell line, can generate multitype in ammatory factors when activated.Thus RAW 264.7 macrophages were selected to investigate the anti-in ammatory effect of luteolin in this study.The results of RT-qPCR and ELISA indicated that the expression of MAPK1, EGFR, HRAS, HSP90AA1, MMP9 and SRC in model group signi cantly increased compared with control group, and luteolin markedly inhibited MAPK1, EGFR, HRAS, HSP90AA1, MMP9 and SRC production at mRNA and protein levels (Fig. 8A-8F and Fig. 9A-9B).

Discussion
In ammation plays a protective role when the body is infected or injured, but excessive in ammatory response will cause a wide range of diseases.Currently, more and more researches have con rmed that in ammation is closely related to the occurrence of various chronic or malignant diseases such as type 2 diabetes, rheumatoid arthritis, asthma, cancer and so on [1, 2].Therefore, there is an urgent need to develop comprehensive strategies for in ammatory disease.A certain amount of studies have been conducted on the anti-in ammatory effect of luteolin, and shown that luteolin exhibits signi cant antiin ammatory effect in a variety of cell and animal models 28,29 .However, the detailed and systematic molecular mechanism by which luteolin inhibits in ammation remains uncertain.
Evaluation of the ADME properties is the rst step in digging for a drug.Studies have shown that one of the most important reasons for the failure of drug development are poor pharmacokinetics and toxic properties 19 .Early evaluation of ADME properties of drugs can signi cantly improve the success rate, and reduce the cost and the occurrence of drug toxicity and side effects of drug development 19 .Obviously, evaluation of ADME properties is of great signi cance to simplify and accelerate the drug discovery process.DL is designed to evaluate how "drug-like" a compound is and the potential of a compound to develop into a drug.The criterion of DL ≥ 0.18 has been widely used to lter out compounds with undesirable ADME-relatedproperties 18 .OB is one of the most critical parameter of oral drugs and the threshold of OB ≥ 30% represents a good indicator of the promising effectiveness of drug delivery to the blood circulation 18 .Moreover, molecular weight (MW) < 500 Daltons, the lipid-water partition coe cient (ALogP) < 5, the number of hydrogen bond donors (Hdon) < 5 and the number of bond acceptors (Hacc) 10 are called "Lipinski's Rule of Five".A compound that complies with "Lipinski's Rule of Five" means that it will have better pharmacokinetic properties and higher bioavailability, and therefore more likely to become a drug 19 .As shown in Table 2, luteolin was satis ed with DL ≥ 0.18, OB ≥ 30% and "Lipinski's Rule of Five" indicating that luteolin has great potential to be developed into a promising drug.
Target shing is the second step in drug mining.The results of target genes of luteolin against in ammation network showed that 226 targets of luteolin against in ammation were obtained.
Furthermore, 9 core genes were screened including MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1.The main function of MMP9 is to degrade and reshape the homeostasis of the extracellular matrix, and plays a critical role in in ammatory response, tissue con guration, regulating matrix-bound growth factor and cytokine expression and cancer 30 .Moreover, research shows that luteolin decrease MMP9 expression to treat ischemic stroke, colon cancer and diabetes 31,32 .MAPK pathway is the intersection of signal pathways such as cell proliferation, in ammation, differentiation, functional synchronization, transformation and apoptosis, and MAPK pathway participates in cell proliferation, differentiation, canceration, metastasis, apoptosis and so on 33 .Luteolin decreases in ammation through inhibiting MAPK1 pathway and thus performs a bene cial treatment in atherosclerosis 34 .CASP3 is the main terminal cleaving enzyme in the process of apoptosis and activation of CASP3 causes apoptosis and in ammation 35 .Studies prove that luteolin can effectively increase CASP3 expression to induced apoptosis in HaCaT cells and cancer cells 36,37 .EGFR, a member of the epidermal growth factor receptor family, plays an important role in tumor cell proliferation, angiogenesis, tumor invasion and metastasis.Moreover, a decease in EGFR expression exerts anti-in ammatory activity in in ammatory diseases such as asthma 38 .It has been con rmed that luteolin inhibits the activation of EGFR to manage glioblastoma, lung cancer, pancreatic cancer and so on [38][39][40][41] .SRC can interact with phosphorylate STAT3, regulate TLR4-induced in ammatory response, regulate tumorigenesis of cancer cells and HIF1α expression 42,43 .Canarium subulatum and boerhavia diffusa L with high content of luteolin perform anti-in ammatory activities by decreasing SRC expression 44,45 .HRAS mutations are more common in bladder cancer and head and neck cancer, and luteolin increases HRAS expression to regulate cell cycle progression, which may be involved in decreasing in ammation response 46,47 .ESR1 is essential for sexual development and reproductive function and also responsible for bone growth and maintaining normal functions of the cardiovascular and nervous systems.ESR1 mutations or abnormal expression are associated with tumor onset and excessive in ammatory response, and study suggests that luteolin might regulate acute in ammation in renal injury through affecting the expression of ESR1 48 .It is worth noting that the CRP/ALB ratio is a relevant biomarker that re ects microvascular permeability, and CRP/ALB ratio has a strong correlation in assessing cohn's disease activity, analyzing the risk of acute myocardial infarction and predicting mortality in hemodialysis patients [49][50][51][52] .Furthermore, monitoring CRP and ALB levels together can better assess the prognosis of bacterial infectious diseases, and CRP/ALB ratio can be used as a new in ammatory prognostic indicator to predict outcomes in patients with hepatocellular carcinoma 53,54 .Interestingly, ALB was con rmed as a potential core gene in the target genes of luteolin against in ammation network.The above results suggest that MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1 may be critical target genes of luteolin against in ammation.
Based on the analysis of gene function, the results revealed that luteolin may regulate the metabolism, energy pathways, signal transduction and activity of receptor protein and series of proteinase to defend in ammation.KEGG pathways suggested that the 31 critical signaling pathways might be the mechanism of luteolin against in ammation, including pathways in cancer, metabolic pathways, PI3K-AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway and so on.The results of GO and KEGG analysis are in line with our prediction that core genes were mainly involved in antiin ammatory biological processes and signaling pathways.Finally, we used RT-qPCR and ELISA to further con rm the accuracy of predicted anti-in ammatory targets of luteolin.Surprisingly, the results of RT-qPCR and ELISA showed that luteolin markedly inhibited MAPK1, EGFR, HRAS, HSP90AA1, MMP9 and SRC mRNA production at protein and mRNA levels.However, we found that the levels of ALB, CASP3 and ESR1 did not increase signi cantly after LPS stimulation, which indicated that we need to further analyze the effect of luteolin on predicted targets expression in vivo and in vitro in proteomics and proteomics studies in the future.

