Investigation of the mechanism of the reduction of anthracycline-induced cardiotoxicity by Qishen Huanwu Capsule based on network pharmacology

Introduction

Cardiotoxicity is one of the most common side effects of anthracycline chemotherapeutics and can manifest as heart failure, coronary artery disease, heart valve disease, arrhythmia and other types of cardiovascular complications. Among these complications, the incidence rate for doxorubicin-induced left ventricular dysfunction is as high as 48% (1). Cardiotoxicity reduces cancer patient compliance with antitumor treatments and increases the mortality of cancer survivors. It has attracted increasingly more attention by medical experts. Preventing or reducing anthracycline-induced cardiotoxicity (ACT) is a hotspot in cardio-oncology research.

Qishen Huanwu Capsule (Ji Yao Zhi Zi Z20050798, batch number 030310) is an in-hospital formulation prepared by our hospital. It is derived from Buyang Huanwu Decoction and consists of Radix astragali (Huangqi), Radix Pseudostellariae (Taizishen), Semen Persicae (Taoren), Flos Carthami (Honghua), Radix Angelicae Sinensis (Danggui), Rhizoma Chuanxiong (Chuanxiong), Radix Paeoniae Rubra (Chishao), Radix Achyranthes Bidentatae (Niuxi), Rhizoma Pinelliae (Banxia) and other Chinese medicines. It can nourish Qi and Yin and remove blood stasis and phlegm. It is mainly used to treat stroke and coronary heart disease with Qi deficiency and blood stasis. The quercetin (2) and kaempferol (3) in Qishen Huanwu Capsule reduce the cardiotoxicity of anthracyclines. Based on network pharmacology, this study explored the main active ingredients, targets and pathways by which Qishen Huanwu Capsule reduces ACT, aiming to provide ideas and theoretical bases for the investigation of the specific mechanism of action by which Qishen Huanwu Capsule in reduces ACT.

We present the following article in accordance with the MDAR checklist (available at http://dx.doi.org/10.21037/apm-20-2204).

Methods

Medical ethics

This study involved bioinformatics analysis only and thus did not require medical ethics approval. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Screening targets of the main components of Qishen Huanwu Capsule

Based on the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php) and Chemistry Database (http://www.organchem.csdb.cn/scdb/default.asp), we searched for all the active ingredients contained in Qishen Huanwu Capsule. Based on the pharmacokinetic parameters of the ingredients, oral bioavailability (OB), drug-likeness (DL), Caco-2 cell permeability (Caco-2), and half-life (HL) were used as the parameters to screen the main active ingredients. The corresponding targets of the main active ingredients obtained by screening were searched in the TCMSP database and the Swiss Target Prediction database (http://swisstargetprediction.ch), and the UniProt database (http://www.uniprot.org/) was used to uniformly convert the target sites into gene names to construct a target library of the main active ingredients of Qishen Huanwu Capsule.

Collection of ACT-related targets

Using “anthracycline-induced cardiotoxicity (ACT)” as the query term, relevant targets were searched in the PharmGkb database (https://www.pharmgkb.org/), OMIM database (https://www.omim.org/), GeneCards database (https://www.genecards.org/), GAD database (https://geneticassociationdb.nih.gov/), and TTD database (http://db.idrblab.net/ttd/). After compilation, the target sites were used to construct an ACT-related target database.

Venn analysis of Qishen Huanwu Capsule targets and the ACT target set

Using the Venny 2.1 online analysis tool (https://bioinfogp.cnb.csic.es/tools/venny/Index.html), we merged the main active ingredient target library of Qishen Huanwu Capsule and the ACT target data set to obtain a common gene set and explored the target sites through which Qishen Huanwu Capsule acts to treat ACT.

Construction and analysis of a protein-protein interaction (PPI) network

The common targets of Qishen Huanwu Capsule and ACT obtained by Venn analysis were input into the STRING (https://string-db.org/) platform to construct a common target PPI network; the organism was set to “Homo sapiens”, and the minimum required interaction score was set to “high confidence (0.7)”. We used R language software to determine the occurrence frequency and draw a histogram. All R packages can be downloaded through R (https://www.r-project.org/) and Bioconductor (https://www.bioconductor.org/).

Gene Ontology (GO) function enrichment of key target genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

We used the ClueGO plug-in of Cytoscape 3.2.1 software to perform GO biological process enrichment analysis on the common targets of Qishen Huanwu Capsule and ACT obtained by Venn analysis; the analysis results were presented as a pie chart. R language software was used to perform KEGG pathway enrichment analysis on the common targets, and the analysis results were presented in the form of bubble diagrams. Cytoscape 3.2.1 software was used to construct a component-target-pathway network to analyze the mechanism of action by which Qishen Huanwu Capsule reduces ACT.

