Analysis of Action Mechanism of Fuxin Decoction on Heart Failure Based on Network Pharmacology

Tao GAO, Meijun LI, Wei LIU, Yitao XUE

1. Zibo Central Hospital of Shandong Province, Zibo 255036, China; 2. Shandong University of Traditional Chinese Medicine, Jinan 250355, China; 3. The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, China

Abstract [Objectives] The purpose was to analyze and predict the mechanism of Fuxin decoction in the treatment of heart failure based on network pharmacology. [Methods] Relevant tools and methods of network pharmacology were used to obtain the active ingredients and action targets of Fuxin decoction and action targets of heart failure, find out the key targets and core clusters of Fuxin decoction on heart failure, and analyze related signal pathways, to explore possible molecular mechanisms. [Results] A total of 53 active ingredients and 224 action targets of Fuxin decoction, 1 010 heart failure-related target genes, and 94 drug-disease directly acting targets were obtained. From the PPI network constructed, 225 key targets and a core cluster composed of 56 nodes and 297 interactions were screened out. Association with tumors, glutamate synapse and other related pathways and related genes such as adenylate cyclase and MAPK were known. [Conclusions] The mechanism of Fuxin decoction in the treatment of heart failure is related to βARs-G protein-adenylate cyclase, PI3K-Akt signaling pathway and HSP90, MAPK and other proteins.

Key words Fuxin decoction, Heart failure, Network pharmacology, Molecular mechanism, Signal pathway

1 Introduction

2 Methods

2.1 Screening and target prediction of active ingredients of Fuxin decoctionThe chemical components contained in Radix Aconiti Carmichaeli, Radix Astragali and Semen Lepidii were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSP). According to the pharmacokinetic ADME parameters, with oral bioavailability (OB) ≥30% and drug similarity (DL) ≥0.18 as criteria, active ingredients were screened out[7-9]. The actions targets of the active ingredients predicted were obtained from the database.

2.2 Diagram construction of "traditional Chinese medicine-ingredient-target network" of Fuxin decoctionUsing network imaging software Cytoscape 3.7.0, the diagram of "traditional Chinese medicine-ingredient-target network" of Fuxin decoction was constructed. In the network diagram, "node" represents the traditional Chinese medicine, ingredient or target, and "edge" represents the relationship between the traditional Chinese medicine and the ingredient, and between the ingredient and the target.

2.3 Search of targets for heart failureWith "heart failure" as keyword, the targets of heart failure were searched in 5 databases: TTD, DrugBank, OMIM, DisGeNET and PharmGKB. All results were collected and duplicates were deleted, and the remaining were the targets of heart failure.

2.4 Screening of directly acting targetsThe targets of the active ingredients of traditional Chinese medicine and the targets of the disease were intersected, and the overlapping targets were selected as directly acting targets.

2.5 Construction of protein-protein interaction network (PPI network)Genes or proteins do not work in isolation. Instead, they often interact with multiple genes or proteins. This kind of interaction between genes or proteins constitutes a complex network (PPI network). The directly acting targets were input into STRING 10.5 database (https://string-db. org) one by one for searching, and the score of reliability was set at 0.7. Proteins or genes related to each target were obtained. Then, all protein or gene interaction data were imported into Cytoscape software to construct an integrated PPI network.

2.6 Screening of key targets and core cluster

2.6.1Screening of key targets. The integrated PPI network constructed was analyzed using NetworkAnalyzer tool of Cytoscape software, and network topology parameters were obtained. Among them, the degree of freedom (Degree), betweenness centrality and closeness centrality are common parameters that reflect centrality. The targets whose Degree value was between the mean and maximum value were selected as the key targets.

2.6.2Screening of core cluster. Cluster analysis was performed using plug-in MCODE of Cytoscape, and the screening criteria were set as follows: Degree Cutoff: 2 and K-Core: 2. The more closely related core cluster in the network was selected.

2.7 KEGG signal pathway analysisThe key targets and core cluster targets were imported into DAVID6.8 database to obtain KEGG signaling pathway (KEGG_PATHWAY) data. The signaling pathways with top fivePvalues were analyzed.

