Dan Huang1, Xiaolong Wen2, Chuansen Lu1, Bo Zhang1, Zongjun Fu1, Yingliu Huang1, Kun Niu3, Fan Yang3. 1. Department of Neurology, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, China. 2. Department of Pharmacy, The Second Affiliated Hospital of Hainan Medical University, Haikou, China. 3. College of Traditional Chinese Medicine, Hainan Medical University, Haikou, China.
Abstract
Background: Compound Danshen Dropping Pills (CDDP) is widely used in clinical treatment of epilepsy. But the underlying active ingredients and molecular mechanisms are unclear. Our study aims to investigate the active components and functional mechanisms of CDDP in treating epilepsy using a network pharmacology approach. Methods: Candidate constituents and targets of CDDP were searched on the Traditional Chinese Medicine Systems Pharmacology database. NCBI and Genecards were used to establish a database of epilepsy targets. Next, used Cytoscape software, the interactive network diagram of "drug-active component-target" was drawn. Based on the STRING database we constructed protein-protein interaction network and analyzed protein-protein interaction relationships. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed for the common targets. Molecular docking provided an evaluation tool for verifying the combination of components and targets, which was performed using Auto-dock. Results: Sixty bioactive components, corresponding to 79 therapeutic targets for epilepsy, were successfully identified. Functional enrichment analysis showed that CDDP plays a pharmacological role in the treatment of epilepsy by regulating serotonergic synapses, morphine addiction, nicotine addiction and other pathways, as well as the NF-κB signaling pathway. Molecular docking analysis showed that representative components may be closely bound to key targets. Conclusions: This network pharmacology study revealed the synergistic effects of multiple components, targets, and pathways of CDDP in the treatment of epilepsy, which will deepen our understanding of the underlying molecular mechanisms of CDDP in the treatment of epilepsy and lay the foundation for further experimental studies. 2022 Annals of Translational Medicine. All rights reserved.
Background: Compound Danshen Dropping Pills (CDDP) is widely used in clinical treatment of epilepsy. But the underlying active ingredients and molecular mechanisms are unclear. Our study aims to investigate the active components and functional mechanisms of CDDP in treating epilepsy using a network pharmacology approach. Methods: Candidate constituents and targets of CDDP were searched on the Traditional Chinese Medicine Systems Pharmacology database. NCBI and Genecards were used to establish a database of epilepsy targets. Next, used Cytoscape software, the interactive network diagram of "drug-active component-target" was drawn. Based on the STRING database we constructed protein-protein interaction network and analyzed protein-protein interaction relationships. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed for the common targets. Molecular docking provided an evaluation tool for verifying the combination of components and targets, which was performed using Auto-dock. Results: Sixty bioactive components, corresponding to 79 therapeutic targets for epilepsy, were successfully identified. Functional enrichment analysis showed that CDDP plays a pharmacological role in the treatment of epilepsy by regulating serotonergic synapses, morphine addiction, nicotine addiction and other pathways, as well as the NF-κB signaling pathway. Molecular docking analysis showed that representative components may be closely bound to key targets. Conclusions: This network pharmacology study revealed the synergistic effects of multiple components, targets, and pathways of CDDP in the treatment of epilepsy, which will deepen our understanding of the underlying molecular mechanisms of CDDP in the treatment of epilepsy and lay the foundation for further experimental studies. 2022 Annals of Translational Medicine. All rights reserved.
