Study on the Mechanism of Angelica sinensis-Phellodendri Chinensis Cortex in the

Shengjing LIU, Jun GUO, Fu WANG, Jiwei ZHANG, Bin YAN, Guanchao DU, Qinghe GAO*

1.Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing 100091, China; 2.Beijing University of Chinese Medicine, Beijing 100029, China

Abstract [Objectives]To explore the mechanism of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of chronic prostatitis(CP)based on the method of network pharmacology.[Methods]The active components and action targets of Angelica sinensis-Phellodendri Chinensis Cortex were screened by(TCMSP), a systematic pharmacological analysis platform of traditional Chinese medicine, combined with literature search.The target was corrected by Uniprot database, and the disease CP target was screened by GeneCards and OMIM database.The common targets of drugs and diseases were screened by R language software, and the visual network map of drugs-active components-targets-diseases was constructed by Cytoscape 3.5.1 software.The common target protein-protein interaction(PPI)network was constructed by using STRING platform.The R language software was used to annotate and analyze the gene function and pathway of the core target through geneontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).[Results]46 active components of Angelica sinensis-Phellodendri Chinensis Cortex were screened, and 212 related targets were predicted, of which 159 were related to disease.These targets were mainly involved in biological processes such as heterologous biological stimulation, oxidation and anti-oxidation, and were mainly concentrated in PI3K-Akt, mitogen-activated protein kinase(MAPK), hypoxia inducible factor-1(HIF-1)and other related signaling pathways.[Conclusions]The multi-component, multi-target and multi-pathway action characteristics of Angelica sinensis-Phellodendri Chinensis Cortex were confirmed by network pharmacology, and the possible mechanism of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP was predicted, which provided a theoretical basis for further experiments to verify its action mechanism.

Key words Angelica sinensis-Phellodendri Chinensis Cortex, Chronic prostatitis, Network pharmacology, PI3K-Akt signaling pathway, MAPK signaling pathway, Guihuang prescription

1 Introduction

Chronic prostatitis(CP)is a common and frequently-occurring disease in clinic.It is reported that 35% to 50% of men have been affected by CP symptoms[1].A survey of 10 600 people abroad showed that the incidence of CP was 8.2%[2].Due to the different survey methods and population, the incidence of CP in China varies greatly, which is between 6% and 32.9%, higher than that reported in foreign literature[3].With the change of environment, society and life style, the incidence of CP has an increasing trend year by year[2].At present, Western medicine mainly treats CP with antibiotics, α-receptor blockers and non-steroidal anti-inflammatory drugs, it is mostly empirical treatment, the therapeutic effect is not good, and there is no effective drug for the treatment of CP[4].CP falls within the category of "turbid semen", "strangury with turbid urine" and "semenuria" in traditional Chinese medicine.Clinical practice has proved that traditional Chinese medicine is one of the important methods for the treatment of CP.

In the early part of this study, through the summary of clinical experience and data mining about the prescription rule of Professor Guo Jun, chief researcher of China Academy of Chinese Medical Sciences and director of male department of Xiyuan Hospital of China Academy of Chinese Medical Sciences, it was found that Angelica sinensis-Phellodendri Chinensis Cortex was the core drug pair for the treatment of CP.And Professor Guo Jun used the "internal elimination method for ulcer" with Angelica sinensis and Phellodendri Chinensis Cortex as the principal drugs, for the formulation of the Guihuang prescription, producing good clinical results[5].Professor Guo Jun believes that Angelica sinensis is warm in nature, and can invigorate the circulation of blood and relieve pain; Phellodendri Chinensis Cortex is cold in nature, and can clear heat and purge pathogenic fire, dryness and dampness, and this medicine is good for promoting blood circulation, clearing heat without harming yin, invigorating the circulation of blood to eliminate stasis and stagnation, and has a better effect on CP of dampness-heat stagnancy type.Modern pharmacological studies have shown that Angelica sinensis has analgesic, anti-inflammatory, anticoagulant effects, and can increase the threshold of pain caused by thermal stimulation[6].Phellodendri Chinensis Cortex has a strong inhibitory effect on Staphylococcus aureus, and has the effects of anti-inflammation[7], immune regulation and anti-oxidation.At present, the research on the mechanism of CP is mainly focused on the regulatory mechanism of inflammatory immune response, in which more and more attention has been paid to the genes and mechanisms that lead to inflammatory response[8].

