Characterization of the bioactive compounds with efficacy against epilepsy from the herb pairs Polygala tenuifolia - Zizyphus jujuba  by modulating CHRNA4/CaMKII signaling pathway: LC-MS/MS combined with network pharmacology analysis and experimental evidence

doi:10.21203/rs.3.rs-2698704/v1

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Characterization of the bioactive compounds with efficacy against epilepsy from the herb pairs Polygala tenuifolia - Zizyphus jujuba by modulating CHRNA4/CaMKII signaling pathway: LC-MS/MS combined with network pharmacology analysis and experimental evidence

https://doi.org/10.21203/rs.3.rs-2698704/v1

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Background

Epilepsy is a typical nervous system disorders identified by the spontaneous recurrence of seizures which injure periods of electroencephalographic activity and behavior. Traditional Chinese Medicine (TCM) herb pairs Polygala tenuifolia and Zizyphus jujuba have been used in treatment of epilepsy in China, while the mechanism of action still remains unclear. This article aims to disclose the substances and potential mechanisms of the anti-epilepsy activity of P. tenuifolia and Z. jujuba extract (PZE) using LC-MS/MS, network pharmacology, ethology and molecular biology methods.

Methods

With the help of the self-built components database, identification of the chemical parameters of PZE was possessed through LC-MS/MS method, and the “ingredient-target-pathway” network of PZE was established through online databeses. Molecular docking was performed using Discovery Studio Visualizer. In the setting of the epilepsy model, pentylenetetrazol (PTZ, 10 mg/kg) was administered intraperitoneally injected for a period of 21 days. Mice were assessed for anxiety-like behavior by Elevated plus maze test, open field test, forced swimming test and tail suspension test. HE staining, western blotting, and immunofluorescence staining were used to detect morphological changes and signal pathway.

Results

Through network analysis, 37 active ingredients were obtained from PZE, SLC6A4, CHRNA4 and MAOA and were found to play a major role in the PPI network. GO and KEGG analyses that display their anti-epilepsy activity. The"Ingredient-target-pathway"network diagram consists of 99 targets, 24 kinds of constituents, and 20 signaling pathways. The values of M15 and M17 show the largest degree. Molecular docking analysis shows the key components screened by network pharmacology have a good interaction with the predicted targets. Animal experiments results showed that: 1) PZE effectively lengthened the latent time of PTZ-induced epilepsy in mice model. 2) PTZ-induced depression-like behavior was strikingly ameliorated by PZE. 3) Hippocampal neurons are significantly shielded by PZE. 4) PZE was shown to play a key role in modulating the CHRNA4/CaMK II signaling pathway in to show anti-epilepsy potency.

Conclusion

This study has successfully identified constituents of PZE through LC-MS/MS methods and predicted the potential targets and CHRNA4/CaMK II as potential signaling pathways of anti-epilepsy effects for PZE, which was proved by animal experiments. The results of this paper are conducive to the systematic elucidating of its mechanism of action and the development of TCM-based anti-epilepsy agents.

Polygala tenuifolia

Zizyphus jujuba

LC-MS/MS

epilepsy

network pharmacology

Epilepsy is a severe and chronic neurological disorder of the brain, resulting in motor, consciousness, sensory and other disorders, its damage is persistent. The life and work of patients is greatly affected by the features of recurrent seizures and transient central nervous system dysfunction. The survey estimates that there are over 70 million patients with epilepsy worldwide, and the number of patients is on the rise every year [1, 2]. The current conventional treatment for epilepsy is antiepileptic drugs, approximately 40% of patients worldwide have achieved the best results, but there are still features of seizures and complications. And many antiepileptics have significant side effects, severely limiting the use of medications. All procedures used to date for fully diagnosed patients can only provide symptomatic relief, and there are no medications that can be used for prevention purposes [3]. Recent studies have demonstrated that the treatment of epilepsy by Chinese medicine. Traditional Chinese Medicine (TCM) offers a new perspective in this area [4, 5]. According to our previous studies [6–8], we have excavated anticonvulsant precursors TCM herb pairs Polygala tenuifolia and Gastrodia elata, which provided the evidence of the anti-epilepsy potency for P. tenuifolia.

For many decades, TCM has been used to effectively treat epilepsy in China. TCM is a treasure house provide invaluable therapia for diseases such as epilepsy. The aim of this paper is to investigate the anti-epilepsy potential of TCM herb pairs P. tenuifolia and Zizyphus jujuba (PZ) through the pharmacology of LC/MS-based networks. Pairs of herbs were recorded in multiple TCM prescriptions using to treating epilepsy including Zaoren Yuanzhi Tang (枣仁远志汤) from Zheng Yin Mai Zhi (症因脉治, 1700 AD), Yangxue Qingxin Tang (养血清心汤) from Wanbing Huichun (万病回春, 1600 AD) and Ding Xian Dan (定痫丹) from Yizong Jinjian (医宗金鉴, 1700 AD). In Chinese patent medicines, the herb pairs P. tenuifolia and Z. jujuba (PZ) were also widely recorded, such as Ding Xin Wan (定心丸, approved number: Z42021255), Xianzheng Zhenxin Wan (痫症镇心丸, approved number: Z33020108) and Zhen Xian Pian (镇痫片, approved number: Z20063511). Traditionally, the roots of P. tenuifolia and the seeds of Z. jujuba were recorded as the medicinal parts for the treatment of insomnia, palpitation and epilepsy for their tonic effects on heart and sedative efficacy the brain. Contemporary pharmacological research has revealed the therapeutic potential of PZ on diseases of the central nervous system diseases such as Alzheimer's disease, Parkinson's disease, neuroinflammation and epilepsy [9–12]. To date, however, the potential mechanism underlying the anti-epilepsy action of PZ remains unclear, and this is the major difficulty in restricting its standardized application.

Network pharmacology is a new system for drug research, particularly suited to medicinal herbs with multi-components like TCM [13]. According to the results of “disease-pathway-target-drug” interaction network, the intervention and influence of drugs on the disease mechanism network can be systematically observed. Therefore, in this article, we investigate the potential mechanism by which PZ exerts its effect on anti-epilepsy targets and pathway using this method. Comparing to other data mining methods, the liquidchromatography-massspectrometry/ massspectrometry (LC-MS/MS) identification of the chemical constitutions and molecular docking analysis of predicted targets make the results more reasonable. Figure 1 describes the workflow of LC/MS and network pharmacology parts of this article. In the present study, we characterized the chemical constitutions of extracts of P. tenuifolia and Z. jujuba (PZE) and summarized the constitunts-targets-pathways relationships of PZE. We were able to construct the possible interaction between targets and PZE's epilepsy-related target network. In addition, we analyzed relative pathways to recapitulate the related mechanism of action of PZE against epilepsy, and the CHRNA4/CaMKП signaling pathway was chosen and proven by molecular biological methods.

Insert Fig. 1.