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3 Results

3.1 Active ingredients and predicted targets of Fuxin decoctionAfter searching in the TCMSP database, for Radix Aconiti Carmichaeli, 21 active ingredients into the blood, 6 ingredients with action targets and 30 predicted targets were obtained; for Radix Astragali, 20 active ingredients into the blood, 17 ingredients with action targets and 462 predicted targets were obtained; for Semen Lepidii, 12 active ingredients into the blood, 9 ingredients with action targets and 321 predicted targets were obtained. After deleting duplicates, 224 targets were retained (Fig.1).

3.2 Database search for heart failure related targetsA total of 57, 146, 185, 778 and 22 targets were retrieved from the TTD, DrugBank, OMIM, DisGeNET, and PharmGKB databases, respectively. After deleting duplicates, a total of 1 010 targets known to be related to the occurrence and development of heart failure were obtained.

3.3 Directly acting target screeningThe 224 action targets of Chinese herbs and 1 010 targets related to heart failure were intersected, and overlapping targets were retained. As a result, 94 directly acting targets were obtained (Fig.2).

3.4 PPI network construction and key targets and core cluster screeningThe directly acting targets were processed according to Method 5, and a PPI network composed of 675 nodes and 3 085 kinds of action relations was obtained. In accordance with Method 6, 225 core targets and a core cluster composed of 56 nodes and 297 interactions were obtained. The specific screening process and results are shown in Fig.3.

3.5 KEGG signal pathway analysis

3.5.1Key target signaling pathway analysis. The five signaling pathways with top fivePvalues were tumor signaling pathway, prostate cancer signaling pathway, chemokine signaling pathway, estrogen signaling pathway and TNF signaling pathway (Fig.4A). HSP90, PI3K, AKT, MAPK and other related genes are closely related to this study (Table 1).

Table 1 Related signaling pathways of key targets

3.5.2Core cluster signaling pathway analysis. The five signaling pathways with top fivePvalues were glutamate synapse pathway, Retrograde endocannabinoid signaling pathway, cholinergic synapse pathway, Morphine addiction pathway, and gap junction pathway (Fig.4B). Adenylate cyclase and guanine nucleotide binding protein (G protein) are found to be closely related to this study (Table 2).

Table 2 Related signaling pathways of core cluster

Note: Green rhombus denotes Chinese herb, purple circle denotes active ingredient, and orange square denotes the target of Chinese herb.Fig.1 Traditional Chinese medicine-ingredient-target network diagram

Note: Blue represents the target of traditional Chinese medicine, red represents the target of disease, and overlapping purple represents the directly acting target of Chinese medicine on disease.Fig.2 Screening of directly acting targets

Note: A denotes PPI network constructed, C1 denotes key targets screened, and C2 denotes core cluster screened. A → B1 → C1 is screening path for key targets, and A → B2 → C2 is screening path for core cluster.Fig.3 PPI network construction and key target and core cluster screening

Note: A. related signaling pathways of core targets; B. related signaling pathways of core cluster.Fig.4 KEGG signaling pathway analysis

4 Discussion

Traditional Chinese medicine believes that the main cause of heart failure is the prolonged heart disease or the loss of blood, Yin and Yang. It leads to dysfunction of the viscera and poor circulation of qi, blood and water. Eventually, it will cause dampness and stasis, and dysphoria. Deficiency of Qi, blood stasis, and water stagnation are the main pathogenesis of heart failure[10]. Radix Aconiti Carmichaeli can warm Tongyang, Mingmen and Kanshui, remove stasis, and promote circulation. According toShennong’sClassicofMateriaMedica, Radix Astragali is a famous tonic, with functions of replenishing qi, raising yang, consolidating superficies for arresting sweating, relieving swelling, relieving sores and promoting muscle growth. AsShennong’sClassicofMateriaMedicarecords, Semen Lepidii is mainly used to treat abdominal mass, accumulation of qi and dietary cold and heat, and it can expel evil and clear waterways. The combination of the three herbs (Fuxin decoction) can warm yang, invigorate qi, relieve asthma, promote diuresis, promote blood circulation, promote menstruation, quickly reduce and relieve the symptoms of heart failure. Modern pharmacological studies have confirmed that the three drugs have the effect of strengthening heart and fighting against heart failure[11-13].

