Edited by: Xiu-Wei Yang,Peking University,Beijing,China
Reviewed by: Yu-Ping Tang,Shaanxi University of Chinese Medicine,China; Dan Yan,Capital Medical University,China
*Correspondence: Yu-ling Ma,
This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Xin Su Ning (XSN) is a China patented and certified traditional Chinese herbal medicine used to treat premature ventricular contractions (PVCs) since 2005. XSN is formulated with 11 herbs, designed to treat arrhythmia with phlegm-heat heart-disturbed syndrome (PHHD) according to Chinese medicine theory. The rational compatibility of the 11 herbs decides the therapeutic outcome of XSN. Due to the multicomponent nature of traditional Chinese medicine, it is difficult to use conventional pharmacology to interpret the therapeutic mechanism of XSN in terms of clear-cut drug molecule and target interactions. Network pharmacology/systematic pharmacology usually consider all the components in a formula with the same weight; therefore, the proportion of the weight of the components has been ignored. In the present study, we introduced a novel coefficient to mimic the relative amount of all the components in relation with the weight of the corresponding herb in the formula. The coefficient is also used to weigh the pharmacological effect of XSN on all relative biological pathways. We also used the cellular electrophysiological data generated in our lab, such as the effect of liensinine and isoliquiritigenin on NaV1.5 channels; we therefore set sodium channel as one of the targets of these two components, which would support the clinical efficacy of XSN in treating tachyarrhythmia. Combining the collected data and our discovery, a panoramagram of the pharmacological mechanism of XSN was established. Pathway enrichment and analysis showed that XSN treated PHHD arrhythmia through multiple ion channels regulation, protecting the heart from I/R injury, inhibiting the apoptosis of cardiomyocyte, and improving glucose and lipid metabolism.
Arrhythmia is a disease featuring the abnormalities of frequency or rhythm of heart excitement caused by abnormal cardiac electrophysiological activities generated by the electrical conduction system of the heart. Symptoms of arrhythmia often include dizziness, breathlessness, and palpitations (
As a complementary and alternative medicine, Chinese medicine plays an increasing role in the treatment of arrhythmias (
Based on the theories of traditional Chinese medicine (TCM), a disease can be categorized from four fundamental dimensions, which is consisted of four pairs of relative concepts including yin–yang, exterior–interior, excess–deficiency, and cold–heat (
Xin Su Ning (XSN) is a multi-herbal medicine patented and launched in China since 2005 for treating cardiac ventricular arrhythmia, especially arrhythmias induced by cardiac ischemia and viral myocarditis (
However, the complexity of the chemical composition of multi-herbal TCM formula brings great difficulties to the pharmacological research. Network pharmacology has been one of the approaches to reveal the complex pharmacological mechanisms behind the multitargeting properties by the multicomponent medicines (
In this study, we tried to introduce a parameter, weight coefficient, to mimic the proportion of all the encompassed components and resort their effects on different targets and pathways. We used the data from multiple bioassay databases, combining with the results of pharmacological assays of high-weight coefficient components; a closer-to-the-fact pharmacological panorama was constructed.
All of the chemical monomer components in the 11 herbs of XSN were retrieved from the book Chemical Components of Source Plants in Traditional Chinese Medicine (
Component weight coefficient:
The weight coefficient is the product of three elements:
Since the activity of the multiple herbal medicine involved in multiple targeting, and each target was usually bound by multiple components. Weight coefficient of one single target in this paper was represented by the sum of all the weight coefficients of the components interacting with this target. Furthermore, weight coefficient of each pathway was also represented by the sum of all the weight coefficients of all targets in this pathway.