Results

Screening of the main active ingredients of Qishen Huanwu Capsule

Qishen Huanwu Capsule comprises Chinese medicines, such as Huangqi, Taizishen, Taoren, Honghua, Danggui, Chuanxiong, Chishao, Niuxi and Banxia. Using the TCMSP database and Chemistry Database and based on OB ≥30%, DL ≥0.18, Caco-2 ≥−0.04, and HL ≥4 as pharmacokinetic parameters (4), 10 active ingredients of Huangqi, 3 active ingredients of Taizishen, 7 active ingredients of Taoren, 10 active ingredients of Honghua, 2 active ingredients of Danggui, 4 active ingredients of Chuanxiong, 4 active ingredients of Chishao, 9 active ingredients of Niuxi, and 4 active ingredients of Banxia were identified. Duplicated ingredients were removed, resulting in a total of 35 main active ingredients, e.g., quercetin, luteolin, kaempferol, hederagenin and beta-sitosterol (Table 1).

Table 1 Qishen Huanwu Capsule: main active ingredients and their pharmacokinetic parameters
Full table

Target prediction

Using the TCMSP database and the Swiss Target Prediction database, we searched the targets of the active ingredients contained in Qishen Huanwu Capsule and retrieved 1,848 targets. After duplicate targets were removed, we obtained a total of 230 targets.

For ACT targets, we used “anthracycline-induced cardiotoxicity” as the search term and retrieved 146 ACT-related targets: 122 in the GeneCards database, 18 in the PharmGkb database, 3 in the OMIM database, and 3 in the GAD database.

We input 230 Qishen Huanwu Capsule targets and 146 ACT targets into Venny 2.1 software to generate a Venn diagram. A total of 36 targets common to both Qishen Huanwu Capsule and ACT were obtained from the intersection of the 2 sets, as shown in Figure 1 and Table 2. These 36 targets could be the potential targets of Qishen Huanwu Capsule, leading to a reduction in ACT.

Figure 1 The Venn diagram of Qishen Huanwu Capsule targets and ACT-related targets. ACT, anthracycline-induced cardiotoxicity.

Table 2 Targets common to both Qishen Huanwu Capsule and ACT
Full table

Diagram showing the network relationship between the active ingredients of Qishen Huanwu Capsule and ACT-related targets

We input the active ingredients of Qishen Huanwu Capsule and the potential targets for ACT treatment into Cytoscape 3.2.1 software to establish a network diagram with close connections between nodes, as shown in Figure 2. The network has 71 nodes and 210 edges. The blue circle represents the main active ingredients of Qishen Huanwu Capsule, and the green circle represents the potential targets of Qishen Huanwu Capsule for the treatment of ACT. The degree of a node in the network refers to the number of edges associated with the node; a higher degree indicates that the compound is associated with more targets. In this network, the average degree of the active ingredients of Qishen Huanwu Capsule is 5.27, and the top 5 compounds in terms of degree are quercetin (degree =30), luteolin (degree =15), kaempferol (degree =14), β-carotene (degree =12), and baicalein (degree =11), suggesting that these compounds are the key active ingredients in Qishen Huanwu Capsule for the treatment of ACT.

Figure 2 The network diagram for Qishen Huanwu Capsule targets and ACT-related targets. ACT, anthracycline-induced cardiotoxicity.

Analysis of the target PPI network

The 36 potential targets obtained from the above screening were imported into the STRING database to obtain a PPI network, and the PPI network was further analyzed using the cytoHubba plug-in in Cytoscape 3.2.1 software. The results are shown in Figure 3. R was used to determine the top 30 targets in regard to frequency, resulting in the bar graph shown in Figure 4. The network in Figure 3 contains 36 nodes and 350 edges, with an average node degree of 19.4; the transition from yellow to red indicates the gradual increase in node degree. The targets with higher protein interaction frequencies are Akt1, MAPK1, and MAPK8, suggesting that these targets may be the key targets of Qishen Huanwu Capsule in the treatment of ACT.

Figure 3 Common target protein interaction network.

Figure 4 Frequencies of common target proteins.

