The Molecular Mechanisms of Suanzaoren Prescription for Anti-Anxiety Were Investigated Based on Network Pharmacology

Anxiety is a common, universal disease caused by psychological and environmental factors. There are medications available to treat anxiety disorders, but these are accompanied by problems such as addiction and withdrawal diculties. Suanzaoren Prescription (SZRP) is often used to treat anxiety disorders in traditional Chinese medicine clinical practice. However, its therapeutic mechanism remains unclear. This work aims to identify potential core therapeutic targets and predict major bioactive compounds by means of network pharmacology, screen compounds that may bind to anxiety targets by molecular docking methods, and validate and evaluate possible therapeutic mechanisms of compounds for anxiety in vitro cell experiments. while TCMSP, ETCM were to obtain target of compounds. The targets of anxiety disorders were obtained through database TTD, OMIM, PharmgKB, CTD, DrugBank and Metacore. The network diagram is drawn by Cytoscape 3.7.2 software and STRING. Biological process and KEGG pathway analysis were conducted by MetaCore and NIMNT. AutoDock Vina software was used to conduct molecular docking. The calcium Flux assay was employed to detect the activation of compounds on CNR1 in cell line CHO-K1/CNR1/Gα15. The 545 compounds in SZRP were collected. We found there were 1050 potential targets of these compounds in SZRP which involved in brain disease, mood disorder, endocrine system disease, bipolar disorder, etc. There were 117 potential targets of SZRP against anxiety (such as DRD2, BDNF, COMT, SLC6A4, HTR1A, etc.), which played the roles in regulation of membrane potential and synaptic transmission, nicotine addiction, retinol metabolism, GABAergic and serotonergic synapse, etc. The results of molecular docking indicated there were the biggest anity between Jujuboside B and DRD2, BDNF, COMT, SLC6A4, Anemarsaponin B_qt and HTR1A, n-methylasimilobine and MAPK3. Based on cell CHO-K1/CNR1/Gα15 and calcium ux technique, we found Chrysophanol, Kaempferol and Emodin have activation effects on CNR1 with EC50 138.3µM, 52.28µM and 56.86µM respectively. These results indicated that the effects of SZRP against anxiety were correlated with synaptic transmission, substance addiction and retinol metabolism, especially targeted on CNR1. It suggested that the therapeutic mechanism of SZRP treating anxiety might be due to the weak action of multiple compounds on multiple pathways and biological processes. on molecular the biggest anity were found between Jujuboside B and DRD2, BDNF, COMT, SLC6A4, Anemarsaponin B_qt and HTR1A, n-methylasimilobine and MAPK3. (6) Chrysophanol, Kaempferol and Emodin have activation effects on CNR1 with EC 50 138.3μM, 52.28μM and 56.86μM respectively.


Materials And Methods
The whole work ow was illustrated in Fig. 1. First, known compounds in SZRP were collected from TCMSP, BATMAN-TCM, ETCM, TCMGenenDIT and TCMID databases. TCMSP, BATMAN-TCM and ETCM were used to obtain potential targets of compounds and conduct enrichment analysis by NIMNT and MetaCore. Secondly, the related targets of anxiety were obtained from MetaCore, OMIM, PhamGkb, TTD, CTD and Durgbank. Then the common targets of SZRP and anxiety were enriched for analysis employing NIMNT and MetaCore databases. Finally, AutoDock Vina were used to predict the compound-target group by molecular docking, and the activity of compounds was detected by the calcium ux assay.

Identi cation of known compounds in Suanzaoren prescription
All compounds in the ve herbs of SZRP were collected from TCMSP

Construction of networks
Three networks were constructed by employing the network visualization software Cytoscape 3.7.2.
These networks were herb-compound network (H-C network), compound-targets network (C-T network) and core potential targets of SZRP against anxiety. Isolated nodes do not appear in the external diagram.
The possible inter-protein interactions (PPIs) were derived from STRING database, which covered almost all functional interactions between the expressed proteins. And PPI network pairs with overall combined scores above 0.4 were selected and the isolated nodes were not shown. The PPI network results of STRING were output in TSV format, and then imported into Cytoscape 3.2.1 to screen the core potential targets.

