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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 4  |  Page : 481-489

Network pharmacology-based study of chinese herbal qixiong formula in treating oligoasthenospermia


Department of Andrology, Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing, China

Date of Submission01-Mar-2020
Date of Acceptance19-Oct-2020
Date of Web Publication16-Dec-2020

Correspondence Address:
Prof. Fu Wang
Department of Andrology, Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing 100091
China
Prof. Jun Guo
Department of Andrology, Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing 100091
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_75_20

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  Abstract 


Objective: The objective is to study the network pharmacology of Qixiong formula (QXF) and explore the mechanism of QXF in the treatment of oligoasthenospermia. Materials and Methods: Using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-traditional Chinese medicine), and an encyclopaedia of traditional Chinese medicine (ETCM) databases as well as data from relevant studies, the effective components and targets of QXF were obtained. Genes associated with oligospermia were screened using GeneCards, OMIM, DisGeNet, DrugBank, and GAD databases. The intersection target was obtained by mapping the target to the gene, and the protein interaction network was created using the STRING database to screen the core target of QXF in the treatment of oligospermia. The intersection target was enriched using gene ontology (GO) and the Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis with the DAVID database. The network of the disease drug target pathway was drawn using Cytoscape software. Results: Overall, 536 active components of QXF and 40 core targets for the treatment of oligoasthenozoospermia were obtained. The analysis of GO and KEGG showed that QXF is mainly involved in oxidative stress, cell motility, nutritional response, and other biological processes. Through the regulation of FOXO, p53, PI3K/Akt, MAPK, mammalian target of rapamycin, Foxo, Wnt, and other signaling pathways, QXF played a role in the treatment of oligoasthenospermia. Conclusion: QXF has multi-component, multi-target, and multi-channel characteristics, providing a new way to study the mechanism of QXF in the treatment of oligoasthenospermia.

Keywords: Network pharmacology, oligoasthenospermia, Qixiong formula, PI3K/Akt, mammalian target of rapamycin


How to cite this article:
Zhao F, Liu SJ, Gao QH, Zhang JW, Du GC, Wang F, Guo J. Network pharmacology-based study of chinese herbal qixiong formula in treating oligoasthenospermia. World J Tradit Chin Med 2020;6:481-9

How to cite this URL:
Zhao F, Liu SJ, Gao QH, Zhang JW, Du GC, Wang F, Guo J. Network pharmacology-based study of chinese herbal qixiong formula in treating oligoasthenospermia. World J Tradit Chin Med [serial online] 2020 [cited 2021 Jan 25];6:481-9. Available from: https://www.wjtcm.net/text.asp?2020/6/4/481/303545




  Introduction Top


Male infertility is the inability of a sexually active, noncontracepting couple to achieve spontaneous pregnancy in 1 year. About 10%–15% of couples do not achieve pregnancy within 1 year and seek medical treatment for infertility. Due to the acceleration of industrialization, increasing environmental pollution, increasing living pressure, changes in living habits, and other adverse factors, such as diseases, male fertility has decreased significantly worldwide, and this incidence is increasing.[1],[2] According to the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen (5th ed.n.), oligozoospermia: <15 million spermatozoa/mL; asthenozoospermia: <32% progressive motile spermatozoa; teratozoospermia: <4% normal forms.[3] Oligoasthenospermia is one of the most important causes of male infertility. It not only causes infertility, but also has a negative impact on the physical and mental health, family life, and even social harmony of the patients. In total, 30%–40% of patients with asthenospermia and oligozoospermia experience a decrease in sperm motility or density; however, there are no abnormalities in other tests, and no clear cause can be found. This is termed as idiopathic oligozoospermia.[4] Possible causes mainly include abnormalities in sperm energy metabolism, abnormal signal transmission channels, and abnormal sperm motility.[5],[6]

Network pharmacology is based on the theories of systematic biology, polypharmacology, and network biology. From the perspective of multi-targets, it uses public information databases and high-throughput omics data analysis as tools, combines a drug-target network with a biosystem network, and builds a drug-target-disease-related network to provide new methods and strategies for drug research and development. In recent years, it has gradually been used as an active ingredient in traditional Chinese medicine (TCM), predicting potential therapeutic targets, and analyzing research on the mechanism of therapeutic action.[7] The network pharmacology method has an advantage in studying the molecular mechanism of TCM compound prescriptions. Therefore, network pharmacology is a reliable method for studying the molecular mechanism of Qixiong formula (QXF).[8],[9]