Conclusion
The potential of luteolin to develop into a drug and the anti-in ammatory mechanism were predicted by using network pharmacology.The anti-in ammatory mechanism of luteolin were possibly related to core genes of MMP9, MAPK1, HSP90AA1, CASP3, ALB, EGFR, SRC, HRAS and ESR1.These results may help guide further research to identify targets for luteolin in various in ammatory diseases.The study also indicates that network pharmacology is a convincing approach for preliminarily identifying the potential of compounds from Traditional Chinese Medicine to develop into drugs and compound-related target genes. Figures Gene name Forward primer (5'-3')Reverse primer (5'-3

Figure 1 Structure
Figure 1

Figure 2 Work
Figure 2

Figure 3 Target
Figure 3

Figure 5 The
Figure 5

Figure 6 GO
Figure 6

Table 2
Pharmacological and molecular properties of luteolin.bymatching280 targets of luteolin with 9015 targets of in ammation (Fig.4A).It is worth noting that 226 overlapping targets represented the targets for luteolin against in ammation.
As shown in Fig.3Aand Fig. 3B, all of 280 targets of luteolin (excluded 20 targets without corresponding gene symbols) and 9015 targets related to in ammation were respectively obtained from PharmMapper, CTD and GeneCards database.The results of Draw Venn diagram suggested that 226 overlapping targets were screened