2.1. Reagents and materials

The dried roots of Polygala tenuifolia Willd. (batch number: 20210328) and seeds of Ziziphus jujuba Mill. (batch number: 20210325) were purchased from Bozhou (Anhui province of China, a city famous for TCM herbs). The samples were authenticated by Professor Qiang Wang (Shaanxi University of Traditional Chinese Medicine). PTZ was purchased from Alfa Aesar, Shanghai, China. Lot: 10180463; Formic acid and methyl alcohol, used in HPLC-QTOF-MS analysis, were obtained from Fisher Sigma-Aldrich (SigmaAldrich, MO, USA), respectively.

For the preparation of extracts, coarse herbal crushing, 80 mesh sieve. Then, 10.0 g powder of P. tenuifolia and 10.0 g powder of Z. jujuba (according to the proportion of Zaoren Yuanzhi Tang) was added in 100.0 mL of 95% ethanol, then the extracts was possessed by reflux extraction for 2.0 h at 80 ℃ and the solution was concentrated in vacuo to abtain the extracts [14]. The extracts were diluted with methanol to a concentration of 10 mg/mL for the HPLC-QTOF-MS/MS analysis.

2.2. The conditition of HPLC-QTOF-MS/MS and Data analysis

HPLC-QTOF-MS/MS analyses were possessed on a Triple TOF™ 5600⁺ system (AB SCIEX, CA, USA). Both the positive and negative ion modes were selected for MS data acquisition of PZE. The separation of the components in PZE were detailly recorded in the Supporting information.

The systematic information on ingredients from PZE was collected and organized by searching the published phytochemical literatures and databases including MassBank and SciFinder scholar. etc [15]. Therefore, a components database containing compound names was established, and chemical formulas of each compound was established to provide a basis for further structural identification.

2.3. Construction and analysis of PZE compounds-disease target interaction network

The chemical formulas of the compounds in PZE were imported into the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to know information about SMILES, and the potential targets were predicted by using SwissTargetPrediction database (www.swisstargetprediction.ch), the details were recorded in the Supporting information.

2.4. Acquisition of potential anti-epilepsy targets and the Protein-Protein interaction (PPI) network construction

The construction of PPI network mainly used database of DisGeNET (http://210.107.182.61/geneSearch/) and STRING (https://string-db.org/), the details were recorded in the Supporting information.

2.5. GO annotation and KEGG enrichment analysis

With the help of Metascape (metascape.org) web-based portal for comprehensive gene annotation and analysis resources [16], we annotated and analysised the Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and the detailed information were recorded in the Supporting information.

2.6. Ingredien -target-pathway network construction

The ingredient-target-pathway network was constructed by introducing PZE targets, pathways and 24 kinds of active ingredients screened out in Cytoscape software. Nodes of different colors mean different types of clusters, while the edges represented the relationships between the nodes.

2.7. Molecular docking

The crystal structure of core target proteins for molecular docking in the PPI network was from PDB database (www.rcsb.org) [17]. The Discovery Studio Client software usage to add hydrogen and partial charges to the proteins and ligands [18]. The results of the docking computations were ranked by the binding affinity (-kcal/mol). We visualized conformations and interactions between the ligands and the target proteins by Discovery Studio Visualizer.

2.9. Animals and group

Our experiment was authorized by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University, fifty young adults male Kunming-strain mice (22–28 g). All mice were divided into five groups at random, and each group contains 10 animals: 1) normal saline group (NS); 2) pentylenetetrazol group (PTZ); 3) treatment group were divided into three group, included high (PZEH 300mg/kg), medium (PZEM 100 mg/kg) and low (PZEL 30mg/kg) dose group.

2.10 Establish mice epilepsy model and sc-PTZ-induced epilepsy

Epilepsy model was established by intraperitoneal (i.p.) injection of 10 mg/kg PTZ for 21 days [19]. PTZ (10 mg/kg) was injected intraperitoneslly 60 min after oral administration of PZE in the treatment group. The NS group was only injected with saline. Subcutaneous (s.c.) injection of 85 mg/kg PTZ solution 60 min after the last treatment. The latent period and incidence of tonic convulsion were observed 30 min after PTZ injection. The latency of each seizure were recorded, and the average values were calculated for statistical analysis.

2.11. The behavioral animal tests

The fortieth day after the beginning of the experiment, four behavioral tests usually employed in studies on laboratory.

2.11.1 Elevated plus maze test (EPMT)

The EPMT experiment is appiled to evaluated anxiety-like behavior and was conducted as described previously [20]. The details of the test were recorded in the Supporting information.

2.11.2. open field test (OFT)

OFT experiment is appiled to evaluated locomotor activity and anxiety-like behavior [21]. The test was analysed for 5 min in a dimly lit room, as described previously [22–24]. The details of the test were recorded in the Supporting information.

2.11.3. Forced swimming test (FST)

FST was possessed referred to the previously research [25]. The details of the test were recorded in the Supporting information.

2.11.4. Tail suspension test (TST)

FST was possessed referred to the previously research [26]. The details of the test were recorded in the Supporting information.

2.12. Histological observation of brain tissue

2.12.1. Hematoxylin and eosin (HE) staining

HE staining was carried out to observe the changes of pathological tissue in brain. The details of the experiment was recorded in the Supporting information.

2.12.2. Immunofluorescence staining

Immunofluorescence staining was possessed to observe the levels of target proteins including CHRNA4 and CaMKП in brain. The details of the experiment was recorded in the Supporting information.

2.12.3. Western blot analysis

Western blotting methods was used to analyze the protein levels of target signaling pathway: CHRNA4/CaMKП/ERK1/2/CREB1-related proteins. The details of the experiment were recorded in the Supporting information.

2.13. Data analysis

The data of results were analyzed through GraphPad Prism 8.0.2 software and described as the mean ± SEM. The differences between control and BPA-treated groups were approximated using one-way ANOVA followed by a Tukey’s test for multiple comparisons. P < 0.05 was deemed to have a statistical signifcance.

3.1. Identification of compounds from PZE

The base peak chromatograms (BPC) of PZE in positive and negative modes are exhibited in Fig. 2. A total of 37 compounds were characterized using a self-constructed database analysis strategy, and the properties of their candidate compounds are shown in Fig. 2. It contained information on retention time, molecular formulas, mass weight, mass errors and fragments of major components, as shown in Table 1.

Insert Table 1. and Fig. 2.

