Jin-Liang Guo, Ma-Li Feng

Shanxi Provincial Institute of Traditional Chinese Medicine, Taiyuan 030012, China

ABSTRACT Objective: To study the mechanism of cnidium cnidium improving sleep by network pharmacology. Methods: The active components of Fructus cnidii and the targets of active components were obtained by systematic pharmacology database and analysis platform(TCMSP).With the help of Uniprot database;the corresponding target genes of active components were searched, and the targets related to insomnia were retrieved in drugbank and GeneCards databases.Cytoscape 3.7.2 was used to construct the hypnotic active componentinsomnia - target network.String database to build PPI network;On the basis of Metascape database, GO enrichment analysis and KEGG enrichment analysis were carried out to predict the mechanism of action. Results: A total of 19 active compounds such as geranyl lignin,osthole, and omellae were screened from Osthole, 55 effective targets were predicted, 962 insomnia related targets were screened, and 27 intersection targets for drug insomnia were made.Among them, PTGS1, PIK3CG, GABRA1, CHRM1, CHRM3, ESR1, etc., may be the key targets of cnidii to improve insomnia. Biological function and pathway enrichment analysis showed that the main active components of Cnidium were involved in chemical synaptic transmission, G-protein-coupled receptor signaling pathway, membrane potential regulation, neurotransmitter transport, monoamine transport, ion transport regulation and other biological processes.Sleep regulation was most likely by regulating Neuroactive ligandreceptor interaction, signaling pathway, Cholinergic synapse, gMP-PKG signaling pathway,it addiction, Serotonergic synapse, and Dopaminergic synapse. Conclusion: The network pharmacology analysis shows that fructus cnidii can improve the characteristics of sleep multicomponent, multi-target and multi-pathway, which provides a certain theoretical basis for the research of mechanism of fructus cnidii regulating sleep.

Keywords:Fructus cnidii Insomnia Network pharmacology Mechanism of action

1. Introduction

Sleep is an indispensable life activity, and the body ensures the normal and stable functions of life through adequate sleep [1].With the development of society and the change of life style, people's pressure is gradually increasing, which leads to the increasing number of insomnia people year by year, and the incidence rate is as high as 30% ～ 35%[2].Long-term lack of sleep, not only cause physiological dysfunction, affect people's physical and mental health and quality of life, serious will also be complicated with hypertension, angina, and other diseases, and even death.For sedative and hypnotic chemicals widely used in clinical practice, they have good short-term efficacy and a single and clear target, but drug resistance and addiction appear after longterm oral administration, with large side effects and it is difficult to fundamentally improve sleep quality [3,4].Therefore, the active ingredients with significant hypnotic effect should be sought from Chinese medicinal materials or natural drugs, and the mechanism of action should be explored from the regulation of biological processes such as synaptic transmission, receptor signaling pathway,membrane potential, and neurotransmitter transport, so as to provide certain theoretical basis for the research and development of modern novel drugs for the treatment of sleep disorders.

Cnidium monnieri (L.) Cuss belongs to the genus Cnidium monnieri (L.) Cuss of Umbelliferae.The dry ripe fruit of.In addition to the traditional known efficacy, modern pharmacology study shows that in the nervous system, antitumor, central inhibition,sedative hypnotic effect is remarkable [5-7], Ma Lan et al. [8] the hangover of cnidium monnieri reflect tolerance and done related to explore, discover cnidium monnieri sedative hypnotic effect significantly, and the hangover reaction and tolerance is relatively small diazepam, obviously it has good clinical application prospects in improving sleep, coumarin of its new efficacy and hypnotic sedative effect and its mechanism from done related research,hypothalamus, hippocampus [9], but because of the limitations on the concrete experiment,At present, the active ingredient and its mechanism of action are not fully understood.

Network pharmacology is an analysis platform based on big data.Through screening the effective compounds in TCM and combining with relevant disease data, the target and mechanism network of drugs acting on diseases are constructed, and relevant data of drug action and mechanism are mined [11,12].A variety of databases and related software are used to visually process data to directly reflect the degree of association between drugs and diseases, predict the target proteins and interacting proteins of drugs in vivo, and analyze the signaling pathways involved in these proteins [13,14].Therefore,this study is based on network pharmacology to explore the possible molecular mechanism of Cnidium Cnidium hypnotic effect through drug-disease-target network.