All the relative targets of each component in the 11 herbs of XSN were retrieved from BindingDB database (
Two networks were constructed: the networks of PHHD target and XSN component targets. All the networks were visualized by Cytoscape 3.7.0 (
All the chemical agents were purchased from Sigma–Aldrich, and all high-weight coefficient components were obtained from Chengdu Herbpurify Co., Ltd. The intracellular buffer contained (in mM): KCl 120, MgCl2 2, CaCl2 1, Na2ATP 3, EGTA 11, HEPES 10, and pH 7.2 corrected with 5M NaOH. The extracellular buffer contained (in mM): NaCl 112, NaH2PO4•H2O 1, KCl 5.4, HEPES 5, NaHCO3 24, glucose 10, MgCl2 1.2, CaCl2 1.8, and pH 7.4 corrected with 5M NaOH. Components experimented in this paper were dissolved in the external buffer, and for the dose–response research, the concentrations of the compounds studied were ranging from 1 to 100 μM in the external buffer solution.
CHL cells stably expressing the α-subunit of human NaV1.5 (SCN5A) were used for electrophysiological assays. Patch clamp assays were carried out at room temperature (∼ 22 to 24°C), and the cells were superfused with the extracellular buffer at a rate of 2 ml/min. Patch pipettes were pulled from borosilicate glass (Harvard Apparatus, UK) using a DMZ-Universal Puller (Zeitz-Instruments, Germany); the average pipette resistance was 3–5 MΩ. The control currents were recorded 5 minutes after the whole-cell configuration was achieved using an Axopatch™ 200B Amplifier (Molecular Devices, USA).
Data was analyzed and illustrated using pCLAMP 10.3 software (Axon Instruments, Inc.) and Origin 9.1. For each protocol and condition, at least five cells were tested. Data values are presented as mean ± standard error of the mean (SEM). The difference between the control and the effect of a compound was statistically tested using Student’s unpaired t-test.
Nine hundred sixty-three monomer components from 11 herbs of XSN were collected from Chemical Components of Source Plants in Traditional Chinese Medicine and TCMSP database (
Chemical properties statistics of components in the 11 herbs of XSN.
Two thousand eight hundred thirty-five component-target relationship data were obtained, covering 487 monomer components and 618 targets (
The targeting spectrum of XSN.
Since Zheng (TCM syndrome) is represented by the series of characteristics with clinical manifestations and symptoms (hereinafter referred to as TCM symptom); all the 10 TCM symptoms of PHHD arrhythmia and their relative modern medicine symptoms (MM symptom) were collected: Xin Ji (palpitations), Xiong Men (respiratory distress), Xin Fan (boredom), Yi Jing (panic attack), Kou Gan (xerostomia), Kou Ku (bitter taste in the mouth), Shi Mian (insomnia), Duo Meng (dreaminess), Xuan Yun (vertigo), and Mai Jie Dai (knotted or regularly intermittent pulse) (
The relative relationship between TCM symptoms and modern medicine symptom.
TCM symptom ID | TCM symptoms | MM symptom ID | MM symptoms | UMLS ID | Synonyms | UMLS ID | HPO ID | Target number |
---|---|---|---|---|---|---|---|---|
SMTS01333 |
|
SMMS00282 | Palpitations | C0030252 | HP:0001962 | 52 | ||
SMTS01384 |
|
SMMS00863 | Chest Heaviness | C0742339 | Respiratory distress | C0013404 | HP:0002098 | 108 |
SMTS01324 |
|
SMMS00381 | Boredom | C0006019 | ||||
SMTS01567 |
|
SMMS00119 | Panic Attack | C0086769 | HP:0025269 | 17 | ||
SMTS00573 |
|
SMMS00144 | Xerostomia | C0043352 | HP:0000217 | 42 | ||
SMTS00580 |
|
SMMS00585 | Halitosis | C0018520 | Abnormality of taste sensation | C4025879 | HP:0000223 | 6 |
SMTS00970 |
|
SMMS00033 | Insomnia | C0917801 | HP:0100785 | 12 | ||
SMTS00211 |
|
SMMS00738 | Nightmares | C0028084 | ||||
SMTS01439 |
|
SMMS00115 | Vertigo | C0042571 | HP:0002321 | 72 | ||
N/A |
|
Irregular heart beat | 0011675 | 317 |
TCM, traditional Chinese medicine; MM, modern medicine; UMLS, Unified Medical Language System; HPO, human phenotype ontology.
