Mei-Hui Chen, Yu-Bo Han, Yan-Bo Sui, Li Liu?

1. Heilongjiang University of Traditional Chinese Medicine,Harbin 150040,China

2. The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine,Harbin 150040,China

Keywords:Radix Astragali-Radix Pseudostellariae Chronic heart failure Network pharmacology Targets Mechanism of action

ABSTRACT Objective: To explore and analyze the effective component, target and mechanism of action of Radix Astragali-Radix Pseudostellariae in the treatment of chronic heart failure (CHF) by network pharmacological research methods. Methods: With the help of TCMSP database,the effective chemical constituents and targets of Radix Astragali-Radix Pseudostellariae were screened out, and CHF-related proteins were obtained by Gene Cards and OMIM database. The target protein interaction network (PPI) was constructed on the String database platform, and using DAVID database to conduct the GO function and KEGG pathway enrichment analysis on the target of the obtained astragalus-pseudostellariae for the treatment of CHF. Results:Radix Astragali-Radix Pseudostellariae were selected as 28 effective chemical components and 104 therapeutic targets for CHF. There were obtained 49 GO functional annotations and 86 KEGG signaling pathways. Conclusion: Radix Astragali-Radix Pseudostellariae can treat CHF through multi-target and multi-pathway.?Corresponding author: LIU Li, Chief Physician, Professor, PhD Supervisor.E-mail: liliu429@163.com.

1. Introduction

Chronic Heart Failure (CHF) is a group of complex clinical syndrome caused by abnormal Heart structure and/or function,which is caused by reduced cardiac output at rest or under load and/or increased cardiac pressure. It can be manifested as dyspnea,palpitation, fatigue, lower limb edema and other symptoms. It is the final stage of various Heart diseases. Its high rates of morbidity,hospitalization and mortality place a heavy burden on society and individuals. The number of people suffering from heart failure in the world is more than 26 million. With the aggravation of the aging of the population in China, the latest results of the epidemiological survey of heart failure show that there are about 13.7 million heart failure patients in China, and the prevalence of heart failure is 1.3%over the age of 35 years old [1].A multicentre randomized controlled trial showed that standard basic therapy combined with oral Chinese medicine Qiliqiangxin capsule could significantly improve the level of NT-proBNP, a major outcome indicator of CHF[2].The Guidelines for Diagnosis and Treatment of Heart Failure in China 2018 also clearly pointed out the intervention role of traditional Chinese medicine, and more and more traditional Chinese medicine treatment is widely used in clinical practice, and the treatment of heart failure by traditional Chinese medicine has the advantages of stabilizing the condition, improving cardiac function and improving the quality of life[3].Traditional Chinese medicine scholars believe that CHF belongs to the categories of "asthma syndrome","deficiency" and "edema", and the pathogenesis can be summarized as "deficiency", "blood stasis" and "water", and the dynamic fluctuation of deficiency and excess determines the development and evolution of heart failure[4].

In the treatment of CHF, Professor Li Liu took tonifying deficiency and strengthening normal body as the guiding ideology,combined with traditional Chinese medicine classics and years of clinical experience, took "enriching qi to warm yang, moving blood to benefit water" as the treatment method, then summarized and applied "Shenqi Yixin Formula" as the empirical prescription for the treatment of CHF, and achieved a good curative effect[5-7].Radix Astragali-Radix Pseudostellariae in the prescription for the sovereign drug, both are the product of Qi, a total of play the work of invigorating the viscera. The two medicine is gentle, with the spleen,lung meridian. Radix astragali has the effect of invigorating qi,strengthening surface and diuresis, protecting myocardial cells and vascular endothelial cells, and inhibiting myocardial hypertrophy and myocardial fibrosis[8-9].Radix Pseudostellariae is beneficial to Qi, invigorating the spleen, generating fluid and moistening the lung,protecting the myocardium, improving oxidative stress, lowering blood lipid, etc.[10-11].At present, the mechanism of Radix Astragalus- Radix Pseudostellariae in the treatment of CHF is still unclear. As a research method of new Chinese medicine, network pharmacology can establish a network model to analyze the target and pathway of Chinese medicine components and diseases, and reveal the interaction relationship between drugs and diseases. Therefore, by taking advantage of network pharmacology, this study explored the potential mechanism of " Radix Astragali-Radix Pseudostellariae "in the treatment of CHF, so as to provide certain theoretical basis for clinical application and further guide drug use.

