Network Pharmacology Systematically Uncovers Multiple Mechanisms of Zhisousan (止嗽散) for Treatment of Chronic Cough

Journal Pre-proof Network Pharmacology Systematically Uncovers Multiple Mechanisms of Zhisousan(nullnullnull) for Treatment of Chronic Cough Ji Yu Zou, Li Jian Pang, Xiao Dong Lv

PII:

S1876-3820(19)31185-0

DOI:

https://doi.org/10.1016/j.eujim.2020.101071

Reference:

EUJIM 101071

To appear in:

European Journal of Integrative Medicine

Received Date:

4 November 2019

Revised Date:

19 February 2020

Accepted Date:

19 February 2020

Please cite this article as: Zou JY, Pang LJ, Lv XD, Network Pharmacology Systematically Uncovers Multiple Mechanisms of Zhisousan(x6b62;x55fd;x6563;) for Treatment of Chronic Cough, European Journal of Integrative Medicine (2020), doi: https://doi.org/10.1016/j.eujim.2020.101071

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Network Pharmacology Systematically Uncovers Multiple Mechanisms of Zhisousan（止嗽散） for Treatment of Chronic Cough

JiYu Zou 1, LiJian Pang 2, XiaoDong Lv 1

1. Liaoning University of Traditional Chinese Medicine,Shenyang 110032, Liaoning,

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China;

2. The Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,

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Shenyang 110847, Liaoning, China.

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The corresponding author:

XiaoDong Lv, ORCID: https://orcid.org/0000-0001-8774-8078

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Address: No.79, Chong Shan East Road, Huang Gu District, Shenyang, Liaoning,

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China.

TEL:13304053456, E-mail:[email protected] and [email protected]

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Author:

JiYu Zou, ORCID: https://orcid.org/0000-0003-4002-507X

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TEL:18004018547, E-mail:[email protected] LiJian Pang, ORCID: https://orcid.org/0000-0002-7300-5019 TEL:18004010055, E-mail:[email protected]

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ABSTRACT Introduction: Zhisousan is a classical prescription consisting of seven Chinese herbal medicines and has been used historically in the treatment of chronic cough. The chemical constituents and targets of Zhisousan were identified using a network pharmacological analysis. Methods: Parameters such as the oral utilization rate and the drug similarity of ingredients in Zhisousan were obtained, effective ingredients of

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traditional Chinese medicine were screened, and targets of effective ingredients of

traditional Chinese medicine were collected by the TCMSP database. Genes related to chronic cough were collected by the GeneCards database, and key targets were

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obtained after intersecting with the targets of Zhisousan. The above targets were

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analysed using the String database, and a protein-protein interaction network (PPI network) was constructed. Finally, GO functional enrichment analysis and KEGG

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pathway enrichment analysis were carried out on key targets. Results: After

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iteratively screening and removal of duplicates, 151 active ingredients and 2022 targets of Zhisousan were obtained. There were 2348 genes related to the chronic

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cough disease and 79 targets for drug and disease identified. PPI network analysis suggested that IL6 (Interleukin-6), VEGFA (Vascular endothelial growth factor A),

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EGFR (Epidermal growth factor receptor) and CASP3 (Caspase-3) may be the key targets of Zhisousan in the treatment of chronic cough. Conclusion: Network pharmacology systemically describes the pharmacological mechanisms of Zhisousan in the treatment of chronic cough are associated with multiple signaling pathways

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such as Hepatitis B, Kaposi sarcoma-associated herpesvirus infection, MicroRNAs in cancer, Non-small cell lung cancers, P53 signaling pathway, and Apoptosis. Key words: Zhisousan; chronic cough; network pharmacology; gene; targets; IL6

INTRODUCTION Air pollution has become increasingly serious, as has the number of days with

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smog which has led to a year on year increase in respiratory diseases, especially among people who smoke. The two-week prevalence rate, the two-week consultation

rate and the number of hospitalizations of respiratory diseases rank first for a long

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time in China [1]. Respiratory diseases are also leading causes of morbidity and

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mortality in the United States [2]. Prevention and early treatment of respiratory diseases is urgent. Cough is the most common symptom in respiratory clinics and Some studies have shown that patients with chronic cough

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community clinics.

