Uncovering the mechanism of quercetin for treating spermatogenesis impairment by a network pharmacology approach

ABSTRACT

Spermatogenesis process plays a major role in male fertility. Quercetin, a flavonoid, is found in daily food or traditional Chinese medicine, which has a significant effect on treating inflammatory diseases. Several studies have shown that quercetin can improve male spermatogenesis. However, the intrinsic mechanism has not been systematically investigated. The network pharmacology approaches were utilized to investigate the mechanisms of quercetin against spermatogenesis impairment (SI). 78 potential therapeutic targets of quercetin against SI were identified. The enrichment analysis showed that the mechanism of quercetin on SI is closely related to its anti-inflammatory pharmacological effects. It is indicated that quercetin could be used for treating SI through inflammation-related targets or pathways including NF-κB, MAPK, JAK-STAT and Toll-like receptor signaling pathways.

KEYWORDS:

• Quercetin
• spermatogenesis impairment
• network pharmacology
• inflammation

Introduction

Infertility is defined as the inability of becoming pregnant after 12 months of unprotected sexual intercourse. It burdens people with physical and psychological stress. About 15% of couples have difficulty in getting pregnant. It is estimated that the malefactor account for 25–30% in infertility, and 30% are caused by the mixed factor (Agarwal et al. 2015; Chu et al. 2019). Therefore, it is essential to ensure that all the factors are properly investigated to rule out possible reversible causes of infertility and the chances of natural fertility will be increased. The most common cause of male infertility is spermatogenesis impairment (SI), which is clinically characterized by oligozoospermia or azoospermia. A large number of clinical studies have reported that SI is closely related to inflammation of the male genital system (La Vignera et al. 2013; Lotti and Maggi 2013). The presence of inflammatory microenvironment can bring about oxidative damage to sperm cells, which results in sperm motility, morphology and fertility disorder (Fraczek et al. 2013).

Complex diseases may result from multiple genes. Therefore they cannot be conquered through a single target (Liu and Sun 2012). Compared with the method of biological experiments, the advantage of virtual experiment is efficient and rapid. Network pharmacology analysis could reveal the drug effect by constructing and analyzing various biological networks (Zhang et al. 2019).

Wuziyanzong Pill is a well-known traditional Chinese medicine (TCM) prescription for male infertility (He et al. 2016). In a previous study, we found that quercetin may play a significant role in the effect of Wuziyanzong Pill through network pharmacology research (Liu et al. 2020). It is perceived that quercetin may have multiple effects against male infertility. Quercetin, a polyhydroxy flavonoid, is widely found in flowers, fruits, and seeds of plants (Akbari Kordkheyli et al. 2019; Andres et al. 2018; Xu et al. 2019). Researches have shown that quercetin has effects of anti-inflammatory, anti-oxidation, anti-tumor effects, as well as significant therapeutic effect on male genital system diseases (Hamza et al. 2015; Lou et al. 2015; Uygur et al. 2014). Therefore, this article will preliminarily explore the mechanism of quercetin in SI through network pharmacological analysis (Figure 1).

Figure 1. Schematic workflow in this study. This report aimed to identify the potential targets through integrating GO biological process, KEGG pathway analyses and network construction.

Materials and methods

Identification of targets of quercetin

The keyword ‘quercetin’ was searched in the DrugBank database (www.drugbank.ca) to collect 3D structure and SMILES number. The potential targets of quercetin were obtained from Binding-DB database (bindingdb.org/bind/index.jsp), Tradition Chinese Medicine Systems Pharmacology Database (TCMSP; tcmspw.com/tcmsp.php), ChEMBL database (www.ebi.ac.uk/chembl/), and STITCH database (stitch.embl.de) using the keyword ‘quercetin’ and its SMILES number. The acquired target genes were then confirmed in UniProt database (www.uniprot.org). The pharmacokinetics properties including oral absorption, distribution, metabolism, excretion (ADME) were retrieved from the TCMSP database.

Collection of SI-related genes

Targets associated with SI were collected by OMIM database (www.omim.org) and GeneCards database (www.genecards.org). The term used in database search is ‘Male Infertility’ and ‘Spermatogenesis’. The SI genes were identified as the intersection of male infertility-related genes and spermatogenesis-related genes.

The intersection genes of targets of quercetin and SI-related genes were identified as potential therapeutic targets of quercetin against SI.

