Mechanism of Pinelliae Rhizoma in Treatment of Insomnia Based on Network Pharmacology

Liang LI, Weibo DAI, Qiaoling CHEN, Wenxian GUO, Jiaci WANG, Ying HU

Zhongshan Hospital of Traditional Chinese Medicine, Zhongshan 528400, China

Abstract [Objectives] To explore the mechanism of Pinelliae Rhizoma in the treatment of insomnia. [Methods] Using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, the effective components of Pinelliae Rhizoma were explored, to predict its targets, and screen out the targets of insomnia through GeneCards, OMIM and other databases. With the aid of Cytoscape software, a "component-target-pathway-disease" network for the treatment of insomnia by Pinelliae Rhizoma was built. Then, a target protein-related interaction network was created through the String database, and biological function annotation and pathway analysis of key targets were performed. [Results] The 13 active components of Pinelliae Rhizoma take action in the treatment of insomnia by intervening 33 targets, 59 GO biological processes, and 10 main biological pathways. [Conclusions] The active components of Pinelliae Rhizoma may treat insomnia by such pharmacological effect as regulating G protein-coupled amine receptor activity, adrenergic receptor activity, catecholamine binding, and neurotransmitter receptor activity.

Key words Pinelliae Rhizoma, Network pharmacology, Insomnia, Active components, Targets

1 Introduction

Pinelliae Rhizoma is dry tuber ofPinelliaternata(Thunb.) Breit., a plant of the Araceae family. It has the effects of eliminating dampness and phlegm, reducing counterflow and checking vomiting, relieving oppression and dispersing hard masses. It is commonly used for wet phlegm, cold phlegm, coughing and phlegm, phlegm palpitations, wind phlegm dizziness, phlegm headache, vomiting, nausea, stuffiness and rigidity in the chest, plum-pit qi; and external treatment of abscess swelling phlegm nodule[1]. According to the different processing methods, there are Raw Pinelliae Rhizoma, Pinelliae Rhizoma Praeparatum, Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine, and Pinelliae Rhizoma A Praeparatum Cum Alumine. Modern research has found that Pinelliae Rhizoma has the effects of lowering qi, eliminating phlegm, and dispelling masses, and has a good effect in the treatment of insomnia in clinical applications. In the treatment of insomnia with Pinelliae Rhizoma as the main medicine, the ancient prescriptions include Xiaobanxia Decoction (restoring deficiency and expelling excess, and communicating yin and yang) and Wendan Decoction (clearing heat and eliminating phlegm, and regulating qi-flowing for harmonizing stomach), and the modern prescriptions include Banxia Houpu Decoction, Banxia Xiexin Decoction, Banxia Baizhu Tianma Decoction,etc.[2]. According to the statistical analysis of the rule of medication for the treatment of insomnia in theDictionaryofTraditionalChineseMedicinePrescription, it is believed that Pinelliae Rhizoma is also one of the high-frequency Chinese medicines prescribed for the treatment of insomnia[3]. These indicate that Pinelliae Rhizoma has become a common traditional Chinese medicine for clinical treatment of insomnia, and there are even reports that large amount use of Pinelliae Rhizoma in the treatment of insomnia has achieved more significant effects[4]. Modern research has revealed the chemical components and pharmacological effects of Pinelliae Rhizoma. Research reports have shown that Pinelliae Rhizoma has pharmacological effects such as sedation, hypnosis, anticonvulsions,etc.[5-8], preliminarily revealing its pharmacological mechanism for treating insomnia. However, due to the complexity of the chemical components in traditional Chinese medicine and the diversity of efficacy, the mechanism of Pinelliae Rhizoma in the treatment of insomnia is still not clear. Network pharmacology can construct a network of "effective components of traditional Chinese medicine-target-signal pathways-treatment mechanism", and can systematically and comprehensively clarify the ways and pathways of effective components of traditional Chinese medicine to treat diseases. With the aid of the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, String, Genecards, Online Mendelian Inheritance in Man (OMIM) database, we constructed a network of active components of Pinelliae Rhizoma and related targets for the treatment of insomnia, and performed biological process and pathway enrichment analysis, in order to Provide theoretical support for clarifying the clinical application of Pinelliae Rhizoma in the treatment of insomnia and the experimental research to be carried out.