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account for about one-third in Chinese specialist clinics [3]. In an epidemiological survey of 1087 college students in Guangzhou, the prevalence of cough among

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college students was close to 11.0%, and the incidence of chronic cough was 3.3% [4]. And cough is one of the most common reasons for patients seeking medical treatment

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[5]. Coughing may occur when inhaling environmental stimuli such as smog, perfume and cold air[6]. Chronic cough has no apparent benefits and seriously affects the quality of life[5]. Traditional Chinese medicine divides cough into two types, namely, exogenous cough and endogenous cough. Its pathogenesis is due to pathogenic factors in the lung 3

when lung Qi is reversed. As early as in "The Yellow Emperor's Canon of Medicine ", there was a special discourse on cough, such as "Su Wen·Cough Theory ":" The lung is interrelated with the skin and hair; The skin and hair are first subjected to the pathogenic factor, and the pathogenic factor then combines with them. When its cold diet enters the stomach, from the pulmonary vein to the lung, the lung is cold. While the lung is cold, which combines external and internal pathogens, so the attack on the

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lungs is a cough." And " internal organs of the body (heart, spleen, liver, lungs and kidneys) make one cough, not only lung.". Later generations of physicians have made

further elucidation. As Zhang Jiebin pointed out, coughing refers to exogenous cough

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and endogenous cough. "Medical Insights" refers to "whenever coughing is cured, it is

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important to do good in the beginning.". Zhisousan, a traditional Chinese medicine prescription, is a classical prescription for the diagnosis and treatment of chronic

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cough[7]. Zhisousan originates from Cheng Zhongling's "Medical Insights", which

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contains seven Chinese medicines: Jiegeng (Platycodon Grandiflorus), Gancao (Glycyrrhizae Radix Et Rhizoma), Baiqian (Cynanchi Stauntonii Rhizoma Et Radix),

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Ziwan (Asteris Radix Et Rhizoma), Jingjie (Schizonepetae Herba), Chenpi (Citri Reticulatae Pericarpium) and Baibu (Stemonae Radix). It is mild and calm, not cold

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but not hot. The prescription name for relieving cough is consistent with the clinical effect of its addition and subtraction in the diagnosis and treatment of cough. And it is the general prescription for coughs. Although it is a general prescription, it is still necessary to verify the cause. And be sure not to see cough to relieve cough, which may delay the illness. 4

The course of cough can be divided into acute, subacute and chronic. Acute cough is of short duration and self-limited, while chronic cough is lingering and difficult to cure[8]. Chronic cough refers to the exclusion of pulmonary parenchymal diseases. Cough is the sole or main symptom for more than 8 weeks. And there is no obvious abnormality in chest X-ray and lung CT[9]. As a common disease, its causes include asthma, cough syndrome of upper respiratory tract, eosinophilic bronchitis

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and gastroesophageal reflux disease[10]. Cough is not only a symptom, but also an

independent disease. It is common in people and can cause the inflammation of the airway to spread. Frequent and severe coughs can lead to respiratory tract bleeding

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and affect the quality of life. Chronic cough can be treated based on by TCM

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syndrome differentiation which is used to guide treatment. Zhisousan is widely used in the treatment of chronic cough. In current clinical practice, Zhisousan may be

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added or combined with the routine western medicine to treat chronic cough. Many

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clinical studies have revealed that the prescription has a good clinical effect on the treatment of chronic cough [11,12]. The related animal experiments of the

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prescription are mostly focused on the treatment of cough. Jiang Dengzhao and others have shown that Zhisousan can regulate the imbalance of Th1/Th2 cytokines in rats