3.2. Identifcation of compounds from PZE

To date, 37 kinds of constituents have been identified from the PZE, among them, xanthones, glycolipids and acids are the main types. All compounds are summarized and recorded in Table 1. Herein, we provide the analytical process of two kinds of representative constituents as the internal standard substances recorded in the Chinese Pharmacopoeia. As we can see from the Fig. 3, polygalaxanthone III (M2) was a classic xanthone from P. tenuifolia with molecular formula of C₂₅H₂₈O₁₅ as inferred from the [M-H]⁻ (m/z: 567.1342). In the second stage mass spectrum detected, the daughter ions at m/z 59.0143 (C₂H₃O₂⁻), 89.0248 (C₃H₅O₃⁻), 271.0254 (C₁₄H₇O₆⁻), 273.0415 (C₁₄H₉O₆⁻), 315.0504 (C₁₆H₁₁O₇⁻), 345.0604 (C₁₇H₁₃O₈⁻), 417.0816 (C₂₀H₁₇O₁₀⁻), 435.0924 (C₂₀H₁₉O₁₁⁻), 447.0915 (C₂₁H₁₉O₁₁⁻) in negative mass mode provided the evidence for the identification of the component.

Insert Fig. 3.

As for the constituents from Z. jujuba, typical compound spinosin (M28) recorded in the Chinese Pharmacopoeia as internal standard substance was detected with the chemical formula of C₂₈H₃₂O₁₅ based on the [M + H]⁺ ion at m/z 609.1784 and [M–H]⁻ ion at m/z 607.1675. In the second stage mass spectrum (Fig. 4), the daughter ions at m/z 297.0752 (C₁₇H₁₃O₅⁺), 327.0854 (C₁₂H₂₃O₁₀⁺), 351.0847 (C₁₆H₁₅O₉⁺), 411.1058 (C₂₂H₁₉O₈⁺), 429.1156 (C₂₂H₂₁O₉⁺), 447.1262 (C₂₂H₂₃O₁₀⁺) in positive mass mode and 427.1041 (C₂₂H₁₉O₉⁻) in positive mass mode give the inspiration of the spectrum analysis.

Insert Fig. 4.

3.3. Acquisition of targets for PZE ingredients and anti-epilepsy

The results of the network construction showed: Screening of 24 core anti-epilepsy components from andidate ingredients by preliminary search. Generally, a total of 527 ingredient-related targets were identified from the SwissTargetPrediction databases. A total of 450 epilepsy-related targets were identified from DisGeNET (Score-gda ≥ 0.05) and GeneCards (Relevance score ≥ 9.21)

Insert Fig. 5.

3.4. Protein-protein interaction (PPI) network analysis results

To obtain the PPI function network, the top targets were entered into the STRING data platform. Of the 55 targets that were correlated, 48 targets had average interactions (with a confidence score ≥ 0.4) in the PPI map generated by the STRING database. The targets of CA5B、CA5A、CA6、PTPRF、PTPN1 and ACP1 targets that were not bound to the lattice were dislodged, and the Cytoscape software was used to own the topology analysis to screen for significant central targets in the network. As shown in Fig. 6, the node size and color settings mirror the size, while the edge thickness and color settings reflect the combined score. When the color of the node closer to red, the corresponding value is higher. The results of the topological analysis displayed that the three targets with a higher degree value among the anti-epilepsy targets of neuronal acetylcholine receptor subunit α-4 (SLC6A4, degree = 25), neuronal acetylcholine receptor subunit α-4 (CHRNA4, degree = 22) and monoamine oxidase A (MAOA, degree = 21), which played a major role in the network.

Insert Fig. 6.

3.5. Molecular mechanism of anti-epilepsy the effect of PZE

We carried out a Metascape enrichment analysis of the 55 duplicate genes for GO and KEGG and achieved 583 biological processes (BP), 48 cellular ingredients (CC) and 68 molecular functions (MF) under the GO entry. The p-value determines the result of the correlation test [53], sort the top 20 of each category by - LogP value from large to small (Fig. 7). The top rank belonged to the vasoconstriction, regulation of tube diameter and blood vessel diameter maintenance enrichment in BP’s, synaptic membrane, dendrite and integral component of synaptic membrane enrichment in CC’s and G protein-coupled amine receptor activity, carbonate dehydratase activity, neurotransmitter receptor activity in MF’s.

The KEGG enrichment analysis of potential anti-epilepsy targets producted 32 signal pathways, which were ranked on the basis of the -log₁₀^p value from small to large, of which 20 pathways were relative to epilepsy (Fig. 8). Of the pathways related to epilepsy, Nitrogen metabolism has a high degree of enrichment, and can be considered as the main anti-epilepsy pathway. The significance of the count and the -log₁₀^p value represent a highly correlated way in which active constitutuents display their antiepileptic activities.

Insert Fig. 7. and Fig. 8.

3.6. Construction of the ingredient-target-pathway network

We adopt a network of 99 nodes and 240 edges by Cytoscape software (Fig. 6). In particular, the nodes consisted of 24 kinds of puffball constituents, 55 kinds of potential PZE targets, and 20 signal paths. It can be seen that each active component corresponds to multiple targets in multiple pathways (Fig. 9), which fully mirrors the multi-ingredient, multi-target and multi-pathway mechanisms of the puffball anti-epilepsy activity. With respect to the component attribute nodes in the network graph, it has been reported that M17 (Tetrahydrocolumbamine, degree value 12) and M15 (O-hydroxybenzoic acid, degree value 11) had the highest degree value; each of these components can be thought of as the central nodes in the network with a major anti-epilepsy effect.

Insert Fig. 9.

3.7. Molecular docking analysis

Under the prediction of molecular simulation docking methods, it can be seen that the key components screened by the pharmacology of the network have a good interplay with the predicted targets. They have a high binding energy and can successfully penetrate into the binding pocket of the target molecule in order to interact with key handicaps in the form of intermolecular hydrogen bonds. The cinnamic acids showed a good interaction with monoamine oxidase A (MAOA) in terms of their Structure-Activity Relationship, xanthones showed favorable interaction with nicotinic-acetylcholine receptor α4 (nAChR4), and saponins displayed preferable interaction with 5-HT transporter (SERT). Molecular construction and docking analysis between 2Z5Y and M16, M18, M20, and M37 were performed; between 6AWN and M17, M22, M25, M35; and between 5KXI and M18, M20, M31, M32-all of which are listed below. The lowest energy predicted complexes are stable intermolecular hydrogen bonding and stacking interactions. Interactions in the complex are decided by steric hindrance and the functional group category. We can see from Table 2 that the docking results are basically in agreement with the structure-function relationship, the substituted cinnamyl group on the phenolic hydroxyl group of vanillyl moiety helped enormously in promoting the binding affinity of the enzyme-inhibitor complexes, the ester linkage played key roles in the interaction with the protein. In terms of the interaction energy, the result displayed that compound M16 and M20 outperformed M18 and M37 in 5Z5Y, M22 and M25 outperformed M17 and M35 in 6AWN, M20 outperformed M18, M31 and M32 in 5KXI. Additionally, M20 displayed the best interaction with the protein 5Z5Y, which was partially different with the result of the enzyme inhibition assay, in general, active 5Z5Y residues comprising TYR 444, ILE 335, ILE 180 can interact with M16, M18, and M37 to a different extent (Fig. 10) [54–56]; in 6AWN that M25 showed the best interaction with the protein than M17, M22 and M25, the active residues of including ASP 98, SER 336, ALA 96, TYR 176, SER 438 can interact with M17, M22 and M25 in different degree (Fig. 10); in 5KXI that M20 demonstrated the best interaction with the protein than M18, M31 and M32, the active residues of including TRP 156, LEU 121, TYR 204, CYS 199 can interact with M18, M31 and M32 in different degree (Fig. 10).