Epilepsy is characterized by spontaneous convulsive behavior and abnormal brain discharge, and it is a very common chronic nervous system disease. Its incidence range is broad, and approximately 0.5–1% of people suffer from epilepsy worldwide (1). There are about 70 million epilepsy patients in the world (2).The pathogenesis of epilepsy is complex, including abnormal excitation of the central nervous system, abnormal discharge of ion channels, genetic factors, and immune system diseases (3). It is considered that the enhancement of cortical excitability caused by the imbalance of brain excitation and inhibition mechanism are vital for pathophysiological of epilepsy (4). Epidemiological investigation has shown that the prevalence of epilepsy globally is close to 1 ‰, and the total number of epilepsy patients in the world has exceeded 50 million (5). Furthermore, epilepsy in the elderly may significantly increase the risk of fracture or abnormal fall injury (6-8). At the same time, epilepsy patients are more prone to sudden death of unknown cause (9). At present, the treatment of epilepsy is mainly pharmacological, which can effectively control seizures, but also increase the risk of suicide or other various concurrent diseases to a certain extent, such as exfoliative dermatitis, polycystic ovary syndrome, etc. (10-12). Therefore, epilepsy has become one of the major diseases endangering human health.At present, antiepileptic drugs such as sodium valproate, carbamazepine, and levetiracetam are mainly used to treat the disease. However, there are disadvantages including poor curative effect, easy tolerance, as well as significant toxicity and side effects. Traditional Chinese medicine can make up for these defects and play an important role in the treatment of epilepsy; it has good curative effect, with long-lasting efficacy and few side effects. Compound Danshen Dropping Pills (CDDP) can reduce inflammatory reactions, control seizures, improve learning and memory function, and repair hippocampal neuron damage. The main components of CDDP are borneol, Salvia miltiorrhiza and Panax notoginseng. It is a traditional Chinese medicine dropping pill developed based on compound Danshen tablets under the guidance of traditional Chinese medicine theory. It has a wide range of clinical applications, such as the treatment of coronary heart disease, acute myocardial infarction and other diseases. In addition, it is widely used in clinical treatment of epilepsy, but its pharmacological mechanism is still unclear (3).Network pharmacology emphasizes the strategy shift from “one target, one drug” strategy to the new method of “network target, multi-component” (13-16). CDDP and other traditional Chinese medicines have the potential to promote the development of multi-component and multi-target synergistic therapy for epilepsy.With network pharmacology, we investigated the underlying anti-epileptic mechanism of CDDP. Firstly, we obtained the potential target genes of CDDP, and then studied the pathways shared by CDDP and the molecular targets of epilepsy. We conducted molecule docking to investigate the interaction between important compounds and targets. Compared with previous studies, we calculated the Root Mean Square Deviation (RMSD) value of docking to ensure the accuracy of docking results. Our results may help to clarify how CDDP can effectively treat epilepsy and promote the developing new drugs.We present the following article in accordance with the STREGA reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-195/rc).
Methods
Target genes related to epilepsy
Epilepsy-related genes were searched through the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) (17) and Genecards (https://www.genecards.org/) (18) databases using “epilepsy” and “Homo sapiens” as the search terms. The targets of both databases were synthesized to delete duplicate values and obtain the relevant epilepsy targets. Seven hundred and twenty-nine targets were obtained from the NCBI, and 2,088 targets were retrieved with Genecards (score ≥1). After excluding the duplicated targets, we obtained a total of 2,234 targets. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
Prediction of protein-encoding genes that are targeted by CDDP
The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) is a frequently used database to study mechanisms of Traditional Chinese medicines (19). We searched the TCMSP for the components of CDDP, including Salvia miltiorrhiza Bge., Panax notoginseng (Burk.) F. H. Chen, Borneol. Each component was obtained by using oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18 as the screening parameters (20).
Potential target genes of CDDP treating epilepsy
Shared target genes between epilepsy and the CDDP-related genes were intersected using a Venn diagram. We considered shared genes to be potential target genes of CDDP treating epilepsy.
The “drug-active component-target” network construction
CDDP active components and common genes were imported into the cytoscape 3.8.1. to build a “drug-active ingredient-target” network.
Construction of the target gene network
With STRING database (http://string-db.org/cgi/input.pl) and defining the species as “Homo sapiens” (21), a protein-protein interaction (PPI) network of intersecting genes of CDDP acting on epilepsy was constructed. The PPI data was downloaded and saved in TSV format. In addition, Cytoscape software (Institute of Systems Biology, the US) and its network analyzer tools were used to determine the accuracy of each target gene when selecting the core target.