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with cough variant asthma and reduce inflammatory cells [13]. In this study, network pharmacological analysis was used to screen the active chemical constituents and targets of Zhisousan, to intersect with genes related to chronic cough diseases, and to analyze the mechanism of Zhisousan in the treatment of chronic cough, so as to facilitate more doctors to use this prescription to treat cough and relieve the pain of 5

patients with chronic cough. METHODS Screening of active ingredient of zhisousan and prediction of corresponding targets Seven traditional Chinese medicines (Jiegeng, Gancao, Baiqian, Ziwan, Jingjie, Chenpi

and

Baibu)

in

Zhisousan

were

respectively

input into

TCMSP

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(http://lsp.nwsuaf.edu.cn/tcmsp.php), the system pharmacological database and

analysis platform of TCM. TCMSP database contains 499 Chinese medicines and

related ingredients, targets, etc. It is a new platform developed by Wang Yonghua's

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team to study the mechanism of traditional Chinese medicine. The active chemical

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constituents of seven traditional Chinese medicines were screened by the two conditions of oral bioavailability (OB) value ≥30% and drug similarity (DL) value

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≥0.18. The corresponding targets of the active chemical constituents of traditional

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Chinese medicines were collected and the symbols of all the corresponding targets were obtained[14].

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Screening of targets of chronic cough disease Through the search with "chronic cough" as a key word in the GeneCards

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database, GeneCards database was used to screen genes related to chronic cough disease, which integrates diseases and human genes and covers a wide range of areas. Selection of core targets of zhisousan for chronic cough The core targets of Zhisousan in the treatment of chronic cough were obtained by intersecting the corresponding targets of the effective components of Zhisousan with 6

the genes related to chronic cough diseases. And the Venn diagram was drawn by R-3.6.1 software. The network construction of Zhisousan-active component-core target-chronic cough Cytoscape 3.7.1 software was used to construct the network of Zhisousan-active ingredient-core target-chronic cough, and to analyze the mechanism of Zhisousan in

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the treatment of chronic cough.

Construction of key target protein interaction network of Zhisousan for chronic cough

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The key targets of Zhisousan for chronic cough were uploaded to the String

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database in the form of symbols for protein-protein interaction analysis. The research species was set as “Homo sapiens”, and the key target protein interaction network of

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Zhisousan for chronic cough was obtained. According to the results, the target

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proteins were analyzed and the core genes were obtained. Analysis of go function enrichment and KEGG signaling pathway enrichment of

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Zhisousan in the treatment of chronic cough Through GO functional enrichment analysis, the key target proteins of Zhisousan

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for chronic cough were annotated. Through enrichment analysis of KEGG signaling pathway, we found out the important signaling pathways related to Zhisousan for chronic cough. Then GO and KEGG were performed. RESULTS Effective components and target prediction of Zhisousan 7

Based on the TCMSP database, 7 active chemical constituents of Jiegeng, 92 of Gancao, 4 of Baiqian, 19 of Ziwan,11 of Jingjie,5 of Chenpi and 32 of Baibu were screened for oral bioavailability (OB) value ≥ 30% and drug similarity (DL) value ≥ 0.18. After iteratively screening and removal of duplicates, 151 active ingredients and 2022 targets were obtained. The number of targets corresponding to the active ingredients is called the degree value. The active ingredients of the first three values

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are quercetin, kaempferol, and luteolin. The quercetin corresponds to a maximum of

77 targets, followed by kaempferol with 34 targets and luteolin with 28 targets. The

active ingredients with higher degree value mentioned above may play a significant

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role in the treatment of chronic cough with Zhisousan, which should be studied in the

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future.

disease-gene of Zhisousan

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The network construction of active components-targets and chronic cough

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Through the GeneCards database, 2348 genes associated with chronic cough were collected. And the Venn diagram(Figure 1) was obtained by R-3.6.1 software