Insert Fig. 10. and Fig. 11.

3.8. Anti-epilepsy Pathway

This study divulges that PZE activation of nAChRs can repair synaptic related protein levels. nAChRs belong to ligand gated ion channel coupled receptors, and form a centrally located ion channel, which primarily modulates Ca²⁺ plasma flow in and out of the cell. Calcium ions, acting as cytoplasmic second messengers, flow into cells via α4 nAChRs and connect with CaM to form an active CaM complex. The CaM complex may conjugate CaMK II, which regulates the spatial conformation of CaMK II and phosphorylates the CaMK II protein. Phosphorylated CaMK II enters the nucleus to catalyse the phosphorylation of Extracellular signal-regulated kinase (ERK) and cAMP responsive element binding protein (CREB). ERK and CREB are downstream of the α4 nAChR signal transduction pathway and serve as transcription factors that regulate neuron growth and development as well as the formation of long term memory (Fig. 12).

Insert Figue 12.

3.9. The efficacy and latency of PTZ

Convulsions were caused by an intraperitoneal (i.p.) injection of PTZ (10 mg/kg) dissolved in saline. Compared with PTZ group, after different degrees of PZE treatment, the latency time of PZEH, PZEM and PZEL was significantly prolonged in Fig. 13.

Insert Figue 13.

3.10. The behavioral animal tests

To assess anxiety-like and depression-like behaviors in PTZ-induced epilepsy mice using the EPMT, OFT, FST and TST. After 15 days of PZE intervention, the EPMT results showed that compared with NS group, although the number and timing of entry into the open arms of PTZ-induced epilepsy model mice was significantly lower than that of NS group mice. In comparison to the PTZ group, the number and d timing of mice entering the open arm was increased in the PZEH, PZEM, and PZEL groups (Fig. 14C&D), the anxiety and depression-like behaviors were improved, it indicated that PTZ could induce anxiety and depression-like behavior in mice. The OFT result indicated that compared with the NS group, there showed striking differences in the total movement distance (Fig. 15E), the number of entering enter central entry (Fig. 15F), the residence time in the central area (Fig. 15G) and the number of feces (Fig. 15H) in mice with epilepsy induced by PTZ, the intervention of PZEH and PZEM group has significant improvement effect on the above indexes. FST result manifested that compared with NS group, the time of immobility of PTZ group was prolonged and the depression was obvious, when given PZE, the immobility time of PZEH, PZEM, and PZEL group decreased, the difference was statistically remarkable (Fig. 16I, P < 0.05). The difference in TST resting time of mice in each group was statistically significant, the resting time of PTZ group was higher than that of NS group. After PZE treatment, the immobility time of PZEH, PZEM, and PZEL group decreased (Fig. 16J). These findings suggested that PZE can ameliorate the depressive response in mice and has antidepressant effects.

Insert Figue 14., Figue 15. and Figue 16.

3.11. Histological observation of brain tissue

3.11.1. Hematoxylin and eosin (HE) staining

HE staining was used to determine the lesion effect of hippocampal neuros in mice exposed to BPA. The cells of the hippocampus cornuammon1 (CA1), and cornu ammon 3 (CA3), dentate gyrus (DG), and Temporal cortex (TeA) of mice in the NS group were arranged in a regular and compact manner, as shown in Fig. 17 by light microscopy. In the NS group, many pyramidal cells appeared smaller with small condensed nuclei compared to the mice. In contrast, the hippocampal structure of mice in the PZEH and PZEM groups was essentially normal, and nerve cell degeneration in the large hippocampal region was less than in the PTZ group.

Insert Figue 17.

3.11.2. Immunofluorescence staining

To investigate the role of CHRNA4/CaMK II in Epilepsy, we first examined the expression of CHRNA4/CaMK II in the hippocampus tissues through immunofluorescence (Fig. 18). Figure 18 is showing that the protein of CHRNA4 and CaMK II were mainly expressed within TeA, CA1 and CA3 regions. Compared with the NS group, the proportion of CHRNA4 positive cells decreased significantly in PTZ group, the level of CHRNA4 in hippocampus increased rapidly in PZEH and PZEM group after PTZ-induced epilepsy, which is approximate to control (Fig. 18K). Oppositely, compared with the NS group, the proportion of CaMK II positive cells increased significantly in PTZ group, The level of CaMK II in hippocampus decreased rapidly in PZEH and PZEM group after PTZ-induced epilepsy, which is approximate to control (Fig. 18L). These experimental results in vitro and in vivo showed that PZE could reduce the epilepsy response and reduce the loss of neuronal cells by inhibiting the CHRNA4/CaMK II signal pathway.

Insert Figue 18

3.11.3. Western blot analysis

To explore the effect of PZE on the CHRNA4/CaMKП signaling pathway, Western blot was used to determine the protein levels of CHRNA4, CaMKП, ERK_1/2, CREB1 in hippocampus tissues (Fig. 19). Further analysis of relative protein expression was performed using the optical density of the band. We found that the protein levels of CaMK II and CREB1 were significantly decreased in the PTZ group and that these increases were blunted by treatment with PZE (Fig. 19N & P, p < 0.01). Upon further comparison, it was found that the protein levels of CaMK II and CREB1 in the PZEH group were significantly higher than those in the PZEL group (p < 0.01). CHRNA4 and ERK_1/2 expression levels in the PTZ group were both higher than those in the NS group, upon further comparison, we found that the protein levels of CHRNA4 and ERK_1/2 in the PZEH group and in the PZEM group were strikingly lower than those in the PTZ group (Fig. 19M & O, p < 0.01). Together, these data suggested that PZE may protect against neuronal injury. The results of western blot analysis reveal that PZE can regulate the expression of the CHRNA4/CaMKП signaling pathway on epilepsy induced by PTZ in mice.

Insert Fig. 19.

Epilepsy is a chronic encephalopathy that is induced by ictal firing of neurons in the brain. Today, the pathogenesis of epilepsy remains poorly defined. Current antiepileptic drugs mainly suppress neuronal excitability via the regulation of neurotransmitters (like glutamate, γ-aminobutyric acid and acetylcholine) or ion channels [57–59] have been shown to play a role in the formation of ion channels. Pharmaceuticals extracted from botanical products have been considered as a therapy for epilepsy with few side-effects [60]. Studies in animal models of plant epilepsy over the last few decades have shown that medicinal plants have the potential to become a richer source of safer and more efficacious antiepileptic drugs. Botanical treatments can offer significant benefits to patients with drug resistant epilepsy by reducing adverse effects and improving quality of life [61].