Cluster analysis
Using Molecular Complex Detection (MCODE), the most important modules of PPI network were identified (22).
Analysis of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
Cytoscape clue GO was used for GO analysis, KEGG analysis of final target. The GO and KEGG pathway analyses were set as P<0.05.
Molecular docking
Molecular docking was intended to validate interactions between the central targets and major active ingredients. We downloaded the protein crystal structure by searching the Research of Cooperative Organization for structural bioinformatics (RCSB) protein database (http://www.pdb.org/). The conformation of the protein was modified using Pymol (Schrödinger, the US) and Autodock1.5.6 (Olson Laboratory of Scripps Institute, the US) software, including the removal of ligand and water, the addition of hydrogen, the optimization of amino acids, and the calculation of charge. Structural files of the key components are then downloaded through TCMSP, and with Chem3D software (Cambridgesoft, the US), their energy is minimized. The natural format of components and proteins was converted to the PDBQT format. Using AutoDock Vina, we performed molecular docking, and Discovery Studio 2019 (Beijing Chuangteng Technology Co., Ltd., China) was used to visualize docking results.
Results
Main components of CDDP
We obtained 336 chemical components of CDDP through TCMSP, including 76 components with OB ≥30% and DL ≥0.18. 65 components were included in Radix Salviae (Danshen), three components were included Borneolum Syntheticum (Bingpian), and eight compounds were included Panax Notoginseng (Burk.) F. H. Chen Ex C. Chow (Sanqi). After eliminating compounds without targets, 60 compounds remained. and had shown the active components and their OB and DL values.
Compound Danshen Dropping Pills (CDDP) screening of bioactive compounds. (A) Venn diagram: based on absorption, distribution, metabolism, excretion (ADME)-related models, 76 bioactive components were screened [blue section stands for the components of oral bioavailability (OB) ≥30%, yellow section stands for drug likeness (DL) ≥0.18]; (B) Venn diagram of CDDP- and epilepsy-related targets; 79 common targets were obtained.
CDDP, Compound Danshen Dropping Pills; WM, molecular weight; OB, oral bioavailability; DL, drug likeness.Compound Danshen Dropping Pills (CDDP) screening of bioactive compounds. (A) Venn diagram: based on absorption, distribution, metabolism, excretion (ADME)-related models, 76 bioactive components were screened [blue section stands for the components of oral bioavailability (OB) ≥30%, yellow section stands for drug likeness (DL) ≥0.18]; (B) Venn diagram of CDDP- and epilepsy-related targets; 79 common targets were obtained.
The targets of CDDP in the treatment of epilepsy
Through TCMSP database, we searched 197 CDDP targets. According to the NCBI and Genecards database, 2,234 epilepsy targets were obtained. Finally, 79 CDDP targets were identified in epilepsy treatment ().
‘Drug-active component-target’ network analysis
We used cytoscape software to construct “drug- ingredient-target” networks. A total of 336 CDDP components were identified from the TCMSP. In this study, 76 bioactive components were screened, 16 of which had no target. Through TCMSP database, 60 components were obtained. The red nodes represented the drugs of CDDP, and the green nodes represented the CDDP active ingredients. Blue nodes represented potential CDDP targets, and the edges indicate the association between ingredients and targets (as shown in ).
Figure 2
Mapping of Compound Danshen Dropping Pills (CDDP)- and epilepsy-related targets, 79 common targets were showed. Purple node denoted for drug, green nodes denoted for bioactive components, and blue nodes denoted for predicted targets.
Mapping of Compound Danshen Dropping Pills (CDDP)- and epilepsy-related targets, 79 common targets were showed. Purple node denoted for drug, green nodes denoted for bioactive components, and blue nodes denoted for predicted targets.