TCM symptom Kou Ku means bitter taste in the mouth, but not bad breath in the mouth; therefore, the MM symptom halitosis retrieved from SymMap was replaced by another keyword: abnormality of taste sensation. Mai Jie Dai included two types of pulses: Jie Mai is knotted or bound pulse, which is slow, relaxed and stops at irregular intervals. Jie Mai represents an irregular beat or palpitation stemming from the heart. Dai Mai means the pulse is with regularly intermittent abnormality, which also suggests the patients with this pulse have advanced heart disease according to modern medicine. Therefore, an alternative keyword irregular heartbeat was used to represent the TCM symptom Mai Jie Dai.
Five hundred two targets in total for PHHD arrhythmia were collected as PHHD-arrhythmia target spectrum; statistics of target count for each TCM symptom were shown in
Statistics of target count for each TCM symptom of PHHD arrhythmia.
To obtain the pharmacological network of XSN on treating PHHD arrhythmia, 40 targets were obtained in the common set of XSN target spectrum and PHHD-arrhythmia target spectrum (
The common genes between XSN target spectrum and PHHD-arrhythmia target spectrum.
Gene_Symbol | TCM_Symptom | Target_Weight_Coefficient | UniProt_ID | ChEMBL_ID | Target_Name | Classification_1 | Classification_2 |
---|---|---|---|---|---|---|---|
CALM1 | Maijiedai, Xuanyun | 790.2976034 | P0DP23 | CHEMBL6093 | Calmodulin | Other | Unclassified protein |
KCNH2 | Maijiedai, Xinji | 13942.14102 | Q12809 | CHEMBL240 | HERG | Ion channel | VGIC |
TP53 | Yijing, Maijiedai, Xinji | 13900.81344 | P04637 | CHEMBL4096 | Cellular tumor antigen p53 | Transcription factor | Other |
ABCC8 | Maijiedai | 13817.52659 | Q09428 | CHEMBL2071 | Sulfonylurea receptor 1 | Transporter | Other |
KCNJ11 | Maijiedai | 13817.40799 | Q14654 | CHEMBL1886 | Potassium channel, inwardly rectifying, subfamily J, member 11 | Ion channel | VGIC |
KCNJ5 | Maijiedai | 169.079052 | P48544 | CHEMBL3038488 | Kir3.1/Kir3.4 | Ion channel | VGIC |
CACNA1B | Maijiedai | 593.6573686 | Q00975 | CHEMBL4478 | Voltage-gated N-type calcium channel alpha-1B subunit | Ion channel | VGIC |
TARDBP | Kougan | 2584.201263 | Q13148 | CHEMBL2362981 | TAR DNA-binding protein 43 | Other | Unclassified protein |
JAK2 | Xuanyun | 2032.705321 | O60674 | CHEMBL2971 | Tyrosine-protein kinase JAK2 | Kinase | Protein Kinase |
TNNC1 | Maijiedai | 2017.925664 | P63316 | CHEMBL2095202 | Troponin, cardiac muscle | Other | Other |
TNNI3 | Maijiedai | 2017.925664 | P19429 | CHEMBL2095202 | Troponin, cardiac muscle | Other | Other |
TNNT2 | Maijiedai | 2017.925664 | P45379 | CHEMBL2095202 | Troponin, cardiac muscle | Other | Other |
GMNN | Xiongmen | 95.88983101 | O75496 | CHEMBL1293278 | Geminin | Other | Unclassified protein |
SMAD3 | Maijiedai | 95.88983101 | P84022 | CHEMBL1293258 | Mothers against decapentaplegic homolog 3 | Other | Unclassified protein |
PPARG | Maijiedai, Xinji | 83.46168023 | P37231 | CHEMBL235 | Peroxisome proliferator-activated receptor gamma | Transcription factor | Nuclear receptor |
GJA1 | Maijiedai | 83.29977996 | P17302 | Gap junction alpha-1 protein | Other | Unclassified protein | |
PTPN11 | Maijiedai | 83.26574007 | Q06124 | CHEMBL3864 | Protein-tyrosine phosphatase 2C | Enzyme | Phosphatase |