2. Materials and methods

2.1 Database

TCMSP(http://lsp.nwu.edu.cn/ tcmsp.php)

Uniprot(https:// www.uniprot.org/)

Drugbank(https://www.drugbank.ca/);

Genecards(https://www.genecards.org/);

Venny(https://bioinfogp.cnb.csic.es/tools/venny/);

String(https:// string-db.org/,Version 11.0);

Metascape(https://metascape.org/gp/index.html#/main/step1).

2.2 Analysis Software

Cytoscape 3.7.2; Omicshar(https://www.omicshare.com/tools/).

2.3 Screening of active ingredients from Cnidium ninidium

The key words "Cnidium ninidium" were input into the TCMSP database, and the oral bioavailability (OB) ＞ 30% and drug-like(DL) BBB＞18 were selected as the screening conditions to retrieve the effective active components of Cnidium ninidium.

2.4 Target screening of effective components of Cnidium Cnidii

Targets TCMSP utilization of retrieval functions, common cnidium fruit, in turn, input effective active ingredient of MOL ID, 200 kinds of main active components corresponding potential targets, remove duplicate values won 55 kinds of targets, Uniprot database will be gained by the target name input, select "reviewed" and "human"species information limited, retrieve the target names corresponding Gene Symbol. Set aside.

2.5 Screening of insomnia related targets

Insomnia was searched in Drugbank and Gene Cards database using the disease name "INSOMNIA", and the target genes related to insomnia were obtained by inducting the screening results and removing duplicate genes.

2.6 Construction of active components of Cnidium Cnidium Cnidii - insomnia - target network

The names of potential drug targets and disease-related targets were respectively uploaded to the Uniprot database, and the "collected"information on the species "Human" was selected to obtain the standard gene names of the targets.After the screening targets will be uploaded to take intersection;The above information was processed and uploaded to Cytoscape 3.7.2 to construct active ingredientdisease-target map of Cnidium ninidium.

2.7 PPI network analysis and core target screening

The protein targets of Cnidia ninidii were imported into the String database. By default, the target with interaction greater than 0.4 was selected, and the hidden free nodes were selected to construct the protein interaction network for Cnidia ninidii to improve insomnia symptoms.The downloaded file information was input into Cytoscape 3.7.2 software, and the corresponding graph size was adjusted according to the importance of nodes, and the final visual map was output to determine the core genes in the protein interaction network.

2.8 Enrichment analysis of hypnotic biological processes and pathways

Will cnidium monnieri treating insomnia of potential target import Metascape platform, species H.s apiens ", select analysis,personalized P ＜ 0.01, fructus cnidii hypnosis targets for gene ontology (GO) analysis and Kyoto encyclopedia (KEGG) gene and genome pathway enrichment analysis, top biological processes using hypnosis Omicshare visualization processing software.

3. Results

3.1 Main active components and action targets of Cnidium Cnidii

TCMSP was used to search for 19 active ingredients, 30% of OB ＞ and 0.18 of DL BBB＞as shown in Table 1. According to the MOL ID, 200 target sites were found, and 55 target information was retained after deletion of duplicate values. Genecards and Ddrugbank retrieval was related to insomnia.Intersection of active ingredient targets of Cnidium ninidium and action targets related to insomnia was made on the online Venn diagram production website.The Venn diagram is shown in Figure 1, and a total of 27 potential action targets were obtained.They are CHRM1, GABRA1, PIK3CG,MAOB, PTGS1, KCNH2, DRD1, CHRM3, GABRA2, CHRM4,ADRB2, SLC6A4, OPRM1, CHRNA7, ESR1, AR, GSK3B,CHRM2, ADRA1b, ADRA2a, SLC6A3, PLAU, ADRB1, HTR2A,ADRA1A, GABRA3.Corresponding to 12 active ingredients,Am midin,XanthoxylinN,(E)-2,3-bis(2-keto-7-methoxy-chromen-8-yl)acrolein, Stigmastero, Prangenidin, o-isovalerylcolum bianetin,o-acetylcolumbianetin, Diosmetin, cniforin A, and cnidimolB, Beta-Sitosterol, AR-Curcumene

Table 1 Active components of Cnidium ninidium screened by OB and DL

Figure 1 Active component of Cnidium ninidium - insomnia - target Venn plot

3.2 Active ingredient - disease - target network

Cytoscape 3.7.2 was used to construct the active ingredient-diseasetarget network plot, as shown in Figure 2.