PPI network analysis
Using Cytoscape 3.8.1 and based on 79 candidate targets, we established a PPI network. There were 79 nodes and 984 edges, where the average node degree was 24.9 and the local clustering coefficient was 0.698 (). MCODE network analysis showed five clusters (), and the scores were 31.737, 3.333, 3, 3, and 3 respectively. These proteins have important function in the PPI network, and the top five proteins were AKT serine/threonine kinase 1 (AKT1), cellular tumor antigen p53 (TP53), interleukin-6 (IL-6), tumor necrosis factor (TNF) (). The 11 compounds with the most targets are shown in .
Figure 3
Common-target network. (A) Protein-protein interaction mapping by string database; (B) interaction network of Compound Danshen Dropping Pills (CDDP)-related targets. The red nodes represented the key targets.
Hub targets and active ingredients. (A) Degree level of each hub-target; (B) number of targets corresponding to important active ingredients.
Common-target network. (A) Protein-protein interaction mapping by string database; (B) interaction network of Compound Danshen Dropping Pills (CDDP)-related targets. The red nodes represented the key targets.Subnetwork graph of protein-protein interaction. (A) Score =31.737; (B) Score =3.33; (C) Score =3; (D) Score =3; (E) Score =3.Hub targets and active ingredients. (A) Degree level of each hub-target; (B) number of targets corresponding to important active ingredients.
Analysis of GO function and KEGG pathway enrichment
Through GO enrichment analysis, we obtained a total of 4,270 GO items, including 4,145 biological processes (BP) items, 11 cell components (CC) items, and 115 molecular functions (MF) items. The results showed that CDDP treatment of epilepsy BP was mainly related to the activation of adenylate cyclase activity, odontogenesis of dentin-containing tooth, including dentin, androgen metabolism, response to toxic substances, response to estrogen, and cellular response to estrogen stimulus. CC was mainly involved in platelet alpha particles cavity, projection of cytoplasm, and dendrites neuronal cytoplasm, etc. MF was mainly involved in the activation of protein kinase B activity, tau protein kinase activity, protein kinase C binding, and adenylate cyclase activity, as shown in .
Figure 6
Enrichment of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Compound Danshen Dropping Pills (CDDP) treating epilepsy. (A) Enriched gene ontology terms; (B) enriched KEGG pathways.
Enrichment of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Compound Danshen Dropping Pills (CDDP) treating epilepsy. (A) Enriched gene ontology terms; (B) enriched KEGG pathways.Based on KEGG pathway enrichment analysis, a total of 103 pathways were obtained. According to the P value, the top 20 items were shown in . These included the serotonergic synapse, morphine addiction, nicotine addiction, NF-κB, and Nod-like receptor signaling pathways.
Molecular docking analysis
Quercetin, luteolin, and tanshinone IIa were selected as the key active ingredients to perform molecular docking with AKT1, IL6, and TP53 (the basic information is shown in ). The key active ingredients and hub targets were verified by molecular docking using the binding energy ≤−5.0 kcal/mol as the standard (18). Docking results (shown in and ) showed that the binding free energy of the key active ingredients of epilepsy and the hub target was far less than −5.0 kcal/mol, and the RMSD was less than 2, indicating that the important active ingredients of CDDP combined well with the hub targets. This indicates that the results of this study are reliable, as shown in .
Table 2
Basic information of the key targets
GC ID
Symbol
Degree
Description
GC14M104769
AKT1
59
AKT serine/threonine kinase 1
GC17M007661
TP53
54
Tumor protein P53
GC07P022725
IL6
53
Interleukin 6
Figure 7
The three targets were as follows: interleukin-6 (IL6, PDB ID: 1alu), AKT serine/threonine kinase 1 (AKT1, PDB ID: 6s9x), and tumor protein p53 (TP53, PDB ID: 3dcy). The top three compounds were luteolin, quercetin, and tanshinone IIa. The binding energy ranged from −10.9 to −5.10 kcal/mol. Structural model of active ingredients with key targets. (A) Structural model of AKTI with luteolin, quercetin, and tanshinone IIa; (B) structural model of IL6 with luteolin, quercetin, and tanshinone IIa; (C) structural model of TP53 with luteolin, quercetin, and tanshinone IIa.