Network pharmacology is a method of multi-target and multi-way biological system network analysis for active drug components based on the theory of system biology[9].In this study, network pharmacology was used to explore the action characteristics of multi-components, multi-targets and multi-pathways of Angelica sinensis-Phellodendri Chinensis Cortex, and to predict the possible action mechanism of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP, so as to provide a theoretical basis for further analysis of its active components and mechanism.

2 Materials and methods

2.1 Screening of active components and targets of Angelica sinensis-Phellodendri Chinensis CortexAll the active components of Angelica sinensis-Phellodendri Chinensis Cortex were searched by TCMSP(http://tcmspw.com/), a systematic pharmacological analysis platform of traditional Chinese medicine.Oral bioavailability(OB)≥ 30% and drug-likeness(DL)≥ 0.18 were selected as screening conditions, and the main active components of Angelica sinensis and Phellodendri Chinensis Cortex which have been studied in CNKI and PubMed databases were looked up.

According to the compounds that have been obtained, the components were extracted by TCMSP database.For the supplementary compounds obtained, first of all, the corresponding SMILES structure of the compound was obtained in the PubChem database(https://pubchem.ncbi.nlm.nih.gov/), and inputted into the pharmmapper database(http://www.lilab-ecust.cn/pharmmapper/)for target fishing.The final prediction target of Angelica sinensis-Phellodendri Chinensis Cortex was obtained after summary and duplicate elimination, and the target was standardized by selecting "Homosapiens" in the Uniprot database(https://www.uniprot.org/).

2.2 Disease targets for CPThe disease target of CP was searched by Genecards database(http://www.genecards.org/), OMIM database(https://www.omim.org/)with the key word of "chronicprostatitis".After collection and arrangement, the corresponding target of CP was obtained.

2.3 Screening of common targets for drugs and diseasesThe R language software(https://www.r-project.org/)was used to intersect the corrected drug targets and disease targets, and the common targets of drugs and diseases were obtained.

2.4 Construction of visual network diagramThe node files of active drug components and common targets of drugs and diseases were imported into Cytoscape 3.5.1 software to build a visual network diagram of drugs-active components-targets-diseases.The topological network was used to show the interaction between the active components of Angelica sinensis-Phellodendri Chinensis Cortex and CP.

2.5 Construction of protein-protein interaction networkThe STRING platform(https://string-db.org/)was used to build protein-protein interaction(PPI)network, and the minimum mutual threshold was set to 0.95.The R language software was used to draw a bar graph of the top 20 genes in terms of frequency of occurrence.

2.6 GO analysis and KEGG pathway enrichment analysisThe David website(https://david.ncifcrf.gov/)was used to convert the gene symbol in the regulatory network of traditional Chinese medicine into gene ID.The R language software was used to annotate and analyze the gene function and pathway of the core target through geneontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The results were presented in the form of bar chart or bubble chart to explore the possible mechanism of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP according to the enrichment degree of core pathway.The above R package was downloaded through R3.6.1 and Bioconductor(https://www.bioconductor.org/).

3 Results and analysis

3.1 Active components and targets of Angelica sinensis-Phellodendri Chinensis CortexThe active components of Angelica sinensis-Phellodendri Chinensis Cortex were screened by TCMSP database with OB ≥ 30% and DL ≥ 0.18 as criteria.A total of 38 active components were obtained after duplicate-elimination.By searching the relevant literature on pharmacological studies of Angelica sinensis and Phellodendri Chinensis Cortex, it was found that ligustilide, butylidenephthalide, glucuronicacid, galacturonicacid, ferulic acid, senkyunolide, vanillicacid and neocnidilide in Angelica sinensis had high biological activity, so they were included in the potential active components for analysis[6,10].The target of the active components was predicted by TCMSP and pharmmapper databases, and corrected by Uniprot database after duplicate-elimination.

After final screening, 46 active components and 212 corrected targets of Angelica sinensis-Phellodendri Chinensis Cortex were obtained.Angelica sinensis mainly included 10 active components, such as volatile oils, organic acids, sterols, and lactones, with 140 action targets.Phellodendri Chinensis Cortex mainly included 37 active components, such as alkaloids, sterols, and flavonoids, with 98 action targets.There was one common active component, β-sitosterol.