The aim of this study was to evaluate the therapic power of PZE on epilepsy. The constituents of PZE were characterized by LC-MS/MS and the potential signaling pathway was predicted by network pharmacology methods, which were seen as a useful tool for connecting ingredients and potential targets. The power of PZE on epilepsy was proven by the PTZ-induced mouse model of epilepsy.

We measured spatial learning and memory after successful establishment of mouse models of epilepsy. Anxiety and depression-like behaviours were enhanced, it indicated that PTZ was able to induce anxiety and depression-like behaviour in mice [62, 63]. The hippocampus is a component of the limbic system and has been shown to play an important role in learning and memory [64]. Hippocampal sclerosis, hippocampal atrophy, and hippocampal sclerosis are the major pathological changes in epilepsy, primarily manifesting as neuronal loss and glial cell proliferation [65]. In the present study, we explored the effect of PZE on mice with epilepsy from hippocampal pathophysiological processes. In this study, HE staining showed that PZE could reduce the apoptosis of neurons and the loss of neurons in the CA1 region of the hippocampus, CA3 region, TeA region, and DG region of epileptic mice. In this experiment, PZE demonstrated both sedative and anticonvulsant effects.

PTZ is a commonly used experimental animal model of inflammation, which is an antagonist of the γ-aminobutyric acid (GABA) receptor and is easy to cross the blood brain barrier. This model is known to be a model for the histopathology and cytopathology of epilepsy [66, 67]. The most common and intuitive parameter for observing antiepileptic activity is latent time. Herein, we provided preliminary evidence for the anti-epilepsy activity of PZE by extending the latent period in the PTZ-induced mouse model to provide pharmacodynamic evidence.

Research on signaling pathways through molecular biology tools are widely used in the investigation of potential therapic mechanisms of medicinal herbs, herein, the CHRNA4/CaMKП/ERK_1/2/CREB1 pathway predicted by network pharmacology was chosen as a witness in this study. In this paper, the results of signaling pathway studies proved the reliability of network pharmacology based on LC-MS/MS as well. Acetylcholine (ACh) is a major neurotransmitter. Depending on the different sensitivities of the signal transducing receptors to muscarinic acid or nicotine, it mediates fast synaptic transmission within ms. According to the location can be divided into: Presynaptic nicotinic Acetylcholine receptorn (nAChR), postsynaptic nAChR and non-synaptic nAChR. Activation of the pre-synaptic nAChRs can lead to increased neurotransmitter release, Postsynaptic nAChRs can rapidly transduce cellular excitability, however, non-synaptic nAChRs play an important role in neuronal excitability and neurotransmitter regulation [68].

nAChR is involved in many physiological functions such as neuronal excitability and neurotransmitter release, and plays a key role in learning and cognitive memory. The nAChRs are widely and patchily distributed in the brain and are closely linked to a range of central nervous system diseases including epilepsy, Alzheimer ' s disease, Parkinson ' s disease and schizophrenia [69]. Its permeability to sodium, potassium, calcium, and other ions, as well as its regulation of excitatory and inhibitory neurotransmitter release. This specific regulation is dependent upon the difference in nAChR expression in excitatory and inhibitory neurons. A large body of research has shown that mutation of nAChR-related coding genes is relative to the occurrence and development of epilepsy. Changes in the gene coding for nAChRs can lead to changes in nAChR-related structure and function, leading to changes in the local brain potential, resulting in the development of epilepsy [70, 71]. When the gene encoding nAChR changes, it may cause changes in nAChR-related structure and function, resulting in changes in brain local potential, resulting in epilepsy.

CHRNA4 consists of six exons, located on chromosome 20q13.33, approximately 17kb, it primarily encodes the α-4 subunit of the neuronal acetylcholine receptor, and its coding region is primarily located in exon 5. The first proven pathogenic ADSHE-related gene is CHRNA4 [72], which has been shown to be associated with cancer. Sporadic sleep-related high-exercise epilepsy previously known as sporadic nocturnal frontal lobe epilepsy previously known as sporadic nocturnal frontal lobe epilepsy has similar clinical features to ADSHE [73].

CaMK II is a multifunctional serine/threonine protein kinase that is found in nearly every subcellular region of the brain and in neurons [74]. CaMK II primarily exists in neuronal tissues, representing 2% of total protein in certain regions such as the hippocampus of rats. Calcium-sensitive CaMK II may be directly activated when Ca2 + in nerve cells is increased. The activated CaMK II can act on a variety of substrates, causing extensive biological effects, such as affecting synaptic reconstruction, neurotransmitter release, hippocampal formation and other plastic changes, which is essential in the occurrence and development of epilepsy.

In summary, PZE significantly extended the latency time of PTZ-induced seizure, simultaneously modulated the expression of proteins related to the nAchR/CaMK II signaling pathway, and ameliorated the impaired learning and memory caused by hippocampal neuronal damage in epileptic mice.

In short, PZE decreased the intensity and duration of PTZ-induced seizures, restrained the activation of microglia and astrocytes, and inhibited the expression of CHRNA4/CaMK II pathways, while activated ERK_1/2/CREB1 pathway, and promoted the ratio of neuronal survival in relative encephalic region. This research uncovers the potential protective potency and mechanisms of PZE on brain damage after developmental epilepsy through the network pharmacology, ethology and molecular biology studies, with the efficacy substances characterized by LC-MS/MS methods. This research proposes a theoretical basis for the development for anti-epileptic agents excavated from TCM.

TCM

Traditional Chinese Medicine

P. tenuifolia and Zizyphus jujuba

LC-MS/MS

liquidchromatography-massspectrometry/massspectrometry

PZE

the extracts of P. tenuifolia and Z. jujuba

CHRNA4

Cholinergic receptor nicotinic alpha 4

CaMK II

Calcium CaM-dependent protein kinase II

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

Normal saline group

PTZ

Pentylenetetrazol group

PZEH

the high dose extracts of P. tenuifolia and Z. jujuba

PZEM

the medium dose extracts of P. tenuifolia and Z. jujuba

PZEL

the low dose extracts of P. tenuifolia and Z. jujuba

EPMT

Elevated plus maze test

OFT

open field test

FST

Forced swimming test

TST

Tail suspension test

Hematoxylin and eosin

BPC

The base peak chromatograms

PPI

Protein-protein interaction

SLC6A4

Neuronal acetylcholine receptor subunit α-4

CHRNA4

Neuronal acetylcholine receptor subunit α-4

MAOA

Monoamine oxidase A

Biological processes

Cellular ingredients

Molecular functions

SERT

Saponins displayed preferable interaction with 5-HT transporter

ERK

Extracellular signal-regulated kinase

CREB

cAMP responsive element binding protein

CA1

Cornuammon 1

CA3

Cornu ammon 3

Dentate gyrus

TeA

Temporal cortex

GABA

antagonist of the γ-aminobutyric acid

Ach

Acetylcholine

nAchR

nicotinic Acetylcholine receptor

Acknowledgments: This work was supported by the National Natural Science Foundation of China (no. 8210142829), Diagnosis and treatment equipment R & D innovation team based on acupoint sensitization (no. 303-202210705), Shaanxi Province Department of Education Fund (no. 21JS020) and Program for Meridian-Viscera Correlationship Innovative Research Team of Shaanxi University of Chinese Medicine (YL-09).\

Author contributions

MN writing and editing the manuscript. ZZ design and revise manuscript. NM and YW do the experiments. JC, MQ, GL and AL guide the experiments. FQ and XY review and editing the manuscript. All authors read and approved the final manuscript.