Figure 8
Binding mode of proteins and ligands. (A) binding mode of AKT serine/threonine kinase 1 (AKT1) with luteolin, quercetin, and tanshinone IIa; (B) binding mode of interleukin-6 (IL6) with luteolin, quercetin, and tanshinone IIa; (C) binding mode of tumor protein p53 (TP53) with luteolin, quercetin, and tanshinone IIa.
Figure 9
Binding site of key targets and active ingredients. (A) binding site of AKT serine/threonine kinase 1 (AKT1) with luteolin, quercetin, and tanshinone IIa; (B) binding site of interleukin-6 (IL6) with luteolin, quercetin, and tanshinone IIa; (C) binding site of tumor protein p53 (TP53) with luteolin, quercetin, and tanshinone IIa.
Table 3
Free binding energy and RMSD of the key active components of CDDP and key targets
Heat map of binding energy (A) and Root Mean Square Deviation (RMSD) (B).
The three targets were as follows: interleukin-6 (IL6, PDB ID: 1alu), AKT serine/threonine kinase 1 (AKT1, PDB ID: 6s9x), and tumor protein p53 (TP53, PDB ID: 3dcy). The top three compounds were luteolin, quercetin, and tanshinone IIa. The binding energy ranged from −10.9 to −5.10 kcal/mol. Structural model of active ingredients with key targets. (A) Structural model of AKTI with luteolin, quercetin, and tanshinone IIa; (B) structural model of IL6 with luteolin, quercetin, and tanshinone IIa; (C) structural model of TP53 with luteolin, quercetin, and tanshinone IIa.Binding mode of proteins and ligands. (A) binding mode of AKT serine/threonine kinase 1 (AKT1) with luteolin, quercetin, and tanshinone IIa; (B) binding mode of interleukin-6 (IL6) with luteolin, quercetin, and tanshinone IIa; (C) binding mode of tumor protein p53 (TP53) with luteolin, quercetin, and tanshinone IIa.Binding site of key targets and active ingredients. (A) binding site of AKT serine/threonine kinase 1 (AKT1) with luteolin, quercetin, and tanshinone IIa; (B) binding site of interleukin-6 (IL6) with luteolin, quercetin, and tanshinone IIa; (C) binding site of tumor protein p53 (TP53) with luteolin, quercetin, and tanshinone IIa.RMSD, Root Mean Square Deviation; CDDP, Compound Danshen Dropping Pills; AKT1, AKT serine/threonine kinase 1; TP53, tumor protein P53; IL6, interleukin 6.Heat map of binding energy (A) and Root Mean Square Deviation (RMSD) (B).
Discussion
Epilepsy is a very common neurological disease, characterized by recurrent epileptic seizures or abnormal behavior, feeling, and even loss of consciousness caused by the abnormal discharge of brain neurons (23). The annual incidence rate of active epilepsy in China is about 3.79 per thousand (24). The prevalence is the highest in 1-year old, and gradually decreases with age. Relevant studies (25,26) have shown that about a quarter of epilepsy cases can be prevented. In patients with epilepsy, about 60–70% of patients can achieve zero seizure through antiepileptic drugs, but the long-term use of antiepileptic drugs may result in some side effects. In addition, a considerable number of epileptic patients are still unable to control seizures through drugs or surgery (27). Based on the above situation, the current research on epilepsy is mainly divided into two aspects: (I) the prevention and treatment of epilepsy, and (II) the health monitoring and nursing of patients with epilepsy. These studies involve multiple fields, including biology, medicine, rehabilitation, electronics, and computer science.In recent years, research into the mechanism of traditional Chinese medicine epilepsy treatment has increased. Numerous studies have shown that traditional Chinese medicine can inhibit the occurrence and development of epilepsy by inhibiting apoptosis, regulating oxidative stress, and inhibiting inflammatory reactions (28,29). Epilepsy has the characteristics of long course of disease, recurrent seizures, and curative difficulty. Traditional Chinese medicine has the characteristics of obvious curative effect, multi-target, multi-channel, as well as minimal toxicity and side effects in the prevention and treatment of epilepsy. Therefore, in-depth study of the signaling pathway mechanisms in epilepsy and the development