GO biological process analysis and KEGG pathway enrichment analysis

We used the ClueGo plug-in to perform GO biological process analysis of 36 common targets, resulting in the pie chart shown in Figure 5. As seen in the figure, the common targets are mainly concentrated in 133 biological processes, including negative regulation of the apoptotic signaling pathway, regulation of reactive oxygen species metabolic processes, and regulation of DNA binding, suggesting that Qishen Huanwu Capsule can reduce the cardiotoxicity of anthracycline chemotherapeutics through mechanisms such as regulating apoptosis, oxidative stress, and DNA damage repair. R was used to perform KEGG pathway enrichment analysis on the common targets. Common targets were mainly enriched in 27 signaling pathways, such as the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and the hypoxia-inducible factor-1 (HIF-1) signaling pathway. The top 20 pathways are shown in Table 3 and Figure 6. The main active ingredients of Qishen Huanwu Capsule, the common targets of Qishen Huanwu Capsule and ACT, and KEGG pathway analysis were used to construct a component-target-pathway network diagram (Figure 7). As shown in Figure 8, a total of 14 genes (HSP90AA1, AKT1, VEGFA, BCL2, CASP9, MYC, EGFR, MAPK1, MDM2, ERBB2, MCL1, EGF, NOS3, and FASLG) were enriched in the PI3K/Akt signaling pathway, suggesting that Qishen Huanwu Capsule can reduce ACT through multiple targets, multiple pathways, and multiple mechanisms.

Figure 5 Biological process pie chart (ClueGo functional analysis).

Table 3 Target pathway enrichment results (top 20)
Full table

Figure 6 KEGG pathway enrichment (top 20).

Figure 7 Component-target-pathway network.

Figure 8 PI3K/Akt signaling pathway.

Discussion

Anthracycline chemotherapeutics, represented by doxorubicin, play an important role in the treatment of various cancers. Unfortunately, side effects such as cardiotoxicity have severely restricted the clinical application of these drugs. The exact mechanism of ACT is still unclear. Current research shows that the molecular mechanism involves multiple aspects such as oxidative stress, apoptosis, autophagy, and type II topoisomerase-related DNA damage (5). Traditional Chinese medicine believes that chemotherapy drugs are heat toxins. Heat toxins are stored in the body and thereby decoct body fluid, consume Qi and injure Yin, resulting in Qi and Yin deficiency. Qi deficiency leads to a lack of strength, resulting in blood stasis, and heat toxins can produce phlegm from body fluid. Therefore, frequent blood and phlegm stasis, Qi and Yin deficiency, and the mutual accumulation of phlegm and blood stasis are the main symptoms of ACT. Qishen Huanwu Capsule is derived from Buyang Huanwu Decoction, in which Huangqi, as the primary active compound, nourishes vitality (Qi vitality facilitates blood circulation); Taizishen, as an important secondary compound, nourishes Qi, promotes body fluid and blood movement, helps Huangqi replenish Qi, and nourishes Yin; Danggui nourishes the blood and promotes blood circulation; Taoren, Honghua, Chishao, Chuanxiong, and Niuxi promote blood circulation and remove blood stasis; and Banxia loosens phlegm and reduces thirst. The whole prescription nourishes Qi and Yin and removes blood stasis and phlegm. Its mechanism and formulation target the main symptoms of ACT.

The PPI network shows that the main active ingredients of Qishen Huanwu Capsule may act through targets it has in common with ACT, such as Akt1, MAPK1, and MAPK8. Akt is a serine/threonine protein kinase and an important downstream target kinase in the PI3K signal transduction pathway. Akt1 is a Akt subtype highly expressed in myocardial tissues, and it has important effects on the growth/hypertrophy, survival/apoptosis, and metabolism of cardiomyocytes. Akt downregulates downstream forkhead box class O 3a (FoxO3a) and inhibits oxidative stress through the Akt/FoxO3a pathway (6); it acts through the Akt/glycogen synthase kinase-3β pathway, upregulates the expression of B-cell lymphoma 2 (Bcl-2), downregulates the expression of caspase-3, and exerts an anti-apoptotic effect (7). Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine protein kinases that can sense extracellular stimuli and trigger a wide range of intracellular responses. Activated MAPKs phosphorylate a variety of target proteins, including transcription factors, such as c-Jun, c-Myc, and activating transcription factor 2, and apoptosis-related proteins, such as Bcl-2 and Bcl-2-associated death promoter, and thus regulate proliferation, differentiation, apoptosis, oxidative stress, inflammation and other cellular activities (8). Doxorubicin, an anthracycline, can activate the MAPK signaling pathway, promote the phosphorylation of apoptotic proteins, induce oxidative stress, and cause myocardial damage (9). It can also degrade inhibitor κBα protein through the MAPK/nuclear factor kappa B signaling pathway, leading to the release of inflammatory mediators and subsequent cardiomyocyte apoptosis (10). Regulating the PI3K/Akt and MAPK signal pathways through Akt1, MAPK1, MAPK8 and other targets is a potential mechanism of action of Qishen Huanwu Capsule in treating ACT.