Funding

National Natural Science Foundation of China (no. 8210142829), Diagnosis and treatment equipment R & D innovation team based on acupoint sensitization (no. 303-202210705), Shaanxi Province Department of Education Fund (no. 21JS020) and Program for Meridian-Viscera Correlationship Innovative Research Team of Shaanxi University of Chinese Medicine (YL-09).

Availability of data and materials

The datasets presented in this review can be found in online repositories

Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable.

Consent for publication: All authors consent to the publication of this work in Chinese Medicine.

Competing interests: The authors declare no competing interests.

Contributor Information

Meng Nian, Email: [email protected]

Zefeng Zhao, Email: [email protected]

Yongqi Wang, Email: [email protected]

Jingxuan Chen, Email: [email protected]

Mingcheng Qian, Email: [email protected]

Guangning Li, Email: [email protected]

Xiaoan Li, Email: [email protected]

Haifa Qiao, Email: [email protected]

Xiaohang Yang, Email: [email protected]

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Table 1

Identified compound composition of PZE.
Peak	Predicted	Formula	t_R(min)	Ion Species	Measured Mass (Da)		MS²lons
M1	Lancerin	C₁₉H₁₈O₁₀	8.743	[M-H]⁻	405.0822	-0.50772	59.0144 (C₂H₃O₂⁻), 71.0143 (C₃H₃O₂⁻), 89.0249 (C₃H₅O₃⁻), 243.0306 (C₁₃H₇O₅⁻), 255.0305 (C₁₄H₇O₅⁻), 257.0462 (C₁₄H₉O₅⁻), 267.0297 (C₁₂H₁₁O₇⁻), 269.0459 (C₁₂H₁₃O₇⁻), 285.0403 (C₁₅H₉O₆⁻), 297.0408 (C₁₃H₁₃O₈⁻), 315.0504 (C₁₆H₁₁O₇⁻), 329.0699 (C₁₆H₉O₈⁻), 345.0624 (C₁₇H₁₃O₈⁻),369.0620 (C₁₉H₁₃O₈⁻), 387.0716 (C₁₉H₁₅O₉⁻)	A	[27]
M2	Polygalaxanthone Ⅲ	C₂₅H₂₈O₁₅	9.630	[M-H]⁻	567.1341	0.04351	59.0143 (C₂H₃O₂⁻), 89.0248 (C₃H₅O₃⁻), 271.0254 (C₁₄H₇O₆⁻), 273.0415 (C₁₄H₉O₆⁻), 315.0504 (C₁₆H₁₁O₇⁻), 345.0604 (C₁₇H₁₃O₈⁻), 417.0816 (C₂₀H₁₇O₁₀⁻), 435.0924 (C₂₀H₁₉O₁₁⁻), 447.0915 (C₂₁H₁₉O₁₁⁻)	A	[28]
M3	Polygalaxanthone V	C₂₆H₃₀O₁₅	11.078	[M-H]⁻	581.1537	-1.33884	273.0417 (C₁₄H₉O₆⁻)	A	[29]
M4	1,7-Dihydroxyxanthone	C₁₃H₈O₄	13.259	[M-H]⁻	227.0373	3.83102	65.0048 89.0247 (C₃H₅O₃⁻), 121.0315 (C₄H₉O₄⁻), 243.0705 (C₁₃H₇O₅⁻)	A	[30]
M5	1,3-Dihydroxy-7-methoxyxanthone	C₁₄H₁₀O₅	14.791	[M + H]⁺	259.0603	-2.37162	/	A	[31]
			14.038	[M-H]⁻	257.0472	1.36305	/	A	[31]
M6	1,3,7-Trihydroxy-2-methoxyxanthone	C₁₄H₁₀O₆	11.078	[M-H]⁻	273.0417	0.57909	/	A	[31]
M7	Irisxanthone	C₂₀H₂₀O₁₁	9.630	[M-H]⁻	435.0925	-1.58721	59.0143 (C₂H₃O₂⁻), 89.0248 (C₃H₅O₃⁻), 271.0253 (C₁₄H₇O₆⁻), 273.0415 (C₁₄H₉O₆⁻), 297.0397 (C₁₃H₁₃O₈⁻), 315.0504 (C₁₆H₁₁O₇⁻), 339.0510 (C₁₅H₁₅O₉⁻), 345.0604 (C₁₇H₁₃O₈⁻), 417.0816 (C₂₀H₁₇O₁₀⁻)	A	[32]
M8	Polygalaxanthone Ⅺ	C₂₅H₂₈O₁₅	9.630	[M-H]⁻	567.1341	0.04351	59.0143 (C₂H₃O₂⁻), 89.0248 (C₃H₅O₃⁻), 271.0253 (C₁₄H₇O₆⁻), 273.0415 (C₁₄H₉O₆⁻), 345.0604 (C₁₇H₁₃O₈⁻), 417.0814 (C₂₀H₁₇O₁₀⁻), 435.0925 (C₂₀H₁₉O₁₁⁻),447.0919 (C₂₁H₁₉O₁₁⁻)	A	[33]
M9	Tenuiphenone C	C₃₆H₆₂O₄	30.755	[M + H]⁺	559.4661	-8.99791	127.0410 (C₆H₇O₃⁺), 139.0409 (C₇H₇O₃⁺),153.0201 (C₇H₅O₄⁺),181.0516 (C₉H₉O₄⁺), 433.4381 (C₃₀H₅₇O⁺)	A	[34]
			30.737	[M-H]⁻	557.4577	-0.45866	437.4357 (C₃₂H₅₇O₂⁻), 513.4667 (C₃₃H₅₃O₄⁻)	A	[34]
M10	Sibiricose A1	C₂₃H₃₂O₁₅	7.395	[M-H]⁻	547.1695	-0.35158	149.0254 (C₅H₉O₅⁻), 179.0712 (C₆H₁₁O₆⁻), 205.0517 (C₁₁H₉O₄⁻), 223.0639 (C₁₁H₁₁O₅⁻), 265.0734 (C₃H₁₃O₆⁻), 295.0835 (C₁₄H₁₅O₇⁻), 325.0944 (C₁₂H₂₁O₁₀⁻), 367.1051 (C₁₇H₁₉O₉⁻),	A	[28]
M11	Tenuifoliside A	C₃₁H₃₈O₁₇	11.401	[M-H]⁻	681.2032	-0.96218	137.0247 (C₇H₅O₃⁻), 179.0359 (C₉H₇O₄⁻), 205.0516 (C₁₁H₉O₄⁻), 223.0619 (C₁₁H₁₁O₅⁻), 237.0777 (C₁₂H₁₃O₅⁻), 239.0567 (C₁₁H₁₁O₆⁻), 263.0568 (C₁₁H₁₃O₆⁻), 281.0672 (C₁₃H₁₃O₇⁻), 443.1181 (C₁₉H₂₃O₁₂⁻)	A	[35, 36]