Figure 2 Active ingredient - disease - target network

Among them, the central "circle" represents the name of insomnia disease, the "green triangle" represents the 12 active components of Cnidium ninidium, and the "red inverted triangle" represents the target of insomnia.The more connecting lines, the larger the graph display, the more intuitive reflection of key node information.Twelve active ingredients were related to 27 targets, among which Stigmasterol, o-acetylcolumbianetin, o-isovalerylcolum bianetin,Xanthoxylin N, Prangenidin and beta-sitosterol acted on more targets, while the targets corresponding to more active ingredients were:PTGS1, PIK3CG, GABRA1, CHRM1, CHRM3 and ESR1 may be the key active components and key action targets of Cnidium ninidium in improving insomnia.

3.3 Construction and analysis of protein interaction networks

The target protein into the String database of common cnidium fruit,species"person", choose the interaction is greater than 0.4 targets to build protein interaction networks, as shown in figure 3, hide the free nodes, edges represent targets, the correlation between the thickness on behalf of the association score is different, if the more coarse,the relevance score is, the greater the different colors to distinguish between different types of evidence.In the protein interaction network, there were 27 nodes and 78 edges, with an average node degree of 5.78 and an average local clustering coefficient of 0.534.The data were imported into Catosape software for further intuitive analysis, and SLC6A4, ADRA1B, HTR2A, SLC6A2, CHRM1,CHRM2, etc. were the core targets of the network, which played a key regulatory role in the protein interaction network.

Figure 3 Target protein interaction network of Cnidium ninidium

3.4 Biofunction and pathway enrichment analysis

Go annotation analysis and KEGG pathway analysis of 27 potential targets of Cnidium ninidium for insomnia were performed using Metascape database, and visual analysis was performed by OmicShare.The results are shown in Figure 4.

Figure 4 GO enrichment analysis

GO annotation analysis consists of three parts, namely biological process (BP), cell component (CC) and molecular function (MF)[15].GO analysis showed that in terms of biological processes,these targets involved chemical synaptic transmission, G-protein coupled receptor signaling pathways,G protein-coupled receptor signalinq pathway for membrane potentialCoupling to cyclic nucleotide second messenger), regulation of membrane potential,neurotransmitter transport, monoamine transport, requlation of ion transport, developmentalGrowth, positive requlation of synaptic transmission, cellular response to organic cyclic compound, memory regulation, regulation of body fluid levels, and requlation of signal receptor activitySignaling receptor activity, organ growth, response to ethanol, and the requlation of calcium ion transmembrane transport.In CC analysis, the first cell components are postsynaptic membrane,membrane raft, glutamatergic synapse, dendrite membrane,cytoplasmic vesicle membrane, and other cell components.In terms of molecular function, G protein-coupled amine receptor activity,adrenergic receptor activity, and neurotransmitter receptor activity involved in requlation of postsynaptic were involvedThe membrane potential, ammonium ion binding, cross membrane transporter activity, beta-catenin binding, cation channel activity, protein domain specificBinding) and other molecular functions.

KEGG pathway analysis was shown, as shown in Figure 5.The leading pathways involved in hypnotic targets of Codnaria sinensis mainly include Neuroactive ligand-receptor interaction,Calcium signaling pathway, Cholinergic synapse, cGMP-PKG signaling pathway, and MorphineAddiction, Serotonergic synapse,Dopaminergic synapse and other signaling pathways.

Figure 5 Enrichment analysis of KEGG pathway

4. Discussion

Insomnia is called "insomnia" in TCM. Yang does not enter Yin,which causes vigilance on night due to deficiency of Yang. Cnidium ninidium has the effect of tonifying kidney and strengthening Yang,and the body of deficiency of Yang reaches the secret of Yin and pacifies Yang by warming kidney and replenishing Yang, and Yang enters Yin and wakes up [16].Modern pharmacological studies have shown that Cnidium ninidium has central inhibitory effect [17].Tong Ligguo et al. [18] analyzed target compounds with sedative and hypnotic activity of Cnidium ninidium by using fingerprint and pharmacodynamic correlation, including osthol and progresin, and newly discovered 5 monomeric components with hypnotic activity,including berberolactone and isogurin.