This research shows that the core cluster of Fuxin decoction is closely related to adenylate cyclase and guanine nucleotide binding protein (G protein). Therefore, it is speculated that the effect of Fuxin decoction is related to the βARs-G protein-adenylate cyclase signaling pathway. After the combination of catecholamine neurotransmitters such as epinephrine and norepinephrine with βAR in heart tissue, the configuration of βAR changes, dissociating Gα from GDP in the inactive GDP-GαGβγ coupled with it. Gα is subsequently combined with GTP to form Gα-GTP, becoming activated and separating from Gβγ subunit, eventually activating adenylate cyclase (AC) in the cell membrane. AC catalyzes ATP to produce cyclic adenosine monophosphate (cAMP). cAMP is used as the intracellular second messenger and it activates protein kinase A (PKA). PKA can phosphorylate a variety of regulatory proteins, thereby regulating the positive time, force and conduction effect of cardiomyocytes[14]. With the development of heart failure, βAR-mediated signal transduction has undergone subtle changes. The main manifestations are the down-regulation of βAR density and the decoupling (desensitization) of G protein-βAR[15]. This change is speculated to be related to G protein-coupled receptor kinases (GRKs). Among them, GRK2 plays an important role in the process of heart failure[16]. The treatment of inhibiting GRK2 is currently a research hotspot in heart failure. Whether Fuxin decoction acts on GRK2 or directly acts on the signaling pathway to strengthen the heart and resist heart failure needs to be explored with further experiments.

Modern medicine believes that ventricular remodeling is the physiological and pathological basis leading to the continuous progression of chronic heart failure[17]. Ventricular remodeling is due to a series of complex molecular and cellular mechanisms that cause changes in myocardial phenotype, structure and function[18]. Current research shows that there are many factors involved, including cytokine activation, neuroendocrine system activation, changes in cell information transmission pathways, abnormal gene expression and interactions between multiple genes.

This research shows that the core targets of Fuxin decoction are closely related to PI3K, AKT, HSP90 and MAPK. The PI3K-Akt signaling pathway is an important pathway involved in the regulation of cell proliferation. Mediated signal pathways regulate cell division, differentiation, apoptosis and other activities, and they play an important role in promoting cell growth and inhibiting cell apoptosis. The effect on heart failure is as follows: after being stimulated by a certain threshold pressure load or factor, myocardial cells are activated, leading to myocardial cell growth and myocardial hypertrophy[19]. Mitogen-activated protein kinase (MAPK) belongs to serine or threonine protein kinase. It can be activated by certain ligands including receptors, growth factors, G protein coupling and some stressors. p38MAPK, c-Jun N-terminal kinase (JNK) belong to the MAPK system. JNK and p38MAPK have been shown to be key regulators of myocardial cell inflammation and apoptosis[20]. Targeted inhibition of JNK and activation of p38MAPK has effectively delayed the progression of heart failure in transgenic research[21]. The continuous contraction and metabolism of the heart require strict control of the quality of related proteins. Misfolded proteins are toxic to cardiomyocytes. Misfolding is more likely to occur in myocardial infarction, heart failure or aging. Molecular chaperones include heat shock protein 70 (HSP 70) and HSP 90 protect the heart from these threats[22]. This study believes that Fuxin decoction may act on the above signal pathways or molecules, to combat ventricular remodeling, delay the progression of heart failure, and improve the long-term prognosis of patients.

In this study, network pharmacology method was applied to predict the complex molecular network of Fuxin decoction in the treatment of heart failure. The mechanism of Fuxin decoction in improving the symptoms of heart failure in the short term and improving the prognosis of heart failure in the long term is explained. The results are partially consistent with those of previous research, and suggest scientific and predictive nature of network pharmacology, providing a reference for further in-depth discussion of mechanism of action. Some mechanisms need to be further experimentally confirmed.