of more effective antiepileptic drugs are the focus of future research.Based on the method of network pharmacology, we clarify the mechanism of CDDP in the treatment of epilepsy. Quercetin is one of the most abundant flavonoids in nature, and has antioxidant, anti-inflammatory, anti-tumor, and antiviral neuroprotective effects (30,31). Quercetin can reduce lipid peroxidation and increase endogenous antioxidant enzymes in the brain, so as to reduce the frequency and severity of seizures (32). It is reported that quercetin can resist apoptosis and protect neurons by increasing X-linked inhibitor of apoptosis protein (XIAP) expression and inhibiting caspase-3 activity in the hippocampus (33). Based on Morris water maze experiment, D’Hooge et al. found that the latency of looking for the platform was prolonged and the residence time in the hidden platform quadrant was shortened, indicating the decline in learning and memory ability (34). Tanshinone IIa is the main fat soluble component of Salvia miltiorrhiza, a diterpene quinone compound. It has pharmacological activities in cardiovascular diseases, anti-tumor, anti-inflammatory, anticoagulant, and improving organ fibrosis (35). Using pentylenetetrazole induced juvenile zebrafish and mouse epilepsy models, it was found that tanshinone IIa may play an anticonvulsant role by activating the γ-aminobutyric acid (GABA) signaling pathway. Tanshinone IIa can also protect against lead-induced neurobehavioral defects in rats (36). In addition, our research group’s previous study found that tanshinone IIa may alleviate epileptic seizures in epileptic rats by regulating the brain-derived neurotrophic factor- Tyrosine Kinase receptor B (BDNF TrkB) pathway, enhance the expression of hippocampal synaptic remodeling proteins, syn and PSD-95, in epileptic rats, regulate synaptic plasticity, and subsequently improve the cognitive function of epileptic rats.Interleukin 6 (IL6) is a commonly used inflammatory factor in the clinic, which can promote the proliferation and functional changes of glial cells and participate in the regulation of neuronal function. Studies have shown that IL6 is a neuroprotective factor, which can protect nerve cells, repair damaged neurons and reduce epileptic brain injury (37,38). P38 is an important member of the mitogen-activated protein kinase (MAPK) family. It causes nuclear translocation after activation and leads to phosphorylation activation for many protein kinases and transcription factors, and it is very important for apoptosis (39). P53 protein is the downstream transcription factor of p38, and mainly acts to cause programmed apoptosis, and negatively regulate cell division and proliferation. It has been found that up-regulation of p53 expression can induce tumor cell apoptosis, and inhibiting the p38/p53 pathway can protect the apoptosis of ischemic stroke neurons (40). It was found that many apoptotic neuronal pyramidal cells gather in hippocampal CA1 area after epilepsy, and the expression of the p53 protein increased in pyramidal cells. However, CDDP also have some limitations. Individual patients will have skin rash, dyspnea, decreased blood pressure, and even shock. Long term use may cause stomach pain, indigestion, loss of appetite and other symptoms.
Conclusions
Our research is the first to investigate the underlying mechanism of cisplatin therapy for epilepsy using web-based pharmacological analysis. The results showed that the multiple pathways, targets, and components of CDDP had synergistic effects in the treatment of epilepsy. Our study can conduct deeper studies upon mechanisms of traditional Chinese medicine treatments for epilepsy, and provides a glimpse into for studying potential mechanisms of TCM treatments in other forms of mental illness using network pharmacology methods. Our study provides a mechanism for CDDP treatment of epilepsy based on the existing database. To guarantee quality and rationality of results, advanced exploratory confirmation in vivo and in vitro is necessary.The article’s supplementary files as
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