Functional Annotation and Enrichment Analysis
Statistical analysis of functional annotation of biological process, KEGG pathway enrichment and disease enrichment analysis of the potential target for SZRP was performed by MetaCore and NIMNT (http://www.idrug.net.cn/NIMNT/). Biological process and KEGG pathway analysis were conducted for the common potential targets of SZRP and anxiety by MetaCore and NIMNT. Selection of enrichment results was statistically signi cant (Pvalue 0.05) and rank top 50 terms.

Molecular docking simulation
In order to study the active components of SZRP for the prevention and treatment of anxiety and to analyze its possible targets, this study used software AutoDock Vina for molecular docking analysis. The conformation sampling adopts the default optimization parameter, executes each operation in singlethread mode, and adopts the default scoring function using AutoDock Vina. The compounds with molecular weight < 500Da in SZRP were selected for docking using AutoDock Vina. Based on our previous studies on SZRP, we selected the core target of SZRP anti-anxiety and compounds into blood and brain for docking using AutoDock Vina.

Determination of compound activity
We selected ve compounds in SZRP, Isomangiferin (B21543), Liquiritigenin (B20416), Kaempferol (B21126), Chrysophanol (B20238) and Emodin (B20240) (The above compounds were purchased from shanghai yuanye Bio-Technology Co., Ltd, HPLC≥98%) to detect the effect on CNR1 receptor with calcium ux method. The positive compound was CP-55940 (Sigma, C1112 FBS) was added into the wells. Then the plate was placed into a 37°C incubator for 60 minutes, followed by a 15 minutes' incubation (Thermo, 3111) at room temperature. At last, 10 μL compound was added into well of the assay plate during reading in FLIPR tetra (Moleacular Devices). For FLIPR reading, the plate containing 5× compound solution was placed in FLIPR. Solutions were added into the cell plate automatically at the 20 seconds and the uorescence signal was monitored for an additional 100 seconds (21 sec to 120 sec.). Data were recorded by ScreenWorks (version 3.1) as FMD les with FLIPR. Data acquisition and analyses was performed using ScreenWorks (version 3.1) program and exported to Excel. The average value of the rst 20 seconds' reading was calculated as the baseline and the relative uorescent units (ΔRFU) intensity values were calculated by subtracting the average value of baseline from the maximal uorescent units (21s to 120s).
Dose response curves were tted with four-parameter-logistic-equation by the software GraphPad Prism 8. The four parameters logistic equation was: Y=Bottom + (T op-Bottom)/ (1+10^ ((LogEC50-X)*HillSlope)) X is the logarithm of concentration. Y is the response.

Results
In order to collect compounds in SZRP and potential targets of SZRP as much as possible, we used the Chinese name, botanical name and common name of herbs in SZRP to search databases and literatures (Table1).

Potential targets of Suanzaoren prescription
The 305 potential target proteins were obtained from the database TCMSP, after mapped using the STRING database and removed the duplicates, there were 111 potential gene targets for SZRP. For each component in herbs, database BATMAN-TCM ranks its predicted candidate targets based on the order of the reduced scores given by the target-predictive algorithm for herb-target interaction prediction. There were potential 847 gene targets for SZRP with gene score 20 from BATMAN-TCM. We collected 408 potential target genes for the compounds in SZRP from database ETCM. The potential targets of 330 compounds were found. Combining targets of these 3 databases, SZRP had a total of 1050 potential targets were obtained after eliminating duplicates (Table 3) (Additional le 3).
A network of compound-targets (H-T network) for SZRP was constructed (Fig. 3). This C-T network had The disease enrichment analysis of 1050 potential targets of SZRP were performed using NIMNT and MetaCore databases in order to investigate disease potentially treated by SZRP.
The results based on MetaCore database showed the top 10 (P value < 0.001 and from small to large) diseases potentially treated by SZRP were chemically-induced disorders; pathological conditions, signs and symptoms; mental disorders; nutritional and metabolic diseases; psychiatry and psychology; metabolic diseases; movement disorders; genetic diseases, inborn; congenital, hereditary, and neonatal diseases and abnormalities; bipolar and related disorders ( Fig. 4) (Additional le 5).
We conducted a comparative analysis of the top 50 diseases potentially treated by SZRP based on databases NIMNT and MetaCore. There were 11 diseases with overlap (Table 4). They were brain disease, mood disorder, endocrine system disease, bipolar disorder, etc.
Anxiety disorders, the most prevalent mental or mood disorders, is considered a polygenic, multifactorial trait wherein the continuum of physiological anxiety up to psychopathology is likely to be shaped by the interplay of central nervous system, endocrine system and immune system. This disease enrichment analysis suggested that SZRP may be used to treat anxiety.