In this screening, 19 active components of Cnidium Cnidii were obtained, 12 of which had potential sleep regulation effect.The active ingredients with more action targets were Stigmasterol,o-acetylcolumbianetin, o-isovalerylcolum bianetin, Xanthoxylin N, Prangenidin, beta-sitosterol;The sedative and hypnotic effects of its total coumarins have been reported in relevant literature [9].The corresponding targets PTGS1, PIK3CG, GABRA1, CHRM1,CHRM3 and ESR1 may be the key active components and action targets of Cnidium ninidium in improving insomnia.PTGS1 is one of the target enzymes of the chemical zopicolone in the treatment of insomnia, and may also be the target target of Cnidium ninidium for improving sleep [19].GABRA1 is a component of the main inhibitory neurotransmitter γ-aminobutyric acid isopentameric receptor in the brain. Inhibitory γ-aminobutyric acid is responsible for the formation of synapses, and it is the target receptor of most sedative and hypnotic drugs, such as diazepam and zopicron [20]. It is speculated that Cnidium ninidium acts on the same receptor.ESR1 is an estrogen receptor protein. The secretion level of estrogen affects sleep, and menopausal women will cause hot flashes, night sweats and other difficulties in falling asleep. Therefore, Cnidium ninidium may regulate menopausal sleep disorders by acting on this protein[21,22].

In this study, through enrichment analysis, we found that 12 major active components of Cnidii nidii acted on 27 key targets, acting on the interaction pathway, calcium signaling pathway, cholinergic synapses, cGMP-PKG signaling pathway serotonin-containing synapses, dopaminergic synapses and other signaling pathways that stimulate nerve tissue.The process involves the GO analysis of chemical synaptic transmission, G protein coupled receptor signaling pathways, coupling cyclic nucleotide of membrane potential secondary information adjustment, membrane potential adjustment,and monoamine neurotransmitter transport transport, ion transport regulation, synaptic transmission are regulation, cell response to cyclic organic compounds, memory regulation, fluid levels regulate the activity of the receptors, signal conditioning, calcium ions across the plasma membrane of regulate biological processes.G-protein conjugated amine receptor activity, adrenergic receptor activity,neurotransmitter receptor activity involved in the regulation of postsynaptic membrane potential, ammonium ion binding chloride ion, transmembrane transport activity, β-catenin, binding cationic channel activity, protein domain specific binding and other molecular functions.Postsynaptic membrane, membrane raft, glutamate synapse, dendritic membrane, cytoplasmic vesicle membrane and other cellular components.SLC6A4, ADRA1B, HTR2A, SLC6A2,CHRM1, CHRM2 and other targets are the key targets of the PPI network, and these targets are also the core regulatory genes.SLC6A4 as serotonin transporters, its main functions in the central nervous system including back in serotonin molecules from the synaptic cleft transshipment presynaptic terminal can reuse to adjust serotonin signal, in regulating serotonin other receptors for serotonin can system availability plays a key role, by adjusting serotonin levels in the body can improve sleep [23-25].As a member of the G-protein-coupled receptor superfamily, ADRA1B is mediated by binding to the G protein that activates the second messenger system of phosphatidlinositol-calcium. ADRA1B is one of the main target receptors of norepinephrine, and Cnidium ninidium may regulate sleep by inhibiting its release [26].HTR2A for 5 - HT G protein coupled receptor [27], can be used as a variety of drugs and receptor of active material and the spirit by guanine nucleotide binding protein triggers signal transduction in regulating downstream effector activity [28], at the same time, the signal can be activated calcium phospholipase and phosphatidyl inositol - the second messenger system, used to adjust phosphatidyl inositol 3 - kinase activity and promote the release of calcium affect neural activity, thus play a role in behavior regulation, including the response to the spirit of active substances and insomnia [29].SLC6A2 is an amine transporter that reduces the degradation of norepinephrine at the synaptic site and prolongs the neurotransmitter's stimulability through the highaffinity sodium dependence of norepinephrine into the presynaptic terminal. Cnidium ninidium may regulate sleep by increasing the duration of norepinephrine action [30,31].Sleep regulation is also involved in the interdependent signaling of specific neurons distributed in brain regions, and the cholinergic pathway is necessary for REM sleep, and experiments by Yasutaka Niwa et al. have shown that the muscarinic acetylcholine receptors CHRM1 and CHRM3 are essential for sleep regulation, especially for REM sleep [32].CHRM2, as a muscarinic acetylcholine receptor, can also regulate sleep [33].

Based on the network pharmacology method, this study predicted the pharmacodynamic substance basis, key targets and signal pathways of Cnidium Cnidium hypnosis, and identified its functional chemical components.By establishing the network diagram of Cnidium ninidium's pharmacological components -insomnia - target, the potential mechanism of Cnidium ninidium's sleep improvement was explored from multiple perspectives, and its insomnia improvement function was played by regulating related biological factors.Although some components and target proteins have been confirmed by relevant experiments, the pathway remains to be confirmed by further basic research and clinical trials, and further experimental verification and support are still needed.