LMNA | Maijiedai | 83.14071435 | P02545 | CHEMBL1293235 | Prelamin-A/C | Other | Unclassified protein |
TSHR | Maijiedai | 83.14071435 | P16473 | CHEMBL1963 | Thyroid-stimulating hormone receptor | GPCR | Family A GPCR |
ATXN2 | Kougan | 12.74911666 | Q99700 | CHEMBL1795085 | Ataxin-2 | Other | Unclassified protein |
SOD1 | Kougan | 1.975815734 | P00441 | CHEMBL2354 | Superoxide dismutase | Enzyme | Oxidoreductase |
SDHA | Yijing, Maijiedai, Xinji, Xuanyun | 1.990418899 | P31040 | CHEMBL5758 | Succinate dehydrogenase (ubiquinone) flavoprotein subunit, mitochondrial | Enzyme | Oxidoreductase |
PON1 | Kougan | 1.732079071 | P27169 | CHEMBL3167 | Serum paraoxonase/arylesterase 1 | Enzyme | Hydrolase |
KYNU | Maijiedai | 1.649920118 | Q16719 | CHEMBL5100 | Kynureninase | Enzyme | Hydrolase |
IFNG | Maijiedai | 1.695300734 | P01579 | CHEMBL3286073 | Interferon gamma | Other | Other |
CHRM3 | Kougan | 0.769622544 | P20309 | CHEMBL245 | Muscarinic acetylcholine receptor M3 | GPCR | Family A GPCR |
SCN5A | Maijiedai, Xinji | 76.0137842 | Q14524 | CHEMBL1980 | Sodium channel protein type V alpha subunit | Ion channel | VGIC |
DAO | Kougan | 1.396943488 | P14920 | CHEMBL5485 | D-amino-acid oxidase | Enzyme | Oxidoreductase |
CPT2 | Maijiedai, Xiongmen | 1.020829585 | P23786 | CHEMBL3238 | Carnitine palmitoyltransferase 2 | Enzyme | Transferase |
CTNNB1 | Xuanyun | 1.020829585 | P35222 | CHEMBL5866 | Catenin beta-1 | Other | Unclassified protein |
NAGS | Xiongmen | 1.020829585 | Q8N159 | N-acetylglutamate synthase, mitochondrial | Enzyme | Transferase | |
TTR | Maijiedai | 1.001902474 | P02766 | CHEMBL3194 | Transthyretin | Other | Other |
COL3A1 | Xuanyun | 0.45282272 | P02461 | CHEMBL2364188 | Collagen | Other | Other |
SLC1A3 | Xuanyun | 0.520835938 | P43003 | CHEMBL3085 | Excitatory amino acid transporter 1 | Transporter | Electrochemical transporter |
HNF4A | Maijiedai | 0.386540369 | P41235 | CHEMBL5398 | Hepatocyte nuclear factor 4-alpha | Other | Unclassified protein |
AKT1 | Maijiedai | 0.26386963 | P31749 | CHEMBL4282 | Serine/threonine-protein kinase AKT | Kinase | Protein kinase |
GAA | Maijiedai | 0.261304876 | P10253 | CHEMBL2608 | Lysosomal alpha-glucosidase | Enzyme | Hydrolase |
LDLR | Maijiedai | 0.132692707 | P01130 | CHEMBL3311 | LDL receptor | Other | Membrane receptor |
EGF | Xuanyun | 0.159065607 | P01133 | CHEMBL5734 | Pro-epidermal growth factor | Other | Unclassified protein |
Based on our previous study, XSN is a class III anti-arrhythmic drug supported by the prolongation of the action potential of cardiac myocytes through blocking hERG channel (
Two high-weight active components: liensinine (LSN, PubChem CID: 160644) and isoliquiritigenin (ISL, PubChem CID: 638278) were detected using electrophysiological approaches. LSN is the No. 35 high-weight component, which blocked human NaV1.5 channel dose-dependently with an IC50 of 3.58 ± 0.36 μM (
Including the pharmacological effects of LSN and ISL, a panoramagram of the integrative pharmacological mechanism of XSN was interpreted (shown as
Panoramagram of the pharmacological mechanism of XSN for treating PHHD arrhythmia. This panoramagram illustrated the connections among the XSN formula, herbs, components, targets, symptoms, and PHHD arrhythmia.