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after intersection with targets of Zhisousan, which contained 79 key targets (Table 1), including IL6(Interleukin-6), VEGFA(Vascular endothelial growth factor A),

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EGFR(Epidermal growth factor receptor), CASP3(Caspase-3), ESR1(Estrogen receptor), MAPK8(Mitogen-activated protein kinase 8), MYC(Myc proto-oncogene protein), FOS(Proto-oncogene c-Fos), and so on. Cytoscape software was used to get the network of the relationship between active ingredients of Zhisousan common targets - chronic cough disease (Table 2, Figure 2). In the network, red 8

represents chronic cough disease, blue represents traditional Chinese medicine prescription Zhisousan, green circles are 79 common targets, purple triangles are active ingredients of Zhisousan, and lines are the interactive relationship. PPI network of Zhisousan in treating chronic cough Relying on the String database, the PPI network of the above intersection targets was obtained (Figure 3). The proteins in the center of the PPI interaction network are

of chronic cough with Zhisousan.

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IL6, VEGFA, EGFR, CASP3 and so on, which may be closely related to the treatment

GO functional enrichment analysis and KEGG signal pathway enrichment

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analysis of Zhisousan in the treatment of chronic cough

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Through GO functional enrichment analysis, 100 related items were obtained, including receptor activity, transcription factor activity, ion binding, transcription

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activator activity, apoptosis, etc. The top items of biological function annotations

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included nuclear receptor activity, transcription factor activity, direct ligand regulated sequence-specific DNA binding, steroid hormone receptor activity, protein

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heterodimerization activity, ammonium ion binding, DNA-binding transcriptional activator activity, RNA polymerase II specificity, etc.

The top 20 entries are listed

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here (Figure 4). KEGG signaling pathway enrichment analysis revealed 114 signaling pathways, including Hepatitis B, Kaposi sarcoma-associated herpesvirus infection, MicroRNAs in cancer, Non-small cell lung cancercies, p53 signaling pathway and Apoptosis and so on. The first 20 pathways are listed here (Figure 5, Figure 6). Finally,

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it is concluded that Zhisousan may treat chronic cough through the above signaling pathway. DISCUSSION Chronic cough has no obvious symptoms except cough. The cause is complicated. The mechanism is unclear and the examination is not abnormal. It is very easy to be misdiagnosed and mistreated. The treatment of TCM based on

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syndrome differentiation is quite effective. Zhisousan, has compatible herbs which have specific characteristics: Jiegeng, Gancao, Baiqian, Ziwan, Jingjie, Chenpi and

Baibu. Jiegeng is slightly warm and not dry. It can disperse wind-pathogen, and

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wind-cold and wind-heat can be used. Ziwan is warm but not hot, moist but not dry. It

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can be used for both external and internal disease. Baiqian has mild temperature but not dryness-heat, that is good at reducing qi and sputum for lung cough medicine. It

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can be used for cold cough, hot cough, new cough and long cough. In the book of

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BenCaoYanYi, "Baiqian is used to keep lung qi and cure cough.". Baibu are bitter and descending, sweet and juicy, warm and moist. It is good at curing cough and upstream

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qi. It is appropriate for all coughs, regardless of exogenous or endogenous coughs. Chenpi regulates the obstruction of lung qi, dries dampness and dissipates phlegm,

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which is described in “MingYiBieLu” as "breathing down, relieving vomiting and coughing.". Jiegeng propagates lung qi. Ziwan, Baiqian and Baibu suppress lung qi, mild and calm. And they all can relieve coughing. Based on the TCMSP platform, 7 active chemical constituents of Jiegeng, 92 of Gancao, 4 of Baiqian, 19 of Ziwan,11 of Jingjie,5 of Chenpi and 32 of Baibu were 10

screened. After iteratively screening and removal of duplicates, Zhisousan had a total of 51 active ingredients and acted on 2202 targets. Among them, the quercetin corresponds to a maximum of 77 targets, followed by kaempferol with 34 targets and luteolin with 28 targets. More than 4000 flavonoids have been found to be very beneficial to human health[15]. Quercetin, a flavonoid, has the effects of antioxidant, anti-allergic, anti-inflammatory, antitussive and antiasthmatic. Studies have shown