2. Materials and methods

2.1 Collection and screening of active ingredients of "Radix Astragali-Radix Pseudostellariae"

Using TCMSP database (https://tcmspw.com/tcmsp.php)respectively retrieval "Radix Astragali" and "Radix Pseudostellariae" to obtain information about the chemical composition of traditional Chinese medicine, the related parameter is set to: the oral bioavailability (OB) > 30%, and the drug-like (DL) > 0.18. After screening, the active ingredients and drug targets of Radix Astragali-Radix Pseudostellariae were obtained.

2.2 Construction of drug-disease-target network

With the keywords of "Chronic Heart Failure", CHF related targets were collected by searching the online human Mendelian genetic database OMIM (http://www.omim.org/) and the human genetic database GeneCards (http://genecards.org/). Use Interactivenn platform (http://www.interactivenn.net/) to capture the common targets of drugs and disease. The common targets (Drug-Disease)was imported into Cytoscope3.7.1 software, and a visual network of drug-disease-target was constructed.

2.3 Establishment of protein interaction network

Using the String database, the drug-disease targets were uploaded,the species was set as "Homo sapiens", the minimum interaction score was 0.400, the target protein interaction network (PPI) was developed, and then the Cytoscope3.7.1 software was used to analyze and sequence the nodes.

2.4 GO and KEGG analysis

The drug-disease results were imported into the DAVID database for gene ontology (GO) analysis and KEGG signaling pathway enrichment analysis based on Kyoto Encyclopedia of Genes and Genes (KEGG). Then, 10 terms of GO and 12 pathways of KEGG were screened for visual analysis according to P value sequencing.

3. Results

3.1 Information on chemical constituents of Radix Astragali-Radix Pseudostellariae

All the chemical components of Radix Astragali-Radix Pseudostellariae were retrieved from TCMSP database. After screening (conforming to OB > 30% and DL > 0.18), a total of 28 effective chemical components were obtained, including 20 of Radix Astragali and 8 of Radix Pseudostellariae. According to the OB value, the basic information of the top 5 active ingredients of each drug was listed, as shown in Table 1.

Table 1 Basic information of chemical composition of Radix Astragali-Radix pseudostellariae

3.2 Construction of drug-disease Target (DDT) network

A total of 106 drug action targets of Radix Astragali-Radix Pseudostellariae were obtained by TCMSP database, and 10,352 CHF-related Gene targets were obtained by searching OMim and Gene Cards database. The obtained drugs were intersected with disease targets, and a total of 104 drug-disease targets were obtained,as shown in Figure 1. The Cytoscape3.7.1 software was used to visualize the regulation network of TCM, and it was found that this network had 104 nodes and 389 edges, as shown in Figure 2.

Figure 1 Drug-disease-Target (DDT) Wayne diagram

Figure 2 Drug-disease-chemical composition-target network

3.3 PPI network construction and visualization analysis results

Enter the String Internet interface, import all drug-disease targets into the database, select "Homo sapiens" as the species, and the lowest interaction score is 0.400. Cytoscape software is used to arrange 104 nodes according to the Degree value. The top 5 targets were IL6, VEGFA, CASP3, MAPK8 and EGFR in turn. As shown in figure 3.

Figure 3 Sequencing results of key targets

3.4 Gene Ontology (GO) Analysis

104 "drug-disease" targets was analyzed by David database, and a total of 49 functional annotations were obtained. Cell Component(CC), Molecular Function (MF) and Biological process (BP)categories were analyzed using OmicShare platform, and P value and enrichment target number were used. The top GO analysis results were sorted. As shown in Figures 4-1 to 4-4.

Figure 4-1 GO function analysis diagram-GO histogram

Figure 4-2 GO function analysis diagram-CC

Figure 4-3 GO function analysis diagram-MF

Figure 4-4 GO function analysis diagram-BP

3.5 Kyoto Encyclopedia of Genes and Genomes (KEGG)enrichment analysis

KEGG enrichment analysis was conducted on 104 DDTs by David platform, and a total of 86 enrichment pathways were obtained.The results showed that the treatment of CHF by " Radix Astragali-Radix Pseudostellariae " was related to multiple signaling pathways.According to the KEGG bubble diagram, the leading pathways were mainly PI3K-Akt, MAPK, TNF, etc., as shown in Figure 5. The Cytoscape software was used to display the visual network of DDT and enrichment pathway, and the network had 140 nodes and 510 edges, as shown in Figure 6.