All the 40 common targets between XSN target spectrum and PHHD-arrhythmia target spectrum were selected to carry out pathway enrichment with Reactome application in Cytoscape. 117 pathways with FDR less than 0.05 were obtained and resort by total weight coefficients (
Pathway enrichment results of 40 targets of XSN on treating PHHD arrhythmia. Top 50 pathways were shown here; and all were sorted by pathway weight coefficient.
To better understand the antiarrhythmic therapeutic efficacy of XSN, cardiac conduction pathway is calculated to hold the highest total weight coefficient in the list of resorting pathway total weight coefficient. It was shown in
Cardiac conduction pathway. Top 1 pathway in the rank of total target weight coefficient, all the target regulated in this pathway were marked in red (
Tan-Re-Rao-Xin Zheng is named as PHHD syndrome in English. Based on the traditional Chinese Medicine theories, PHHD is defined as that heat having existed in the body for a period of time without distributing leads to the body's fluids being concentrated as Phlegm. The concept
Physical symptoms of PHHD are usually consist of the followings (Yongdun et al., 2005;
XSN capsule originated from the famous compound formula Huanglian Wendan (HLWD) Decoction recorded in 150 years ago, which has the effect of removing phlegm, blood stasis, and fire toxin. Huanglian, Banxia, Fuling, Gancao, and Zhishi are from the original formula HLWD decoction; Changshan, Lianzixin, Qinghao, and Kushen were added to make XSN formula more inclined to the circulation system. Therefore, XSN was not only a combination of anti-arrhythmic monomer components, but also a complex therapeutic system to regulate biological/pathological process relative to PHHD syndrome.
The compatibility principle of the herbs in XSN also reflects another key property of Chinese herbal medicine: amount/proportion of herbs in compound formula. For a better understanding of the pharmacological mechanism of XSN, we introduced a novel parameter,
In this study, we used network pharmacological approach to visualize the complex pharmacological mechanism of XSN on treating PHHD arrhythmia. All the data were collected from databases and books to assure the quality and reliability of all the relationships in the pharmacological network. Based on our previous study, XSN can be classified as class III antiarrhythmic drug, and with some pharmacological property of class I drug (
Including the pharmacological effects of LSN and ISL, we drew a panoramagram of the integrative pharmacological mechanism of XSN with 475 components targeting 617 targets. Among the 617 targets, 40 targets were relative to PHHD arrhythmia. To explain the mechanism, we carried out pathway enrichment instead of subnetwork analysis, 116 pathways were obtained, and top 50 were shown as
As an antiarrhythmic drug, XSN showed multiple therapeutic effects. From the standpoint of TCM treatment, both tip and root causes can be reflected in the pathways, which can also be considered as quick-acting and long-acting mechanisms. First, ion channels play essential role in terminating abnormal electrical activity in arrhythmic heart. Based on the results from the analysis above, the mechanism on cardiac electronic activity may involve such targets: TNNI3, CACNA1S, SCN5A, KCNH2, KCNJ11, and CALM1. NaV1.5, CaV1.2, and hERG are three key ion currents in cardiac electric conduction during depolarization and repolarization. Troponins I (encoded by TNNI3) is integral to cardiac muscle contraction, which is also used as diagnostic and prognostic indicators in the management of myocardial infarction and acute coronary syndrome. Calmodulin 1 (encoded by CALM1) mediates the regulation of plenty of enzymes, ion channels, aquaporins, and other proteins acting with calcium-binding. Secondary, as long-acting mechanism, XSN was used to treat arrhythmia caused by coronary heart disease and viral myocarditis. Protection of myocardium, such as protection of I/R injury, antithrombosis and anti-apoptotic effect should be the essential effects of XSN. Intracellular sodium accumulation is a key pathophysiological mechanism in myocardial ischaemia/reperfusion (I/R) injury. Furthermore, it is thought that, Nav1.5, alongside NHE and other transporters, contributes to this sodium overload that can be reduced with application of lidocaine, suggesting that inhibition of INa may attenuate I/R injury. Previous work has shown XSN is cardioprotective in I/R injury induced in isolated rat hearts. Given the effect of ISL in inhibiting Nav1.5, it is possible that LSN and ISL reported in the present study are responsible for some of XSN’s cardioprotective actions in this way.