Targets related to anxiety
We collected targets related to anxiety from six database (Table 5). A total of 67 gene targets related to anxiety were found in OMIM. 7 gene targets for anxiety were found in PharmGkb. The 41 therapeutic targets for anxiety disorders from TTD were converted by STRING. The 50 genetic targets for anxiety disorders were found in CTD. Durgbank has 73 protein targets for anxiety disorders, which are converted to 73 by STRING. The 133 genes associated with anxiety were found in MetaCore. After integrating targets from the six databases and removing duplicates, there were 249 targets related to anxiety (Table  5) (Additional le 6).
We collected 1050 potential targets for compounds in SZRP and 249 targets associated with anxiety disorders. There were 117 targets after overlap between these types of targets (Table 6) (Additional le 7).
We constructed the PPI network related to SZRP for anti-anxiety employing 117 common potential targets ( Fig. 5) (Additional le 8). This PPI network had the average shortest path length 2.06, average degree 23.03 and average neighborhood connectivity 29.24. The target with the highest degree is DRD2, whose degree was 54. Through the double median of Betweenness, Closeness and Degree three parameters, 11 core targets were obtained after four screening. The 5 targets were ranged from the largest to the smallest in order of Degree value, and were DRD2, BDNF, COMT, SLC6A4, HTR1A.

Functional annotation of biological process
The results of NIMNT-based GO-BP enrichment analysis showed that the biological processes of the top 10 (P value < 0.05 and from small to large) included, regulation of postsynaptic membrane potential, regulation of membrane potential, monoamine transport, G protein-coupled receptor signaling pathway coupled to cyclic nucleotide second messenger, response to ammonium ion, response to xenobiotic stimulus, modulation of chemical synaptic transmission, regulation of trans-synaptic signaling, catecholamine transport, regulation of amine transport ( Fig. 6) (Additional le 9).
The enrichment analysis results of Process Networks based on MetaCore showed that the biological processes of the top 10 (P value < 0.05, arranged from small to large) ( Fig. 6) (Additional le 9) include, chemical synaptic transmission, anterograde trans-synaptic signaling, trans-synaptic signaling, synaptic signaling, regulation of postsynaptic membrane potential, cell-cell signaling, regulation of membrane potential, response to organic cyclic compound, system process, regulation of biological quality.
After comparative analysis of the biological processes of the top 20 (P value < 0.05, arranged from small to large) in the two databases, there is 3 biological process after taking the intersection. They were regulation of postsynaptic membrane potential, regulation of membrane potential, and synaptic transmission, GABAergic.
MetaCore based Pathway enrichment analysis results showed that the top 10 (P value < 0.05, arranged from small to large) ( Fig. 7) (Additional le 10) pathways included, tinnitus-associated changes in auditory pathway, protein folding and maturation_insulin processing, mediated direct regulation of xenobiotic metabolizing enzymes, mediated direct regulation of xenobiotic metabolizing enzymes, mediated direct regulation of xenobiotic metabolizing enzymes, mediated direct regulation of xenobiotic metabolizing enzymes, nicotine signaling (general schema), retinol metabolism, estradiol metabolism, serotonin modulation of dopamine release in nicotine addiction.
There were 4 pathways in total after taking the intersection of the top 20 (P value < 0.05, arranged from small to large) pathways in the two databases. The common pathways were nicotine addiction, retinol metabolism, GABAergic and serotonergic synapse.

Molecular docking simulation
According to our previous studies, the components of SZRP into blood and brain were Spinosin, Ononin, There were many studies 19-21 indicated that cannabinoid receptor 1 (CNR1) and mitogen-activated protein kinase 3 (MAPK3) played the key role in the pathophysiological mechanism of anxiety. Therefore, we executed molecular docking of compounds with molecular weight 500Da in SZRP employing AutoDock Vina. Results showed there were the biggest a nity between CNR1 and Isomangiferin, MAPK3 and n-methylasimilobine ( Fig. 8) (Table 7) (Additional le 11).