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that quercetin can improve the airway inflammation of asthmatic mice and is widely

used in the treatment of respiratory diseases[16,17]. The active ingredients with more

targets mentioned above may play a key role in the treatment of chronic cough with

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Zhisousan.

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There are 79 common targets between the active components of Zhisousan and genes related to chronic cough, including IL-6, VEGFA, EGFR, CASP3, etc. As a

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proinflammatory factor to remove foreign bodies and participate in inflammatory

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reaction, IL-6 is an important mediator of inflammation synthesis in acute phase and plays a central role in cytokines IL-6, IL-8 and TNF(tumor necrosis factor) cooperate

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with each other and participate in inflammation. VEGFA is a kind of cell growth factor produced by vascular endothelial cells, which can increase vascular

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permeability, induce vascular growth through its receptor VEGFR, promote the proliferation of pulmonary vessels, and thicken the tracheal wall[18]. Firstly, the lung tissue is rich in blood. Secondly, under the action of various factors, such as inflammatory reaction, smoking, hypoxia, TNF-alpha, IL-6 and so on, it can promote the expression of VEGF and its receptor. Therefore, VEGFA can be expressed in large 11

quantities to maintain the normal structure and function of pulmonary vessels and repair damage. Studies have shown that VEGFA is a downstream target of hypoxia‐ inducible factor‐1α(HIF-1α). HIF-1αcan regulate the expression of VEGFA at the transcriptional level in hypoxia[19]. Clinical studies have shown that there is a certain correlation between VEGF and EGFR pathway[20]. Epidermal growth factor (EGF) is an important component of cell signaling pathway, which can make cell

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proliferate and inhibit cell apoptosis. Apoptosis can lead to the loss of cells in tumor and normal tissues, which is a unique way of cell death. CASP3 is closely related to

apoptosis and can cleave a series of substrates[21]. CASP3 is specifically expressed in

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lung cancer tissues[22]. And the 79 common targets are intricate, so the link between

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the targets may also be one of the mechanisms of Zhisousan in the treatment of chronic cough.

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KEGG enrichment analysis revealed hepatitis B, Kaposi sarcoma-associated

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herpesvirus infection, MicroRNAs in cancer, non-small cell lung cancercies, p53 signaling pathway, apoptosis and other signaling pathways. According to statistics, the

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prevalence rate of hepatitis B virus infection and tuberculosis co-infection is very high in China. Mr. Yang et al. have shown that there are potential susceptible sites of

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hepatitis B co-infection in patients with tuberculosis. IL-6 participates in the initial stage of viral infection. The virus damages the liver to a certain extent, and anti-tuberculosis drugs also cause drug-induced liver damage to a certain extent, which make treatment more difficult. Cough symptoms associated with hepatitis B and tuberculosis are not easy to treat[23]. MicroRNAs is a non-coding RNA molecule 12

with about 22 nucleotides, which can regulate gene expression at the post-transcriptional level. In recent years, a variety of respiratory diseases have been found to involve in MicroRNA, which can regulate their biological processes[24], such as viral infectious respiratory diseases, pulmonary tuberculosis, asthma, COPD, pulmonary fibrosis, lung tumors, etc. Panganiban et al. have shown that circulating miRNAs may be a non-invasive biomarker for the diagnosis of asthma and allergic

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rhinitis[25]. MicroRNAs is also the target of non-small cell lung cancer. In

microRNAs, miR-34a is directly regulated by p53 and plays an important role in inhibiting cancer[26]. And non-small cell lung cancer is closely related to cell