Figure 5 Bubble diagram of KEGG enrichment analysis

Figure 6 Drug-disease-target-pathway network

4. Discussion

CHF is a slowly progressing disease, which refers to the gradual development of the signs and symptoms of heart failure in addition to the existing chronic heart disease. CHF is related to inflammatory response, oxidative stress and regulation of vascular endothelial function. The most important pathophysiological mechanism of CHF is ventricular remodeling. However, myocardial cell apoptosis,myocardial hypertrophy and myocardial fibrosis are the basic features of ventricular remodeling. The majority of CHF patients are elderly and the disease is prolonged, which consumes the body's vital qi, and the heart qi is deficient. Radix Astragali is an important medicine to replenish qi and has the function of tonifying qi and warming yang. A large number of studies have confirmed that Astragalus membranaceus and its active components have obvious heart-strengthening effect and can improve heart failure[12]. Radix Pseudostellariae is a supplement for both Qi and blood.It can replenish Qi and nourish Yin instead of heat, and can improve CHF caused by acute myocardial infarction, and has the effect of protecting myocardium[10]. Based on network pharmacology study found, Radix Astragali-Radix Pseudostellariae contains a variety of active ingredients, can be applied to multiple targets, multiple pathways, common regulating biological function of CHF.

In this study, a total of 28 chemical components were obtained by screening statistics, among which formononetin, kaempferol,quercetin and β-sitosterol were the main compounds. During the development of CHF, oxidative stress, inflammatory factors and AngⅡ can induce apoptosis of cardiomyocytes and initiate compensatory structural remodeling. Further development of CHF will lead to pathological remodeling such as cardiac hypertrophy and decreased cardiac compliance, and eventually develop into heart failure. Large accumulation of reactive oxygen species(ROS) can promote oxidative stress disorder in patients and cause damage and death of myocardial cells, which is closely related to the pathological process of ventricular remodeling in heart failure.Yu Xue[13] established a hypoxic and reoxygenated model of H9C2 cardiomyocytes, and found that formononetin can reduce the ROS level in cardiomyocytes, improve cell adhesion status, increase cell survival rate, and have a certain protective effect on cardiomyocytes.TLR4 is the subtype of TLR with the highest expression level in cardiomyocytes, and plays an important role in inflammatory response by binding with ligand to activate downstream NF-κB and induce the production of inflammatory factors[14]. Studies have shown[15] that kaempferol can significantly improve the activity of H9C2 cells, which may be related to the inhibition of TLR4/NF-κB signaling pathway, and reduce cell apoptosis by anti-inflammation of cardiomyocytes, thus playing a protective role of cardiovascular.Quercetin, as a flavonoid compound, has a variety of biological effects such as antioxidant stress, inhibition of inflammatory response, inhibition of fibrosis, and has a significant protective effect on the heart, liver, kidney and other organs[16], and can significantly reduce the phosphorylation level of Akt in AngⅡ induced cardiac mast cells, thereby inhibiting its activity[17]. In addition, quercetin can also increase the bioavailability of endothelial cells and improve the function of vascular endothelial cells by inhibiting NADPH oxidase mediated eNOS phosphorylation, which has a protective effect on the heart[18]. JN[19] found that β-sitosterol can inhibit IL-6 activity, reduce the release of inflammatory factors such as TNF-α and IL-1β, improve the inflammatory response, and participate in the alleviation of oxidative stress and inhibition of cell apoptosis by upregulation of SOD and down-regulation of ERK1/2 levels[20]. It can be seen that the related chemical components of Radix Astragali-Radix Pseudostellariae have a certain positive effect on the treatment of CHF.

According to PPI network diagram, " Radix Astragali-Radix Pseudostellariae " plays an effect on CHF through multiple targets.After analysis, the most interaction targets were interleukin 6 (IL-6),vascular endothelial growth factor A (VEGFA), CASP3, mitogenactivated protein kinase 8 (MAPK8), epidermal growth factor receptor (EGFR), etc. Inflammatory factors such as IL-1, IL-6 and TNF-α have a direct effect on the development of CHF, among which IL-6 is an important inflammatory mediator. Correlative experiments have confirmed that IL-6 stimulates the expression of transforming growth factor (TGF-β), increases the synthesis of extracellular matrix collagen, reduces the degradation of collagen,causes myocardial fibrosis, leads to or aggravates ventricular remodeling, and promotes the occurrence of CHF[21-22]. VEGFA is a major member of the VEGF family and plays an important role in the regulation of angiogenesis. After binding to VEGF-R1 and VEGF-R2 receptors in cardiac tissue, VEGFA induces proliferation,differentiation and migration of endothelial cells through multiple biological pathways, and participates in vascular remoderings of CHF and myocardial angiogenesis[23]. On the other hand, VEGFA is produced by cardiomyocytes stimulated by inflammation,mechanical action and cytokines, and changes in cardiac energy metabolism affect the development of CHF[24-25]. Cardiomyocyte apoptosis is the key to the transformation of compensatory myocardial remodeling to heart failure, and is regulated by a variety of factors, among which CASP3 and MAPK8 are the more important proteins. Casp3 can trigger the Caspase cascade and induce cell apoptosis. Studies have found that[26] by regulating the expression of Bcl-2 in the heart tissue of rats with heart failure,reducing the expression levels of Bax and Caspase 3, thereby inhibiting the apoptosis of cardiomyocytes and achieving the effect of improving the cardiac function of rats. When MAPK8 is activated,BMF, a pro-apoptotic protein, is phosphorylated in epithelial cells and enters mitochondria to produce biological effects and initiate the apoptotic process, thus leading to changes in heart structure[27]. Ang II-induced vascular smooth muscle cell hypertrophy is associated with the trans-activation of epidermal growth factor receptor EGFR,a transmembrane glycoprotein that plays a key role in vascular remodeling by inducing ER stress[28]. In addition, the activation of EGFR can promote the phosphorylation of receptor tyrosine kinases,leading to the initiation of a series of signaling pathways, and then regulate the proliferation and differentiation of cardiomyocytes[29].These potential targets are involved in the pathophysiological process of CHF through multiple pathways.