M12	Tenuifoliside B	C₃₀H₃₆O₁₇	9.189	[M-H]⁻	667.1879	-1.06376	137.0247 (C₇H₅O₃⁻), 191.0356 (C₁₀H₇O₄⁻), 205.0508 (C₁₁H₉O₄⁻), 239.0570 (C₁₁H₁₁O₆⁻), 367.1037 (C₁₇H₁₉O₉⁻), 443.1193 (C₁₉H₂₃O₁₂⁻), 461.1287 (C₁₉H₂₅O₁₃⁻)	A	[35]
M13	Benzoic acid	C₇H₆O₂	16.100	[M + H]⁺	123.0450	-5.3472	77.0391 (C₆H₅⁺), 105.0347 (C₇H₅O⁺)	A	[37]
			6.308	[M-H]⁻	121.0302	1.59343	77.0401 (C₆H₅O⁻)	A	[37]
M14	1,5-Anhydro-D-sorbitol	C₆H₁₂O₅	6.464	[M + H]⁺	165.0693	-2.55948	/	A	[38]
			8.629	[M-H]⁻	163.0616	-0.55827	71.0141 (C₃H₃O₂⁻), 101.0232 (C₄H₅O₃⁻), 147.0073 (C₆H₁₁O₄⁻), 149.0263 (C₅H₉O₅⁻), 163.0405 (C₆H₁₁O₅⁻), 163.0734 (C₆H₁₁O₅⁻)	A	[38]
M15	O-Hydroxybenzoic acid	C₇H₆O₃	30.653	[M + H]⁺	139.0400	-2.24204	/	A	[39]
			9.188	[M-H]⁻	137.0247	3.80504	93.0352 (C₆H₅O⁻)	A	[39]
M16	4-Hydroxy-3,5-dimethoxylcinnamate	C₁₂H₁₄O₅	8.106	[M-H]⁻	237.0772	1.47702	/	A	[40]
M17	Tetrahydrocolumbamine	C₂₀H₂₃NO₄	6.427	[M + H]⁺	342.1683	-2.24204	/	A	[41]
			5.590	[M-H]⁻	340.1561	0.65858	252.0437 (C₁₆H₁₄NO₂⁻), 282.1145 (C₁₇H₁₆NO₃⁻), 310.1093 (C₁₈H₁₆NO₄⁻)	A	[41]
M18	3,4,5-Trimethoxycinnamic acid	C₁₂H₁₄O₅	8.106	[M-H]⁻	237.0772	1.47702	161.0463 (C₁₀H₉O₂⁻)	A	[42]
M19	1,3-Dihydroxy-4,5-dimethoxyxanthone	C₁₅H₁₂O₆	18.408	[M + H]⁺	289.0712	-1.74459	217.0503 (C₁₂H₉O₄⁺), 229.0515 (C₁₃H₉O₄⁺)	A	[42]
			12.501	[M-H]⁻	287.0567	1.62827	/	A	[42]
M20	1,6-Dihydroxy-3,5,7-Trimethoxyxanthone	C₁₆H₁₄O₇	15.485	[M + H]⁺	319.0812	-1.03534	/	A	[42]
			14.677	[M-H]⁻	317.0682	0.51318	287.0205 (C₁₇H₇O₇⁻)	A	[42]
M21	5,6,7-Trimethoxycoumarin	C₁₂H₁₂O₅	6.478	[M + H]⁺	237.0895	16.2122	165.0489 (C₉H₉O₃⁺), 191.0843 (C₁₀H₇O₄⁺),205.0642 (C₁₁H₉O₄⁺),207.0794 (C₁₁H₁₁O₄⁺),209.0948 (C₁₁H₁₃O₄⁺), 219.0795(C₁₂H₁₁O₄⁺)	A	[42]
			11.648	[M-H]⁻	235.0625	-0.33897	/	A	[42]
M22	Alphitolic acid	C₃₀H₄₈O₄	22.786	[M-H]⁻	471.3471	-1.48703	/	B	[43]
M23	Maslinic acid	C₃₀H₄₈O₄	22.826	[M-H]⁻	471.3471	-1.48703	/	B	[43]
M24	Epiceanothic acid	C₃₀H₄₆O₅	21.523	[M-H]⁻	485.3274	-0.81051	423.3265 (C₂₉H₄₃O₂⁻)	B	[43]
M25	Zizyberanalic acid	C₃₀H₄₆O₄	22.560	[M-H]⁻	469.3316	-1.21452	/	B	[43]
M26	Betulinic acid	C₃₀H₄₈O₃	24.445	[M-H]⁻	455.3520	-1.55475	/	B	[43]
M27	Vicenin-2	C₂₇H₃₀O₁₅	11.418	[M-H]⁻	593.1517	-1.48353	/	B	[44]
M28	Spinosin	C₂₈H₃₂O₁₅	10.722	[M + H]⁺	609.1784	-3.10585	297.0752 (C₁₇H₁₃O₅⁺), 327.0854 (C₁₂H₂₃O₁₀⁺), 351.0847 (C₁₆H₁₅O₉⁺), 411.1058 (C₂₂H₁₉O₈⁺), 429.1156 (C₂₂H₂₁O₉⁺), 447.1262 (C₂₂H₂₃O₁₀⁺)	B	[44]
			9.921	[M-H]⁻	607.1675	-0.34371	427.1041 (C₂₂H₁₉O₉⁻)	B	[44]
M29	6-Feruloylspinosin	C₃₈H₄₀O₁₈	12.477	[M + H]⁺	785.2228	-3.61507	/	B	[44]
			11.648	[M-H]⁻	783.2139	-0.30584	235.0625 (C₁₂H₁₁O₅⁻), 427.1026 (C₂₂H₁₉O₉⁻)	B	[44]
M30	1,7-Dimethoxyxanthone	C₁₅H₁₂O₄	17.609	[M + H]⁺	257.0812	0.93414	199.0399 (C₁₂H₇O₃⁺), 213.0557 (C₁₃H₉O₃⁺), 227.0350 (C₁₃H₇O₄⁺), 241.0505 (C₁₄H₉O₄⁺)	A	[45]
M31	1-Hydroxy-3,7-dimethoxyxanthone	C₁₅H₁₂O₅	17.687	[M + H]⁺	273.0781	-53.21667	/	A	[46]
M32	1-Hydroxy-3,6,7-trimethoxyxanthone	C₁₆H₁₄O₆	19.264	[M + H]⁺	303.0862	-0.6369	259.0607 (C₁₄H₁₁O₅⁺), 271.0599 (C₁₅H₁₁O₅⁺), 287.0554 (C₁₅H₁₁O₅⁺)	A	[47]
M33	Propyl benzoate	C₁₀H₁₂O₂	27.557	[M + H]⁺	165.0915	2.75397	149.0975 (C₉H₉O₅⁺)	A	[48]
M34	Stigmasterol	C₂₉H₄₈O	28.541	[M + H]⁺	413.3748	-6.14861	/	A	[49]
M35	α-Spinasterol	C₂₉H₄₈O	28.541	[M + H]⁺	413.338	-6.14861	/	A	[50]
M36	1,2,3,6,7-Pentamethoxyxanthone	C₁₈H₁₈O₇	16.325	[M + H]⁺	347.1111	-2.78277	285.0758 (C₁₆H₁₃O₅⁺), 289.0708 (C₁₅H₁₃O₅⁺), 303.0863 (C₁₆H₁₅O₆⁺), 317.0651 (C₁₆H₁₃O₇⁺), 331.0800 (C₁₇H₁₅O₇⁺)	A	[51]
M37	N-Nornuciferine	C₁₈H₁₉NO₂	1.838	[M + H]⁺	282.1525	-0.98118	236.1473 (C₁₆H₁₄NO⁺)	B	[52]