Enrichment analysis

The Metascape (metascape.org) is an online database for annotating gene and enrichment analysis.(Zhou et al. 2019) The targets of quercetin against SI were respectively uploaded to Metascape database for the Gene Ontology (GO) biological processes enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation.

Network construction

STRING database (string-db.org) can be employed to explore the interrelationship between genes (Szklarczyk et al. 2011). VarElect database (varelect.genecards.org) is used to analyze the correlation between the target gene and a specific phenotype or disease (Stelzer et al. 2016). To get the inner association of targets, the potential quercetin targets and targets of quercetin against SI were submitted to STRING database after selectin ‘Homo sapiens’ for organism option. VarElect database was employed to explore the relationship between the targets of quercetin against SI and the phenotype ‘spermatogenesis’. The protein–protein interaction (PPI) network and the target–pathway network were constructed by Cytoscape (www.cytoscape.org) (Chandran and Patwardhan 2017; Huang et al. 2017). The CytoHubba, a plug from Cytoscape, was used to predict the core nodes and subnetworks in the network (Chin et al. 2014).

Results

ADME parameters of quercetin

The parameters of quercetin were explored in TCMSP database. Oral bioavailability (OB) is the foremost parameter to evaluate pharmacokinetic properties. Drug-like (DL) is a parameter to assess the medicinal property of a compound. Generally, OB ≥ 30% and DL ≥ 0.18 have been reported as the standard for druggability screening (Pei et al. 2016; Shen et al. 2017; Zhang et al. 2016). As shown in Table 1, OB and DL of quercetin were assessed by TCMSP database, which means quercetin has good oral absorption and drug-like properties.

Table 1. Pharmacological properties of quercetin.

Name	MW	AlogP	Hdon	Hacc	OB (%)	DL	RBN
quercetin	302.25	1.5	5	7	46.43	0.28	1

MW, molecular weight; Alog P, lipid/water partition coefficient; Hdon, number of hydrogen-bond donors; Hacc, number of hydrogen-bond acceptors; OB, oral bioavailability; DL, drug likeness; RBN: number of rotatable bonds.

Lipinski’s rule of five stipulates that the main parameters of a compound should be met by the relative the molecular weight (MW) < 500, the lipid/water partition coefficient (Alog P) < 5, the number of hydrogen-bond donors (Hdon) < 5, the number of hydrogen-bond acceptors (Hacc) < 10 and the number of rotatable bonds (RBN) ≤ 10 (Liu et al. 2016). As listed in Table 1, the parameters reveal quercetin may be an attractive candidate for drug development.

Predicting quercetin-associated targets and spermatogenesis impairment associated targets

262 pharmacological targets of quercetin (Table S2) and 1191 disease conditioning genes (Table S3) of SI were collected. The intersection of the two parts is considered as potential 78 targets of quercetin against SI (Table S1).

The PPI network of potential targets of quercetin against SI

STRING online tool was employed to construct the PPI network. In the theory of topology, the degree centrality index is a key indicator to measure the importance of a node in the network (Chin et al. 2014).

Previous studies have demonstrated that natural flavonoids not only act through a single target but also through multiple targets and pathways(Li et al. 2017). We constructed PPI network of the potential targets of quercetin against SI by using the data from STRING database and Cytoscape software (Figure 2a). The network of the potential targets contained 78 nodes and 1076 edges. The Cytohubba plug was employed to analyze the top 10 hub genes in this network. They are TP53, AKT1, VEGFA, ESR1, MYC, TNF, EGFR, EGF, CCND1, MAPK1 (Figure 2b).

Figure 2. (a) PPI network of the potential therapeutic targets of quercetin against SI. The area of nodes indicates the degree of the target in the network. The warmer of the target color, the higher of the degree; (b) PPI network of the therapeutic hub targets of quercetin against SI. The more reddish of the target, the higher of the degree. The network networks were constructed by Cytoscape 3.7.1.