3.2 Screening of disease targets for CPThe disease targets of CP were obtained by searching GeneCards and OMIM databases, and 7 376 and 23 targets were selected respectively.A total of 7 397 CP disease treatment targets were obtained after duplicate-elimination.

3.3 Screening of common targets for drugs and diseasesThe R language software was used to find the union of the corrected drug targets and disease targets so as to obtain common targets, and the number of common targets was 159.

3.4 Construction of visual network diagramThe active drug components, drug and disease common targets were imported into Cytoscape 3.5.1 software to construct the drug-active component-target-disease visualization network, including 191 nodes(159 targets, 30 active components, 1 for disease and 1 for drug).The orange represents the drug, the green represents the active component, the blue represents the interaction target between the drug and the disease, the dark green represents the disease, and the connected edge represents the interaction between the drug, the component and the disease target(Fig.1).

Fig.1 Drug-active component-target-disease interaction network

3.5 PPI interaction network constructionThe common target was inputted into the STRING platform to build the common target PPI network, and the data with confidence level higher than 0.95 were selected to get the PPI network diagram(Fig.2).

Fig.2 Common target PPI network

The R language software was used to draw a bar graph of the top 20 targets in terms of frequency of occurrence(Fig.3).Through PPI network analysis, it was found that tumor suppressor gene(TP53), epidermal growth factor receptor(EGFR, ESR1, EGF), nuclear receptor coactivator protein 1(NCOA1), specific cyclin D1(CCND1), interleukin 6(IL-6), apoptosis signal molecules(CASP3, CASP8)and so on had high protein interaction frequency, and were the node proteins of the whole network.

Fig.3 Core gene of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP

3.6 GO analysis and KEGG pathway enrichment analysisEn-richment analysis was performed on differential genes of Angelica sinensis-Phellodendri Chinensis Cortex, and the genes were annotated and classified from biological process(BP), cellular component(CC)and molecular function(MF), and the histogram of gene enrichment was obtained(Fig.4).

Fig.4 GO enrichment analysis histogram of key targets for active components of Angelica sinensis-Phellodendri Chinensis Cortex in treating CP

The KEGG pathway enrichment analysis of the common target was carried out, and the bubble diagram of pathway enrichment was obtained(Fig.5).In the column chart and bubble chart, the height of the column chart or the size of the circle represents the number of genes enriched, and the color represents the significant degree of enrichment.The smaller thePvalue, the higher the significant degree, and the redder the color.After excluding a wide range of pathways, the top 10 signaling pathways were selected for analysis(Table 1).After literature search combined with previous research, three core signaling pathways most likely related to Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP were selected, and the number, name and source of core genes were analyzed(Table 2).

Fig.5 Bubble diagram of KEGG pathway enrichment

Table 1 Comparison of enrichment results of Angelica sinensis-Phellodendri Chinensis Cortex target pathway(top 10)

Table 2 Comparison of enrichment results of core signaling pathway targets

4 Discussion

In this study, 46 active components of Angelica sinensis-Phellodendri Chinensis Cortex were obtained by preliminary screening, among which the main active component of Angelica sinensis was ligustilide.Pharmacological studies have confirmed that it can inhibit the expression of a variety of inflammatory factors and has anti-inflammatory effects.Experiments have proved that ligustilide can improve prostate pathology and pain sensitivity in CP model rats[11-12].Among the active components of Phellodendri Chinensis Cortex, palmatine hydrochloride(OB=64.6%)has the highest oral availability.Pharmacological studies have confirmed that it has anti-bacterial, anti-viral, and anti-fungal and anti-cancer effects[13].Among the active components, worenine(DL=0.87)and coptisine(DL=0.86)are the most potent compounds, which have the effects of anti-inflammation and anti-oxidation[14].InShennong’sClassicofMateriaMedica, it is said that Angelica sinensis can be decocted to cure all kinds of sores[15].TheCompendiumofMateriaMedicasays that Phellodendri Chinensis Cortex can be used to treat sores in the mouth and tongue[16].The contents of ancient books are consistent with the experimental results to a certain extent, having a certain reference value.In addition to the targets with higher values, there are many components and targets related to other diseases in Angelica sinensis-Phellodendri Chinensis Cortex drug pair, which is worthy of further exploration.