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proliferation, apoptosis and metastasis. Therefore, it can be seen that the signaling

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pathways such as microRNAs, apoptosis and non-small cell lung cancer also interact with each other. Human immunodeficiency virus (HIV) infection can reduce the

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immunity. Currently, HIV-related lung disease is the main cause of death in HIV

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infected people[27]. Non-infectious pulmonary diseases are the main chronic complications in patients with HIV. About 20% of the HIV patients accelerated the

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change of emphysema in the lungs compared with the normal group of the same age[28]. Mr. Tan's related cases suggest that Kaposi's sarcoma of the lung has

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symptoms similar to pneumonia, such as coughs, shortness of breath, blood in sputum and so on.

Inflammatory or tumorous lung infiltration or solitary pulmonary nodules

were showed. It may also involve most of the bilateral lung lobes without tumor changes. At this time, attention should be paid to whether the patient has a history of HIV infection[29]. P53 can detect and repair mitotic damaged cells. Abnormal 13

expression of p53 is closely related to tumors. P53 is a tumor suppressor that can accelerate cell cycle arrest, apoptosis and senility. Studies have shown that lung cancer is associated with many cell proliferation and apoptosis genes, such as p53, miR-34a, inhibitor of apoptosis protein and so on[26,30]. These pathways and the interaction with these pathways may also be closely related to the treatment of chronic cough with Zhisousan.

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In this study, network pharmacology was used to analyze the relationship

between Zhisousan and chronic cough disease as a whole, so as to clarify the mechanism of Zhisousan in the treatment of chronic cough disease. Considering the

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limitations of the emerging network pharmacology, we will continue to study the

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mechanism of Zhisousan in the treatment of chronic cough disease. Moreover,source, performance and dosage of traditional Chinese medicine in clinical practice have not

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been taken into account. And many other variable factors still need to be further

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improved in order that more doctors might use Zhisousan to treat chronic cough disease. Hope to improve Zhisousan into Chinese patent medicine to facilitate the use

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of patients and alleviate the pain of patients with chronic cough. In this study, network pharmacology and traditional Chinese medicine are

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combined to provide a reference for the clinical application of Zhisousan in the treatment of chronic cough. It can be used to treat chronic cough by adding and subtracting traditional Chinese medicine into the basic prescription by more doctors. And the direction of experimental study on the treatment of chronic cough with Zhisousan is also provided in terms of relevant targets and signaling pathways in the 14

future.

Declaration of Competing Interests The authors declare that they have no competing interests. Author Contributions JiYu Zou designed research，analyzed data，drafted and edited manuscript;

the manuscript, and approved of the final submission.

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Funding

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All authors contributed to data accumulation. All authors contributed to revisions of

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The authors were supported by grants from the High Level Innovation and Entrepreneurship Team of Liaoning Province "Xing Liao Ying Cai Plan"

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(XLYC1808011) and the Key Projects of Liaoning Science and Technology

Author Statement

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Department (20170540626).

I have made substantial contributions to the conception or design of the work, or

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the acquisition, analysis, or interpretation of data for the work. I have drafted the work or revised it critically for important intellectual content.

I have approved the final

version to be published. And I agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 15

All persons who have made substantial contributions to the work reported in the manuscript, including those who provided editing and writing assistance but who are not authors, are named in the Acknowledgments section of the manuscript and have given their written permission to be named. If the manuscript does not include Acknowledgments, it is because the authors have not received substantial

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contributions from nonauthors.

Conflict of Interest

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The authors declare that they have no competing interests.

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Funding Source

The authors were supported by grants from the High level innovation and entrepreneurship team of Liaoning Province "Xing Liao Ying Cai Plan" (XLYC1808011) and the Key Projects of Liaoning Science

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and Technology Department(20170540626).