GO biological process analysis of the therapeutic targets

Visualization data from Metascape online database demonstrated that the biological progress of therapeutic targets of quercetin against SI was associated with multi-process (Figure 3). The top five processes according to the significance of P value are ‘apoptotic signaling pathway’, ‘response to inorganic substance’, ‘developmental process involved in reproduction’, ‘cytokine-mediated signaling pathway’ and ‘reactive oxygen species metabolic process’. Meanwhile, these genes also related to ‘positive regulation of cell migration’, ‘regulation of cellular protein localization’ and ‘regulation of growth’. These results reflect the broad pharmacological effects of quercetin, which is consistent with previous research. In addition, the enrichment results showed that quercetin may intervene in ‘cytokine-mediated signaling pathway’, ‘cellular response to organonitrogen compound’, ‘response to lipopolysaccharide’, ‘response to oxygen levels’, ‘regulation of mitotic cell cycle’ and ‘intrinsic apoptotic signaling pathway’. The terms of ‘cytokine mediated signaling pathway’ and ‘response to lipopolysaccharide’ are often closely associated with the inflammatory response. In the process of cell proliferation and apoptosis, biological processes such as ‘regulation of mitotic cell cycle’ and ‘intrinsic apoptotic signaling pathway’ are often involved. On the other hand, the terms ‘response to oxygen levels’ and ‘cellular response to organonitrogen compound’ are commonly observed during intracellular redox reactions and oxidative stress. The results provided evidence that the effects of quercetin in the treatment of reproductive disorders may be related to regulate the inflammatory response.

Figure 3. GO biological process analysis of the therapeutic targets of quercetin against SI. The y-axis shows significantly enriched biological processes, the x-axis shows the p-value of categories.

KEGG pathway analysis of the therapeutic targets of quercetin against SI

Previous study showed that the effects of quercetin are related to inflammatory response (Li et al. 2016). Inflammation is a protective response to eliminate harmful stimuli. It leads to the restoration of normal tissue structure and function. In order to elucidate the pharmacological mechanisms of quercetin for treating SI in inflammatory response pathways, we analysed the KEGG pathway by online tool Metascape (p < 0.05). The results of inflammatory-related pathways were presented in Figure 4(A). To reveal the correlation between quercetin anti-inflammatory targets and signaling pathways, we constructed quercetin targets associated classical inflammatory pathways network by Cytoscape software (Figure 4b).

Figure 4. (a) Annotation of classical inflammation KEGG pathway of the targets of quercetin against SI. The y-axis shows significantly enriched pathways categories of the therapeutic target, the x-axis shows the p-value. (b) Therapeutic target-inflammation-related pathways network. The arrows are targets, squares are inflammation-related KEGG pathways. The orange arrows are hub therapeutic targets. The network networks were constructed by Cytoscape 3.7.1.

Discussion

Compared with the biological experiment methods, the advantages of network pharmacology are low cost, high efficiency, and speed. The results of network pharmacology and systems biology could reveal the complex mechanism of drugs by constructing an interactive network between targets and diseases. Enrichment analysis also makes demonstrates pharmacologic mechanism more clearly and understandably (Boezio et al. 2017; Hong et al. 2017; Ning et al. 2017; Pang et al. 2018; Xu et al. 2021).

Male infertility still be a worldwide difficulty which causes physiological and psychological challenges for patients. SI is one of the most important factors of fertility function disorders. The common pathogenic factors also include neurological disease, infection, trauma, iatrogenic injury, anatomical or genetic abnormalities, gonadal hormones, and sperm antibody development (Kliesch 2017). The drugs for treating male infertility includge dopamine receptor agonists, gonadotropin-releasing hormone, and human chorionic gonadotropin (Katz et al. 2017). Previous research have also shown that genitourinary inflammation is closely related to azoospermia and oligozoospermia in male patients (Agarwal et al. 2018; Calogero et al. 2017; Fraczek et al. 2013). The inflammatory microenvironment can lead to oxidative damage in sperm cells and result in impaired sperm motility, morphology and fertility (Rusz et al. 2012; Silva et al. 2018).

TCM could be used for treating male infertility through improving sperm quality and adjusting immune function (Jiang et al. 2017). In TCM theory, male reproductive disorders are mainly coursed by kidney deficiency, qi deficiency, blood deficiency, blood stasis, and damp heat. The clinical treatment includes tonifying kidney-Qi, nourishing blood, removing phlegm and clearing away heat and dampness (Zhou et al. 2019). Studies showed that quercetin can be found in many traditional Chinese medicines for clearing away heat and tonifying kidney-Qi including plantaginis semen, Schisandra chinensi. These herbs have been reported to alleviate the impairment of spermatogenesis for treating male infertility(Karna et al. 2019; Qin et al. 2000).