Notes: A: P. tenuifolia; B: Z. jujuba.

Table 2

Molecular docking score of the key active compounds.
Target (PDB)	Com.	CDOCKER interaction energy (Kcal/mol)	Key interactions
MAOA (2z5y)	M16	-44.7752	ASN 181 (H-bond), ALA 68 (H-bond), PHE 208 (van der Waals), LEU 337 (van der Waals), ILE 335 (van der Waals), ILE 180 (van der Waals), TYR 444 (van der Waals), TYR 407 (Pi-Pi-Stacked)
	M18	-40.7385	TYR 69 (H-bond), ALA 68 (H-bond), PHE 208 (van der Waals), LEU 337 (van der Waals), ILE 335 (van der Waals), ILE 180 (van der Waals), TYR 444 (van der Waals)
	M20	-46.7966	TYR 444 (H-bond), ASN 181 (H-bond), CYS 323 (Pi-Sulfur), PHE 208 (van der Waals), LEU 337 (van der Waals), ILE 335 (pi-alkyl), ILE 180 (pi-alkyl), TYR 407 (Pi-Pi-Stacked)
	M37	-34.5098	GLA 215 (H-bond), ILE 207 (H-bond), ASN 181 (H-bond), PHE 208 (van der Waals), LEU 337 (van der Waals), ILE 335 (van der Waals), ILE 180 (van der Waals), TYR 444 (Pi-Pi-Stacked), TYR 407 (Pi-Pi-Stacked)
SERT (6awn)	M17	-49.3710	THR 439 (H-bond), ASN 177 (H-bond), ALA 169 (van der Waals), ILE 172 (Amide-Pi-Stacked), ALA 173 (Carbon H-bond), VAL 501 (van der Waals), SER 336 (Carbon H-bond), NA 707 (van der Waals), ALA 96 (Carbon H-bond), ASP 98 (Pi-Anion), TYR 176 (Pi-Sigma), SER 438 (van der Waals)
	M22	-50.4302	TYR 95 (H-bond), GLU 493 (H-bond), ALA 169 (van der Waals), ILE 172 (pi-alkyl), ASP 98 (pi-alkyl), TYR 176 (pi-alkyl), SER 438 (van der Waals)
	M25	-58.9826	GLA 100 (H-bond), LEU 99 (H-bond), ASP 98 (H-bond), ILE 172 (pi-alkyl), VAL 501 (van der Waals), SER 336 (van der Waals), NA 707 (van der Waals), ALA 96 (van der Waals), TYR 176 (pi-alkyl), SER 438 (van der Waals)
	M35	-56.7521	ARG 104 (H-bond), GLU 494 (H-bond), ILE 172 (pi-alkyl), ALA 169 (pi-alkyl), ALA 173 (pi-alkyl), VAL 501 (van der Waals), ASP 98 (pi-alkyl), TYR 176 (Pi-Sigma), SER 438 (pi-alkyl)
nAChR4 (5kxi)	M18	-27.5261	LYS 79 (H-bond), TRP 156 (van der Waals), LEU 12 (van der Waals), THR 157 (Carbon H-bond), CYS 200 (Pi-Sulfur), TYR 204 (Pi-Anion), CYS 199 (van der Waals)
	M20	-39.0269	GLU 202 (H-bond), ALA 201 (H-bond), TRP 156 (Carbon H-bond), LEU 121 (pi-alkyl), THR 157 (Carbon H-bond), CYS 200 (Pi-Anion), TYR 204 (Pi-Long pair), CYS 199 (Sulfur-X), TRP 57 (van der Waals)
	M31	31.0103	GLU 202 (H-bond), LYS 79 (H-bond), TRP 156 (van der Waals), LEU 121 (van der Waals), THR 157 (Carbon H-bond), CYS 200 (Pi-Sulfur), TYR 204 (Pi-Long pair), CYS 199 (van der Waals)
	M32	-37.7832	GLU 202 (H-bond), LYS 79 (H-bond), TRP 156(Carbon H-bond), LEU 121 (van der Waals), THR 157 (Carbon H-bond), CYS 200 (Pi-Sulfur), TYR 204 (Pi-Long pair), CYS 199 (van der Waals)

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Characterization of the bioactive compounds with efficacy against epilepsy from the herb pairs Polygala tenuifolia - Zizyphus jujuba by modulating CHRNA4/CaMKII signaling pathway: LC-MS/MS combined with network pharmacology analysis and experimental evidence

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Abstract

Figures

1. Introduction

2. Methods

2.1. Reagents and materials

2.2. The conditition of HPLC-QTOF-MS/MS and Data analysis

2.3. Construction and analysis of PZE compounds-disease target interaction network

2.4. Acquisition of potential anti-epilepsy targets and the Protein-Protein interaction (PPI) network construction

2.5. GO annotation and KEGG enrichment analysis

2.6. Ingredien -target-pathway network construction

2.7. Molecular docking

2.9. Animals and group

2.10 Establish mice epilepsy model and sc-PTZ-induced epilepsy

3. Results

3.1. Identification of compounds from PZE

3.2. Identifcation of compounds from PZE

3.3. Acquisition of targets for PZE ingredients and anti-epilepsy

3.4. Protein-protein interaction (PPI) network analysis results

3.5. Molecular mechanism of anti-epilepsy the effect of PZE

3.6. Construction of the ingredient-target-pathway network

3.7. Molecular docking analysis

3.8. Anti-epilepsy Pathway

3.9. The efficacy and latency of PTZ

3.10. The behavioral animal tests

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

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