2 Materials and methods

2.1 Screening of active blood components of Pinelliae RhizomaThrough the TCMSP database and analysis platform (TCSP, http://lsp.nwu.edu.cn/index.php), we searched Banxia (Pinelliae Rhizoma), and conducted mining of all component data of Pinelliae Rhizoma in the database, and collected a total of 116 compounds. Based on the pharmacokinetic parameters, we set oral bioavailability (OB)≥30% and drug-like (DL)≥0.18 as screening conditions, screened the collected compounds for pharmacokinetics, the obtained compounds were the active blood components of Pinelliae Rhizoma, and these compounds were taken as the research object[9].

2.2 Screening of Pinelliae Rhizoma-insomnia co-action targetUsing the TCMSP database, we searched for the target information of the active components of Pinelliae Rhizoma, and inquired the human gene name corresponding to the target through the UniProt database (https://www.uniprot.org/), and then through the Genecards database (https://www.genecards.org/), OMIM database (https://www.omim.org/), we searched related targets for insomnia. After combining the two databases and removing the duplicates, we used the R language (https://www.rproject.org/) to intersect the targets and disease targets related to the active components of Pinelliae Rhizoma, and obtained the target of the active blood components of Pinelliae Rhizoma to treat insomnia, and plotted the Venn diagram.

2.3 Construction of drug-components-targets-disease networkWith the aid of Cytoscape 3.7.2 software (http://www.cytoscape.org), we imported Pinelliae Rhizoma-insomnia’s common active components and common protein target genes into the system, conducted visualized processing, and constructed drug-components-targets-disease network. The drug Pinelliae Rhizoma, active components and key targets in the network were denoted by nodes, and the connections between drug-components, components-targets, and target-disease were represented by edges.

2.4 Construction of the key target protein-protein interaction (PPI) networkWe first imported the common target genes of Pinelliae Rhizoma and insomnia into the String database (Version 11.0) (https://string-db.org), selected the "Multiple Proteins" function, set the species to be human (Homosapiens), and obtained the protein-protein interaction network, set the scoring value > 0.4, constructed the PPI network relationship diagram, and obtained the node degree value and other information reflecting the relationship between the targets.

2.5 Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysisUsing R language, we installed Bioconductor platform related installation packages (DOSE, clusterProfiler, pathview, ClusterProfiler,etc.), and then used perl tool to convert the key target name of Pinelliae Rhizoma for treating insomnia to entrez ID, setpvalue=0.05,qvalue=0.05 as conditions, performed GO enrichment analysis and KEGG enrichment analysis, output the results, and drew barplot histograms and dotplot bubble charts.

3 Results and analysis

3.1 Screening of active blood components of Pinelliae RhizomaThrough the TCMSP database, we retrieved 116 active components of Pinelliae Rhizoma, and then screened them with the criteria ofOB≥30% andDL≥0.18, and obtained 13 potential active components of Pinelliae Rhizoma, as listed in Table 1.

Table 1 Active blood components of Pinelliae Rhizoma and their OB and DL values

3.2 Prediction of the potential targets of the active components of Pinelliae Rhizoma in the treatment of insomniaThrough the TCMSP database target prediction model, we predicted the target information of the effective compounds of Pinelliae Rhizoma, predicted and screened 179 target points of the active components of Pinelliae Rhizoma, and annotated the gene ID through UniProt. Through Genecards and OMIM database, we searched for insomnia target genes,obtained genes with correlation score greater than 5. After combining the two databases and removing the duplicates, we obtained a total of 2 249 disease targets. Then, we mapped the target genes of Pinelliae Rhizoma and insomnia, obtained 33 common targets of Pinelliae Rhizoma and insomnia, and plotted the Venn diagram by R language (Fig.1). Common targets of Pinelliae Rhizoma and insomnia areNR3C2,PTGS1,CHRM1,ADRB1,HTR3A,ADRA2C,ADRA1B,ADRB2,ADRA1D,OPRM1,SLC6A4,AR,AKT1,VEGFA,FOS,BAX,MMP9,TP63,HIF1A,MPO,AHR,IGF2,ADRA1A,GABRA1,MAP2ADH1C,ADRA2A,SLC6A2,SLC6A3,PLAU,MAOB,MAOA, andESR1, indicating that Pinelliae Rhizoma can treat insomnia through multiple targets.