Cell experiments for validating binding a nities of compounds and targets
According to the results of molecular docking, the mean absolute value of the combined score of the three subtypes of CNR1 (5tgz, 5xra, 5xr8) with compounds was greater. And based on compounds being purchased and reported or not, we selected ve compounds to veri ed the activity of targeting on CNR1 by experiments. These compounds were Liquiritigenin, Isomangiferin, Kaempferol, Emodin and Chrysophanol. The calcium Flux assay was used to detect the activation of these 5 compounds on CNR1

Discussion
In this study, we found that 545 chemical constituents in herbs of SZRP might target on 1050 biomolecules, and play a role in the treatment of brain disease, mood disorder, etc. The effects of SZRP on anti-anxiety may be via regulating membrane potential and synaptic transmission, etc., which involve in 117  The brain derived neurotrophic factor (BDNF) is a critical contributor to neuronal growth, development, learning, and memory 47. BDNF expression associated with the pathophysiology of anxiety 48,49 . Clinical studies have revealed that GAD has been showed lowered plasma BDNF levels 50-52. BDNF also may play a central role in the pathogenesis of bipolar disorder 53. In our previous experimental study of SZRP, it was found that SZRP can improve the BDNF mRNA expression level in insomnia rat, thus improving neuronal damage, improve the symptoms of insomnia 54,55 . And SZRP can also up-regulate the expression of BDNF genes, and has an anti-depressant effect 56.
Catechol-O-methyltransferase (COMT) inactive catecholamine neurotransmitters and catechol hormones, also shortens the biological half-lives of certain neuroactive drugs. Some ndings demonstrated that COMT Val (158) Met polymorphism is associated with risk of GAD via reduced resting parasympathetic nervous control 57 and COMT SNPs also modulated the association between antenatal maternal anxiety 58. Experimental research found that COMT KO mice showed increased anxiety 59.
Sodium-dependent serotonin transporter (SLC6A4) is sodium-dependent serotonin transporter, whose primary function in the central nervous system involves the regulation of serotonergic signaling. SLC6A4 are associated with vulnerability to affective disorders. Clinical case analysis found that, the serotonin transporter gene SLC6A4 involved in the etiology of anxiety-related 60,61 . It was found in some experimental studies that serotonin transporter expression was associated positively with anxiety-like behavior 62, 63 .
5-hydroxytryptamine receptor 1A HTR1A was G-protein coupled receptor for 5-hydroxytryptamine (serotonin) and also function as a receptor for various drugs and psychoactive substances. HTR1A played a role in the regulation of dopamine and 5-hydroxytryptamine levels in the brain, and thereby affect neural activity, mood and behavior. The study showed that mice with 5-HT1AR gene knockout showed anxiety-like behavior 64. 5-HT1AR neurotransmission-related dysfunction has been associated with several psychiatric disorders, including anxiety 65, in 5-HT neurons to repress HTR1A expression and drive adult anxiety-like behaviors 66. SZRP can reduce time for voluntary activities and regulate the expression of 5-HT1AR in the hippocampus of rats, thus improving the sleep state of sleep-deprived rats 67-69.
Mitogen-activated protein kinase 3 MAPK3 is mitogen-activated protein kinase 3, which acts as an essential component of the MAP kinase signal transduction pathway, and thus suppress cancer cells 70.
MAPK3 is also associated with mental illness, such as autism spectrum disorder 71. In this study, biological process enrichment analysis of potential target for shared target indicated that the regulation of membrane potential and GABAergic synaptic transmission might be some mechanism of SZRP anti-anxiety. Regulation of membrane potential refers to the regulation of the potential difference between the two sides of the membrane. Regulation of membrane potential have been implicated in diverse pathophysiological processes, including mood regulation etc. According to experimental research reports, the medial habenula ( an epithalamic structure implicated in anxiety-like behavior) , neurons are capable of ring transient, high-frequency action potential bursts mediated by T-type channels in C57BL/6J mice 82. But an impaired maintenance of long-term potentiation in the hippocampus and display enhanced anxiety-like behavior of Np65 KO mice 83. However, membrane potential increased and anxiety-like behavior were signi cantly relieved after the treatment of G-1(the GPER agonist) 84. Similarly, postsynaptic membrane potential and GABAergic synaptic transmission are closely related to anxiety disorders. Mechanisms underlying the anxiolytic effects of metformin by up-regulated the surface expression of GABAA receptors and increased miniature inhibitory postsynaptic currents 85. The study showed that in EPM model rats treated by SZRP, the OE and OT were signi cantly increased, and the content of GABA was extremely signi cantly elevated 86 and the mRNA expression level of GABAA receptor was up-regulated 14. After being treated by SZRP, the content of GABA in hypothalamus, brainstem and brain tissue increased in insomnia model rat with chronic sleep deprivation, insomnia model rat with liver and blood de ciency and elderly insomnia model rats 87-90. In insomnia rats with fatigue model, SZRP could down-regulate the levels of GABA in the cerebral cortex and hypothalamus 91.