According to the 46 active components selected, 212 related targets were predicted.Through the intersection with disease targets, it was found that there were 159 common targets, indicating that Angelica sinensis-Phellodendri Chinensis Cortex had a wide coverage and good drug targeting in the treatment of CP.Through PPI network analysis, the genes with high frequency of protein-protein interaction were obtained, including cancer-related gene(TP53), epidermal growth factor(EGFR, ESR1, EGF), inflammatory gene(IL-6), apoptosis signal molecule(CASP3, CASP8)and so on.Current studies have shown that the expression level of epidermal growth factor EGFR and EGF in CP is significantly higher than that in normal prostate tissue, which is positively correlated with the pathogenesis of CP[17-18].Cytokine IL-6 can activate lymphocyte differentiation and enhance the role of natural killer cells, and the level of IL-6 in seminal plasma of patients with CP can be used as a predictive index of CP, which has a certain value in the diagnosis of CP[19].

The results of GO analysis showed that the most likely biological pathways related to differential genes mainly involved heterologous biological stimulation response, steroid response, antibiotic response, oxidation-related response and so on.Molecular functions mainly involve membrane rafts, membrane regions, membrane microdomains, presynaptic and postsynaptic membrane components, transcription factor complexes, nuclear chromatin and so on.Cellular localization is mainly related to DNA binding to transcriptional activation, ubiquitin-like protein ligase binding, transcription factor activity, steroid receptor activity and so on.

The results of pathway enrichment analysis suggested that Angelica sinensis-Phellodendri Chinensis Cortex might involve multiple signaling pathways in Table 1, and it was more closely related to PI3K-Akt signaling pathway(22 targets), MAPK signaling pathway(15 targets)and HIF-1 signaling pathway(13 targets)in Table 2.The analysis of these three signaling pathways showed that the multi-components of Angelica sinensis-Phellodendri Chinensis Cortex were involved in the mechanism related to the treatment of CP, which reflected the therapeutic characteristics of traditional Chinese medicine and the synergistic effect of Angelica sinensis and Phellodendri Chinensis Cortex in the treatment.PI3K-Akt signaling pathway is involved in the regulation of cell proliferation, differentiation and apoptosis, and is closely related to inflammation, tumor and reproductive system diseases.Inflammatory pain and neuropathic pain can be alleviated by inhibiting the activation of PI3K-Akt signal transduction pathway and the release of upstream and downstream modulators[20-21].MAPK signaling pathway is an important system that mediates intracellular and extracellular signal transduction, and its family signaling pathways include P38MAPK, ERKs, JNK/SAPK and ERK5/BMK1, which participate in the process of cell proliferation, differentiation and apoptosis, and are closely related to inflammation and tumor[22-23].Among them, p38MAPK signaling pathway is more closely related to the regulation of inflammation and stress response, and is considered to be the intersection point and common pathway of cellular information transmission[24].At present, some experiments and studies have shown that the inhibition of p38MAPK pathway can decrease the pain threshold of mechanical and thermal pain in rats, and regulate IL-6, TNF-α and other cytokines to inhibit the aggregation or activation of inflammatory cells[25-26].Angelica sinensis-Phellodendri Chinensis Cortex may involve the regulation of PI3K-Akt, MAPK, HIF-1 and other signaling pathways, and can be used to treat CP through the regulation of cell proliferation and apoptosis, oxidation and anti-oxidation and other biological processes.

Through the analysis method of network pharmacology, this study screened the active components and action targets of Angelica sinensis-Phellodendri Chinensis Cortex pair, constructed a network diagram for analysis, and further predicted the key regulatory targets and related pathways of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP, reflecting the multi-component, multi-target and multi-path characteristics of Angelica sinensis-Phellodendri Chinensis Cortex in the treatment of CP, providing a reference for the treatment of CP and providing a theoretical basis for further experiments to verify its action mechanism.

The Chinese version of this paper has been published in Journal of Basic Chinese Medicine, January 2021,Vol.27.No.1.