Fig.1 Intersection of target points of Pinelliae Rhizoma’s active ingredients and targets of insomnia

3.3 Construction of drug-components-targets-disease network for Pinelliae Rhizoma in the treatment of insomnia

With the aid of Cytoscape 3.7.2 software, we made a visual processing of the relationship network for active blood components of Pinelliae Rhizoma in the treatment of insomnia, as shown in Fig.2.

Fig.2 Drug-components-targets-disease network for Pinelliae Rhizoma treating the insomnia

3.4 PPI network analysis results of the key target of Pinelliae Rhizoma in the treatment of insomniaWe imported 33 common target genes of Pinelliae Rhizoma and insomnia into the String database, set the confidence score > 0.4, and obtained the PPI protein interaction network diagram (Fig.3). we hided the free proteins that appear, the blue line represents the evidence of gene co-evolution, the red line represents the evidence of gene fusion, and the yellow line represents the evidence of text mining. This network includes 33 nodes, 146 edges, and an average degree of 8.85. The interaction proteins with scores ≥ 0.98 are VEGFA-HIF1A, AKT1-ESR1, VEGFA-MMP9, AKT1-HIF1A, ESR1-FOS, and VEGFA-IGF2. Using R language, we extracted the information bar graph of the key targets in the network (Fig.4). The results indicate that the proteins FOS, AKT1, HTR3A, MAOA, SLC6A4, and VEGFA have higher degrees, indicating that these proteins play a significant role in the network.

Fig.3 Key target PPI network

Fig.4 Information bar graph of key targets

3.5 Biological function and pathway analysis of key targets

Taking R language as the platform, using the package in Bioconductor and the provided GO annotation information and the KEGG pathway database, we performed the signal pathway enrichment analysis. GO annotation results show that a total of 59 GO biological processes have been obtained. The top biological functions are mainly G protein-coupled amine receptor activity (8 targets) and adrenergic receptor activity (7 targets), catecholamine binding, neurotransmitter receptor activity, DNA binding transcription activation activity, RNA polymerase II specificity (5 targets each), chloride transmembrane transport protein activity, inorganic anion transmembrane transport activity, RNA polymerase II transcription factor Binding, anion transmembrane transporter activity, metal ion transmembrane transport activity, pathway activity, passive transmembrane transporter activity (4 targets each), indicating that the active components of Pinelliae Rhizoma can treat insomnia through regulating various biological pathways (Fig.5).

Fig.5 GO biological process enrichment analysis of key targets of Pinelliae Rhizoma and insomnia

KEGG pathway enrichment is based on thePvalue and the number of genes as screening conditions. The results show that genes are significantly enriched in endocrine resistance pathways, mitogen-activated protein kinase signaling pathways (MAPK), tyrosine metabolism pathways, and drug metabolism-cells pigment P450 pathway, EGFR tyrosine kinase inhibitor resistance pathway, phenylalanine metabolism pathway, histidine metabolic path-way, glycine, serine and threonine metabolism pathway, tryptophan metabolism pathway, arginine and proline metabolic pathway,etc. (Fig.6).

Fig.6 KEGG pathway enrichment analysis of key targets of Pinelliae Rhizoma and insomnia

4 Discussions

The results of this study show that the key compounds in Pinelliae Rhizoma can affect insomnia through multiple target genes and multiple signaling pathways. Through search, it is found that 24-ethylcholest-4-en-3-one, carvedin, baicalein, baicalin, β-sitosterol, stigmasterol, (E)-11-eicosenoic acid, coniferin are the main active components of Pinelliae Rhizoma for treating insomnia. Among them, 24-ethylcholest-4-en-3-one, β-sitosterol, and stigmasterol are phytosterols and have antioxidant, anti-inflammatory, immune regulation, and central nervous protection effects[10]. β-sitosterol also has significant anti-anxiety and anti-convulsant effects[11]; stigmasterol can improve learning and cognition by enhancing the mediators of the cholinergic neurotransmission system[12]. Baicalein and baicalin are flavonoids and can protect nerve cells through various mechanisms such as anti-oxidative stress, anti-inflammatory reaction, anti-neuronal excitotoxicity, and inhibition of nerve cell apoptosis[13-15]. Baicalein also shows anti-epileptic and anti-depressant properties, and can improve the brain cognitive function[16-17]. Through PPI analysis, we obtained 33 core protein genes:FOS,AKT1,HTR3A,MAOA,SLC6A4,VEGFA,ADRA1B,ADRA1A,ADRA1D,ADRA2A,MAOB,SLC6A2,SLC6A3,ADRA2C,ADRB2,MMP9,OPRM1,ADRB1,AR,ESR1,HIF1A,MAP2,IGF2,AHR,CHRM1,MPO,PLAU,BAX,ADH1C,GABRA1,NR3C2,PTGS1, andTP63.