Oxidative stress is caused by the unbalanced response of reactive oxygen species and endogenous antioxidants to injury, and it is one of the main mechanisms of cardiotoxicity caused by anthracycline chemotherapeutics. Quercetin, kaempferol, isorhamnetin and other ingredients in Qishen Huanwu Capsule can reduce ACT by inhibiting oxidative stress. Quercetin upregulates the expression of 14-3-3γ, increases the level of superoxide dismutase (SOD) and glutathione peroxidase in cardiomyocytes, reduces the level of malondialdehyde (MDA) and reactive oxygen species, inhibits oxidative stress, improves mitochondrial function, and reduces doxorubicin-induced damage to cardiomyocytes (2). Kaempferol binds to the promoter region of the pro-apoptotic gene Bcl-2-associated X protein (Bax), inhibits the p53 signaling pathway and the extracellular signal-regulated kinase-dependent MAPK pathway activated by doxorubicin, and reduces the oxidative stress, apoptosis and injury, and mitochondrial dysfunction caused by doxorubicin (3). Isorhamnetin increases SOD, catalase and glutathione peroxidase activity through the MAPK signaling pathway, reduces MDA activity, and reduces the oxidative stress caused by doxorubicin (11). The PI3K/Akt, MAPK, P53 and other signaling pathways were all enriched with the abovementioned active ingredients, as determined by KEGG analysis.

Anthracyclines can cause myocardial damage by inducing cardiomyocyte apoptosis. The expression and regulation of the Bcl-2 family are key factors of apoptosis. Bcl-2 is an apoptosis inhibitor protein, and Bax is a pro-apoptotic protein. These proteins regulate apoptosis through the PI3K/Akt pathway and P53 pathway (12). In Qishen Huanwu Capsule, quercetin, kaempferol, baicalein, β-carotene, β-sitosterol, wogonin, and acacetin act on the Bcl-2 family. Baicalein antagonizes cardiotoxicity caused by doxorubicin by upregulating the expression of Bcl-2, downregulating the expression of Bax, reducing the ratio of Bax/Bcl-2, and inhibiting cardiomyocyte apoptosis (13). Quercetin and β-sitosterol directly or indirectly regulate CASP9 and CASP3 in the CASP cascade reaction, which may be an important mechanism by which Qishen Huanwu Capsule regulates apoptosis (14). Luteolin downregulates the expression of p-phosphatase and tensin homolog deleted on chromosome ten (PTEN), p-Akt, p-extracellular signal-regulated kinase, p-mammalian target of rapamycin (mTOR), and p-glycogen synthase kinase-3β in H9C2 rat cardiomyocytes and reduces H9C2 cell apoptosis induced by doxorubicin through the PTEN/Akt and extracellular signal-regulated kinase pathways (15).

Autophagy plays an important role in cardiotoxicity induced by anthracyclines (16). Autophagy is a type of protein degradation pathway that depends on lysosomes. It mainly acts to degrade damaged organelles and misfolded proteins in cells and is widely involved in a variety of physiological and pathological processes (17). The basal level of autophagy is of great significance for maintaining homeostasis, whereas overactivated autophagy causes cell death due to the excessive degradation of intracellular components (18). The PI3K/Akt pathway activates the downstream effector molecule mTOR to inhibit autophagy, whereas FoxO3, a member of the FoxO family, activates autophagy by upregulating Atg or autophagy-regulatory genes. HIF-1α upregulates the expression of BCL2-interacting protein 3 and Beclin-1 to transform microtubule-associated protein light chain 3 (LC3)-I into LC3-II, thus inducing autophagy (19). In this study, based on KEGG analysis, the PI3K/Akt, FoxO and HIF-1 pathways that regulate autophagy were all enriched. In addition, the main active ingredient of Qishen Huanwu Capsule, baicalein, upregulates the expression of membrane-associated RING-CH 5 in cardiomyocytes through the KLF4-MARCH5-Drp1 pathway, inhibits mitochondrial division caused by H₂O₂ and ischemia-reperfusion, enhances mitochondrial autophagy and reduces cardiomyocyte apoptosis caused by anthracyclines (20). These results suggest that Qishen Huanwu Capsule can reduce ACT by regulating autophagy.

This study systematically predicted, through network pharmacology, the potential mechanism by which Qishen Huanwu Capsule reduces ACT. We analyzed and constructed the “Active Ingredients of Qishen Huanwu Capsule-ACT-related targets” network diagram for Qishen Huanwu Capsule-mediated reduction in ACT and identified 35 main active ingredients of Qishen Huanwu Capsule and 36 targets common to both Qishen Huanwu Capsule and ACT. These results reflect the possible mechanism by which Qishen Huanwu Capsule reduces ACT and provide ideas and theoretical bases for further verification of its pharmacological mechanism.

Acknowledgments

Funding: Traditional Chinese Medicine Scientific Research Project of Hebei Provincial Administration of Traditional Chinese Medicine (Project Number: 2020502).

Footnote

Reporting Checklist: The authors have completed the MDAR checklist. Available at http://dx.doi.org/10.21037/apm-20-2204

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/apm-20-2204). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are 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. This study involved bioinformatics analysis only and thus did not require medical ethics approval. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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