Triterpenoids
It is not reported that SZRP may regulate membrane potential in anxiety.
In additional, pathways enrichment analysis of potential target for shared target of SARP and anxiety suggested that pathway nicotine addiction, retinol metabolism, serotonergic synapse might be regulated by SZRP. Nicotine addiction that means nicotine dependence and addictive behavior. However, nicotine addiction and nicotine withdrawal can lead to anxiety-like behavior 92-94. Experimental research report that activation of the neuronal PPARgamma the receptor for the thiazolidinedione class of medications prevents the expression of signs of nicotine withdrawal 95 and increased expression of CRF (Corticotropin-Releasing Factor) and CRF-receptors in certain areas of the brain after the development of intraperitoneal nicotine administrations in rats 96. Interestingly, the hippocampus seems to be closely linked to nicotine 97-99 and is also known to be associated with anxiety 100-102. Retinol (vitamin A metabolism refers to retinol comes out of the liver and binds to retinol-binding proteins, which are transported to various tissues of the body through the blood for normal metabolism. Retinol metabolism is involved in oxidative processes 103 , 104 in body, and is also associated with anxiety. Retinol induced anxiety-like behavior in adult rats chronically subjected to vitamin A supplementation 104 , 105. Serotonin (5-HT) is an important neurotransmitter, which is high in the cerebral cortex and synapses. Serotonin in the brain controls emotional regulation, cognitive function and other functions. Neurotransmitters such as serotonin and synapses are known to play an important role in the development of anxiety 106. It was found that the integrity of serotonergic synapses and anxiety related phenotype in mice 107. It was worth noting that only a few studies had considered relationship between pre-and post-synaptic serotonergic transmission. Anxiety caused by serotonergic synapse was due to an imbalance of serotonergic synapse regulated mRNAs expression, such as 5-HTR 2A is upregulated 108, 5-HTR 1A down-regulated 109.
It was found that SZRP exhibited binding a nity for serotonin receptors on the basis of Ultraperformance liquid-chromatography/electrospray-ionization synapt high-de nition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) combined with pattern recognition approaches 110. A clinical study reported that the treatment of SZRP increased the serum 5-HT content in patients with liver blood de ciency insomnia 111. In the EPM rat model of anxiety, the content of 5-HIAA in the hippocampus and thymus of SZRP group was signi cantly higher than that of the model group 11 , 16 , 86. And the content of 5-HT and 5-HIAA in serum, hypothalamus, hippocampus, cerebral cortex and brain stem increased in model rats of

Conclusions
In this study, the therapeutic components and mechanism of SZRP for anxiety disorders were investigated by using network pharmacology combined with experimental veri cation. The main ndings were as follows: (1) the 525 compounds and 1050 potential targets in SZRP were collected, and SZRP might be used to treat brain disease, mood disorder, endocrine system disease, bipolar disorder, etc. (2) the 249 potential targets of anxiety were obtained, and 117 possible targets of SZRP against anxiety were found, for example, DRD2, BDNF, COMT, SLC6A4, HTR1A and other biological molecules. However, there are limitations to this study. The ingredients in SZRP and anxiety-related genes may not be global or full. We did not verify all the a nity between compounds and targets employing experiments. In conclusion, this study provided theoretical basis and clues for the study on the pharmacological mechanism of SZRP against anxiety. The work ow of network pharmacology approach to investigate the mechanisms of Suanzaoren prescription against anxiety.

Figure 4
The top 50 diseases enriched for the potential targets of the Suanzaoren prescription. A. Diseases enrichment from NIMNT; B. Diseases enrichment from MetaCore; Select the top 50 for visualization. Xaxis shows the -lg (P value) of the terms; Y-axis shows the different diseases (P<0.001). * mean that the disease is related to nervous system or emotional disorder.

Figure 5
The protein-protein interaction network related to Suanzaoren prescription for anti-anxiety. This network had 116 nodes and 1336 edges (The one isolated hide disconnected node was not shown in this network). The red node is one with top 11 degree.

Supplementary Files
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