Acknowledgements

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Thanks to the TCMSP database created by Prof. WANG YH's team, which provides us with great convenience for our data retrieval. Thanks Liaoning University of

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Traditional Chinese Medicine for its support.

APPENDIX The

letter

A

was

used

instead

of

"(2S)-2-[4-hydroxy-3-(3-methylbut-2-enyl)phenyl]-8,8-dimethyl-2,3-dihydropyrano[2 16

,3-f]chromen-4-one". The

letter

B

was

used

instead

of

"(E)-1-(2,4-dihydroxyphenyl)-3-(2,2-dimethylchromen-6-yl)prop-2-en-1-one". The

letter

C

was

used

instead

of

"(2S)-6-(2,4-dihydroxyphenyl)-2-(2-hydroxypropan-2-yl)-4-methoxy-2,3-dihydrofuro [3,2-g]chromen-7-one". letter

D

was

used

instead

of

ro of

The

"3-(2,4-dihydroxyphenyl)-8-(1,1-dimethylprop-2-enyl)-7-hydroxy-5-methoxy-coumar in". letter

E

was

used

-p

The

instead

of

The

letter

F

re

"3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-8-(3-methylbut-2-enyl)chromone". was

used

instead

of

letter

G

na

The

lP

"5,7-dihydroxy-3-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromone". was

used

instead

of

"2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6-(3-methylbut-2-enyl)chromone". letter

H

was

used

instead

of

ur

The

"(E)-3-[3,4-dihydroxy-5-(3-methylbut-2-enyl)phenyl]-1-(2,4-dihydroxyphenyl)prop-2

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-en-1-one". The

letter

I

was

used

instead

of

instead

of

"1,3-dihydroxy-9-methoxy-6-benzofurano[3,2-c]chromenone". The

letter

J

was

used

"1,3-dihydroxy-8,9-dimethoxy-6-benzofurano[3,2-c]chromenone". 17

The

letter

K

was

used

instead

of

instead

of

"(2R)-7-hydroxy-2-(4-hydroxyphenyl)chroman-4-one". The

letter

L

was

used

"(2S)-7-hydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)chroman-4-one". The

letter

M

was

used

instead

of

"2-[(3R)-8,8-dimethyl-3,4-dihydro-2H-pyrano[6,5-f]chromen-3-yl]-5-methoxyphenol

The

letter

N

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". was

used

instead

of

"8-(6-hydroxy-2-benzofuranyl)-2,2-dimethyl-5-chromenol".

-p

The letter O was used instead of "3'-Hydroxy-4'-O-Methylglabridin".

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The letter P was used instead of "7,2',4'-trihydroxy － 5-methoxy-3 －

[1]

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23

GLOSSARY The Yellow Emperor's Canon of Medicine It's the earliest extant medical canon in China, establishing a unique theoretical system for TCM and laying a solid foundation for the theoretical and clinical development of TCM. Su Wen· Cough Theory

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It's an article in The Yellow Emperor's Canon of Medicine, which is devoted to cough specially. Medical Insights

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It's a medical book written by Cheng Zhongling, a famous doctor of the Qing

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Dynasty. Smog

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It's a mixture of smoke and fog. It not only exacerbates respiratory diseases, but

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also causes it.

Jiegeng (Platycodon Grandiflorus), Gancao (Glycyrrhizae Radix Et Rhizoma),

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Baiqian (Cynanchi Stauntonii Rhizoma Et Radix), Ziwan (Asteris Radix Et Rhizoma),

Jingjie

(Schizonepetae

Herba),

Chenpi

(Citri

Reticulatae

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Pericarpium), Baibu (Stemonae Radix) They are seven traditional Chinese medicines in Zhisousan, with Latin names in

brackets. TCMSP It's a unique system pharmacology platform of Chinese herbal medicines that 24

captures the relationships between drugs, targets and diseases. Oral bioavailability (OB) The speed and degree at which oral drugs are absorbed into the human circulation. Drug similarity (DL) It's the similarity between a compound and a drug.

ro of

Symbol It's a standardized form of target name. PPI network

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It's a protein-protein interactions network that is produced by the String database.

re

IL6 (Interleukin-6)

acute phase response.