In order to study the role of targets in gene function and signaling pathways, we performed GO biological function enrichment analysis. We found that the above-mentioned PPI core genes can play a role in the treatment of insomnia through affecting the G protein-coupled amine receptor activity, adrenergic receptor activity, catecholamine binding, neurotransmitter receptor activity, DNA binding transcription activation activity, RNA polymerase II specificity, chlorine transmembrane transporter activity, inorganic anion transmembrane transport activity, RNA polymerase II transcription factor binding, anion transmembrane transport protein activity, metal ion transmembrane transport activity, channel activity, and passive transmembrane transport protein activity. KEGG pathway enrichment analysis results indicate that the target genes of Pinelliae Rhizoma for treating insomnia are mainly enriched in endocrine resistance pathway, mitogen-activated protein kinase signaling pathway (MAPK), tyrosine metabolism pathway, drug metabolism-cytochrome P450 pathway, EGFR tyrosine kinase inhibitor resistance pathway, phenylalanine metabolism pathway, histidine metabolic pathway, glycine, serine and threonine metabolism pathway, tryptophan metabolism pathway, arginine and proline metabolic pathway,etc., and these pathways are basically closely connected with the occurrence and development of the insomnia.

Insomnia is closely related to the body’s internal secretion. Disorders of endocrine function lead to abnormal hormone secretion, triggering autonomic nervous system dysfunction and leading to insomnia[18]. The MAPK pathway is involved in the regulation of cell proliferation, differentiation, transformation and apoptosis. The MAPK pathway can regulate the expression of brain-derived neurotrophic factor (BDNF) genes. BDNF plays an important role in the survival, growth and function of brain neurons. Insomnia can cause decrease of the BDNF level, which in turn leads to impaired cognitive function[19-20].

Phenylalanine is an essential amino acid of the body and is related to the pathogenesis of central nervous system diseases. Through phenylalanine hydroxylase, phenylalanine is converted to tyrosine by, and then metabolized to catecholamines, which is closely related to the synthesis of norepinephrine and dopamine[21-22]. The metabolism of glycine, serine and threonine plays an important role in ensuring the development of the brain and the normal function of the brain central nervous system[23]. The metabolism of arginine and proline is an important component of collagen, joint and tendon synthesis, and is closely related to the body’s metabolic level. In the target metabolomics study of the sedative and hypnotic effect of the traditional Chinese medicine Jiaotai Pill, the results showed that through down-regulating the metabolism of phenylalanine, tyrosine and tryptophan, dopamine metabolism, glutamine metabolism and other pathways and regulating the neuroprotective factor BDNF, it can improve the metabolic disorders of insomnia model animals, which is consistent with the enrichment results of this study[24-25].

In summary, through network pharmacology, we explored the mechanism of the main active components and target genes of Pinelliae Rhizoma and insomnia. Results indicate that the components such as 24-ethylcholesterol-4-en-3-one, carvedin, baicalein, baicalin, β-sitosterol in Pinelliae Rhizoma may mediate endocrine resistance pathways, MAPK pathways, and tyrosine metabolism Pathway, phenylalanine metabolic pathway,etc., play a role in regulating G protein-coupled amine receptor activity, adrenergic receptor activity, catecholamine binding, neurotransmitter receptor activity, DNA binding transcription activation activity and other pharmacological effects. The above results can provide new ideas for the treatment of insomnia. However, since the information collected by the current database is not comprehensive, the screening process of active components and their targets will be affected by the subjective attitude of researchers. Besides, there are also many factors affecting the metabolism of active components of drugs in the body. The results of this study will have certain limitations, and the mechanism of Pinelliae Rhizoma in the treatment of insomnia still needs to be verified by many more experiments.