The GO analysis of " Radix Astragali-Radix Pseudostellariae " in the treatment of CHF showed that Astragalus-Pseudostellariae Radix participated in its biological process through a variety of forms,mainly involving cell apoptosis, vascular endothelial cell migration,oxidative stress response, inflammation reaction, etc. Multiple signaling pathways were obtained by KEGG enrichment analysis,and the main pathways included PI3K-Akt, MAPK, TNF, etc. PI3KAkt is an important signal transduction pathway in the body. As an upstream signal, PI3K can activate and promote the phosphorylation of downstream Akt and participate in the signal transduction process.By regulating a variety of downstream proteins, PI3K has an impact on angiogenesis, myocardial cell apoptosis autophagy, energy metabolism, Ca2+ circulating protein and inflammatory response and other activities. It is closely related to the occurrence and development of CHF [30-31]. The decrease of PI3K expression level and Akt phosphorylation level has positive effect on the improvement and recovery of cardiac function. The relevant experiments[32]showed that the active ingredients in Radix Astragali could inhibit the activity of TBK1/PI3K/ Akt by upregulating SiKE and prevent cardiac hypertrophy. MAPK is an important transmitter of signal transduction from the extracellular to the nucleus. It is widely found in a variety of cells and is involved in a variety of physiological and pathological processes such as cell growth, proliferation,differentiation, apoptosis, stress, inflammation and autophagy.When the MAPK signaling pathway is activated, Phosphorylation of extracellular regulated protein kinases (ERKs), c-Jun aminoterminal kinases (JNK) and p38 mitogen-activated protein kinase(p38MAPK) regulates gene expression and causes hypertrophy and apoptosis of cardiomyocytes, leading to cardiac hypertrophy and fibrosis[33-34]. Inhibitory ERKs, JNK and p38MAPK pathways can alleviate myocardial cell apoptosis, reduce the level of myocardial autophagy, reduce myocardial injury, normalize the level of myocardial cytokines, and improve ventricular systolic function[35-37]. Activation of TNF signal transduction pathway can promote cell growth and differentiation, induce cell apoptosis and inflammatory response, and participate in ventricular remodeling in various ways. Myocardial cells themselves cannot produce TNF-α, but when CHF is present, TNF-α can be expressed in cells, activate NF-κB transcription to the nucleus, induce inflammatory factors such as IL-6 and cyclocxygenase (COX), stimulate cardiomyocyte hypertrophy and apoptosis, lead to tissue remotening, and aggravate the condition of CHF [38-40]. Therefore, this study believes that" Radix Astragali-Radix Pseudostellariae " may exert biological effects through multi-target, multi-pathway and multi-signal pathway coordination, and achieve the purpose of delaying the development of heart failure in many aspects, such as inhibiting cell apoptosis,alleviating inflammatory response, and opposing ventricular remodeling.

Radix Astragali-Radix Pseudostellariae are tonic products with stable drug properties. This study, based on the network pharmacology build visualization network diagram, found " Radix Astragali-Radix Pseudostellariae " medicine for effective chemical composition contains 28, 104 targets, 86 pathway with CHF potential relevance, its mechanism of action may inhibit apoptosis,anti-inflammatory and antioxidant stress synergy in the treatment of CHF, It will provide a basic reference for the future exploration of therapeutic genes and the in-depth study of the mechanism of" Radix Astragali-Radix Pseudostellariae " or traditional Chinese medicine in the treatment of CHF.