Besides of calcium overload, another reason of myocardium injury was caused by apoptosis of cardiomyocyte. The p53 tumor suppressor is one of the major apoptosis signaling pathways. It regulates a wide variety of genes involved in apoptosis, growth arrest, or senescence in response to genotoxic or cellular stress. The p53 can promote apoptosis through interactions with Bcl-2 family proteins in the cytoplasm. In addition, HIF1 alpha was also downregulated by the top1 component berberine (
Inflammatory response is well recognized as a critical contributor for the development and complications of atherosclerosis cardiovascular disease (ASCVD), including myocardial infarction (MI), heart failure, and stroke, which involve complex interactions between multiple biological processes.
Besides of cardiovascular pathways, several diabetic pathways were also obtained. In TCM system, disease with diabetes-related symptoms is called “
Beyond of these targets, several targets including fatty acid synthase (FASN), pyruvate kinase L/R (PKLR), acetyl-CoA carboxylase (ACC, alpha unit encoded by ACACA), transketolase (TKT), free fatty acid receptor 1(FFAR1), glucagon-like peptide 1 receptor (GLP1R), and so on were also regulated by XSN. These targets are involved in the bioprocess of glycolysis, fatty acid synthesis, and pentose phosphate pathway, which are key steps of glucose and lipid metabolism. Through these mechanisms, XSN may improve fatty acid storage and convert it into glycogen. Lipid metabolism disorder has been reported being related to the TCM heat syndrome (
In summary, the mechanism of XSN on treating PHHD arrhythmia may act as follows: on quick-acting aspect, XSN balanced the ion current of the heart by regulating multiple ion channels to terminate cardiac arrhythmia; on the long-acting aspect, XSN protects the heart from I/R injury, inhibits the apoptosis of cardiomyocyte, and improves glucose and lipid metabolism. Part of the mechanism of XSN on treating PHHD syndrome by sharing pathways with insulin secretion or glucose and lipid metabolism also suggest the potential therapeutic effect of diabetes and diabetic cardiopathy.
All datasets generated for this study are included in the manuscript/
Y-LM and TW conceived and designed the study; TW and HS performed the electrophysiological experiments; XW discussed, analyzed and standardized the TCM symptoms; UP and ML analyzed and discussed the biological functions of the pathways; CC provided experimental equipment and technical support for cell culturing; TW and Y-LM wrote the paper.
This work was supported by grants from the Chinese Medicine Research Fund, University of Oxford. The grant was funded by Shaanxi Momentum Pharmaceutical Co.,Ltd.
The authors declare that this study received funding from Shaanxi Momentum Pharmaceutical Co., Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.
We thank Prof. Yan Zhu (Tianjin University of Traditional Chinese Medicine) for providing the stable transfected cell lines and Dr. Hongbin Yang (East China University of Science and Technology) for technical support on AdmetSar 2.0 system.
The Supplementary Material for this article can be found online at:
XSN component library.
XSN component target relationships.
XSN Target Weight Coefficient type.
PHHD-arrhythmia target spectrum.
XSN PHHD Pathway.