Quercitrin is a kind of flavonoid found in various vegetables, fruits, and herbs. Normally, quercitrin is converted into quercetin by enzyme in the body (Li et al. 2018). Research showed that the pharmacological effect of quercetin including anti-inflammatory, anti-oxidation, anti-tumor, and immune regulation. (Akbari Kordkheyli et al. 2019; Uygur et al. 2014; Zhang et al. 2011). And quercetin can intervene spermatogenesis disorder based on these pharmacological effects (Li et al. 2010).

In this study, we collected targets of quercetin and targets associated with SI from online databases. The effect of the drug on disease is not only related to a single target but also affected by multiple biological progresses and signal pathways. Based on this theory, we annotated GO progress and KEGG pathway analysis by Metascape online tool and constructed PPI network by Cytoscape software. The results showed that the therapeutic targets of quercetin against SI may mainly focus on the inflammatory process, oxidative stress, cell proliferation, apoptosis, the cell migration and extracellular stimulatory response, which consist with recent studies (Mao et al. 2018; Yelumalai et al. 2019; Yoshimoto et al. 2017).

During the inflammatory response, transcription factors such as nuclear factor-κb (NF-κB) and mitogen-activated protein kinase (MAPK) in leukocytes are activated, and a series of inflammatory mediators such as IL-1, TNF-α and vasoactive amines are produced (Kuprash and Nedospasov 2016). These cytokines will initiate inflammatory cascades and participate in various damage processes. Studies also showed that these cytokines and related pathways have a great impact on spermatogenic capacity (Haervig et al. 2018; Lotti and Maggi 2013; Seshadri et al. 2011; Xie et al. 2019). The JAK-STAT pathway is widely involved in apoptosis, inflammation, and immune system regulation (Isomaki et al. 2015). Research showed that the JAK-STAT pathway could induce testicular germ cell proliferation, promote testosterone production, and affect spermatogenesis process (Loza-Coll et al. 2019; Smendziuk et al. 2015; Yi et al. 2017). JAK-STAT pathway also promotes somatic cyst stem cell renewal in the adult Drosophila testis niche (Ma et al. 2014). Studies also showed that the time of Drosophila embryonic gonad shaping and the activation of JAK-STAT signaling pathway simultaneously occur (Herrera and Bach 2019). These studies demonstrate that the JAK-STAT signaling pathway has great significance in germ cell differentiation. Research showed that male reproductive inflammation also actives Toll-like receptor pathway to affect sperm parameter (Chehrei et al. 2017; Zhan et al. 2013). Inflammatory responses mediated by Toll-like receptor are entirely dependent on JNK MAPK pathway and promote chemotactic factors secretion by activating NF-κB pathway to induce inflammation (Wang et al. 2011). The damaged spermatogenic cells could induce inflammatory genes expression through activating the Toll-like receptor pathway (Zhang et al. 2013). In a nutshell, all the classical inflammation-related signaling pathways influence the processes of spermatogenesis from various aspects.

NF-κB, MAPK, JAK-STAT, Toll-like receptor signaling pathways have synergistic or antagonistic interactions to complete signaling transduction process together (Dhawan and Richmond 2002; Lim and Cao 2001; Shen and Stavnezer 1998; Wang et al. 2011; Wazea et al. 2018). We found that quercetin may affect spermatogenesis impairment by modulating these four signaling pathways in our study. TP53, AKT1, MYC, TNF, EGFR, EGF and MAPK1 may be hub targets of quercetin against SI. As shown in the above results, these targets are involved in NF-κB, MAPK, JAK-STAT, Toll-like receptor signaling pathways. The present study also reveals a high probability of quercetin for treating SI by four classical inflammation-related signaling pathways. But at the same time, the present research also has some limitations. In this study, the virtual research methods such as network pharmacology analysis are employed to explore the mechanism of quercetin, but further biological experiments are also needed to confirm and explore the above mechanism. We will further explore the mechanism of the effect of quercetin against SI through experimental verification in the future research. In conclusion, quercetin acts on the SI process through multi targets and multi anti-inflammatory pathways. The NF-κB, MAPK, JAK-STAT, Toll-like receptor signaling pathways may be the most important pathways.

Conflict of interest

The authors declare that they have no conflict of interest.

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by National Natural Science Foundation of China: [Grant Number 81973584].