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Cytokine with a wide variety of biological functions. It is a potent inducer of the

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VEGFA (Vascular endothelial growth factor A) Growth factor active in angiogenesis, vasculogenesis and endothelial cell growth.

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Induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels.

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EGFR (Epidermal growth factor receptor) Receptor tyrosine kinase binding ligands of the EGF family and activating

several signaling cascades to convert extracellular cues into appropriate cellular responses. CASP3(Caspase-3) 25

Participate in the activation cascade of caspases responsible for apoptosis execution. ESR1(Estrogen receptor) Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues.

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MAPK8(Mitogen-activated protein kinase 8)

Serine/threonine-protein kinase involved in various processes such as cell proliferation, differentiation, migration, transformation and programmed cell death.

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MYC (Myc proto-oncogene protein)

FOS (Proto-oncogene c-Fos)

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Transcription factor that binds DNA in a non-specific manner.

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Nuclear phosphoprotein which forms a tight but non- covalently linked complex

Jo

ur

na

with the JUN/AP-1 transcription factor.

26

ro of

-p

re

lP

na

ur

Jo Fig1

27

ro of

-p

re

lP

na

ur

Jo Fig2

28

ro of

Fig4

-p

re

lP

na

ur

Jo Fig3

Fig5

29

ro of -p re lP na

Fig6

Serial

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Table 1 The key targets of Zhisousan in the treatment of chronic cough Key

count

targets

1

IL6

49

2

VEGFA

49

3

EGFR

48

4

CASP3

46

Jo

number

30

Serial

Key

ESR1

44

6

MAPK8

44

7

MYC

43

8

CCND1

41

9

FOS

38

10

ERBB2

36

11

PPARG

32

12

MDM2

31

13

APP

29

14

NOS3

28

15

CASP8

27

16

NR3C1

26

17

CAV1

25

18

ICAM1

25

na

ur

19

MCL1

25

PGR

25

21

AHR

24

22

CASP9

24

23

RB1

23

24

HIF1A

22

Jo

20

-p

5

re

targets

lP

number

ro of

count

31

Serial

Key

TIMP1

22

26

CYP3A4

21

27

CCNB1

20

28

CRP

20

29

ESR2

20

30

PARP1

20

31

IGFBP3

19

32

NFE2L2

19

33

NFKBIA

19

34

APOB

18

35

CHEK1

18

36

MAPK10

18

37

NQO1

18

38

PLAU

18

na

ur

39

VCAM1

18

IGF2

17

41

CYP1A1

16

42

CYP2B6

16

43

GSTP1

16

44

RAF1

16

Jo

40

-p

25

re

targets

lP

number

ro of

count

32

Serial

Key

BCL2

15

46

CHEK2

15

47

LDLR

15

48

ACHE

14

49

IRF1

14

50

PON1

14

51

BIRC5

13

52

CYP1B1

13

53

PRKCA

13

54

RASSF1

13

55

ERBB3

12

56

PCNA

11

57

SELE

11

58

ADRA2C

9

na

ur

59

ALOX5

9

CHRM2

9

61

GSTM1

9

62

PTGS1

9

63

ABCC1

8

64

DRD2

8

Jo

60

-p

45

re

targets

lP

number

ro of

count

33

Serial

Key

POR

8

66

PTGER3

8

67

TP63

8

68

NR1I3

7

69

CHRM1

5

70

COL3A1

5

71

HTR3A

5

72

MGAM

5

73

TYR

5

74

CHRNA2

3

75

ECE1

3

76

F7

3

77

RXRB

3

78

CHRM3

2

na

ur PRSS1

1

Jo

79

-p

65

re

targets

lP

number

ro of

count

34