Reinforcing the kidneys and promoting blood circulation have demonstrated a good effect on the treatment of male infertility.[10],[11] QXF is an empirical prescription of the Xiyuan Hospital of China Academy of Chinese Medical Sciences. The composition of the prescription is Astragalus membranaceus (Huang-Qi), Ligusticum wallichii (Chuan-Xiong), Radix Rehmanniae Praeparata (Shu-Di-Huang), Chinese yam (Shan-Yao), Cornus officinalis (Shan-Zhu-Yu), Semen Cuscutae (Tu-Si-ZI), fruit of Chinese wolfberry (Gou-Qi-Zi), Achyranthes bidentata (Niu-Xi), Fructus Amomi (Sha-Ren), Coptis chinensis (Huang-Lian), Epimedium herb (Xian-Ling-Pi) and Caulis (Ji-Xue-Teng). It can invigorate the kidneys and spleen, promote blood circulation, and promote sperm development. QXF can significantly improve forward motile sperm Progressive motility (PR) and PR + nonforward motile sperm (NP) grade spermatozoa.[12] However, the mechanism of its multi-component, multi-target, and multi-pathway treatment of IAS is not comprehensive. This study further clarifies the possible mechanism of QXF and provides the basis for subsequent related studies, with the aim of providing a more theoretical basis for the treatment of oligoasthenospermia.


  Materials and Methods Top


Collection and screening of QXF

TCMSP Version: 2.3 Center for Bioinformatics, Northwest University, Xian, Shaanxi, China[13], (http://tcmspw.com/tcmsp.php) was used to collect the main chemical compositions of A. membranaceus, L. wallichii, R. Rehmanniae Praeparata, Chinese yam, C. officinalis, Epimedium herb, Semen Cuscutae, fruit of Chinese wolfberry, Caulis spatholobi, A. bidentata, Fructus Amomi, and C. chinensis. Combined screening methods commonly used in network pharmacology and related studies include predicted oral bioavailability (OB) ≥30% and Drug-Likeness Evaluation ≥0.18, which were used in this study as screening criteria when OB <30%. However, some unselected chemical constituents with biological activity and pharmacological effects identified by fingerprints and other methods are also included in the candidate active ingredients.[14],[15] The drug potential targets were imported to the UniProt database (https://www.uniprot.org/), and the search condition was set to Organism: Homo sapiens (Human) to obtain the corresponding canonical protein names of the target. BATMAN-TCM (last update: January 2016) is an online bioinformatics analysis tool specially designed for studying the molecular mechanism of TCM, which contains information from databases of TCM and diseases, including TCMID, OMIM, and TTD. It can directly analyze the potential targets of TCM components. The above 12 TCMs were retrieved separately, and the active ingredients and targets with a comprehensive score of more than 25 points were screened. ETCM (Last update: January 2016) is a database that collects a large variety of information, such as that relating to herbs, TCM compounds, TCM chemical compositions, drug targets, and related diseases. Targets with QED scores above 0.8 were selected. The obtained data were entered into an Excel spreadsheet, and a drug-active ingredient-target data table was constructed.

Disease target and target collection

Genecards (Version 5.1.7 https://www.genecards.org/) is a comprehensive biomedical database that contains rich biomedical data on genes and their products.[16] The OMIM (updated in March 2020) database (https://omim.org/) contains the characteristics of human genes and related diseases and focuses on the molecular relationship between gene mutations and dominant expressions.[17] DisGeNet (Version 7.0) is a database that collects information on genes and mutation sites related to human diseases and integrates database information, such as UniProt and Orphanet. The DrugBank (Version 5.1.7) database contains comprehensive molecular information that integrates bioinformatics and chemical informatics resources and provides detailed drug data with drug target information and their mechanisms, including pharmacochemistry, pharmacology, pharmacokinetics, ADME, and interaction information. The Genetic Association Database (date of download: 06/25/2020) contains information regarding disease targets. The terms “asthenospermia,” “oligospermia,” “spermatogenic dysfunction,” “deficiency of sperm motility,” and “low sperm motility” were entered into these databases to query for oligospermia related genes and record them using Excel spreadsheets. Finally, the duplicated items were summarized and eliminated to obtain the target data, and a disease-gene data table was constructed.

Collection of core targets and establishment of protein-protein interaction networks

Mapping the data table of QiXiong Fang-target protein-target gene and the target protein in the disease gene data table, the intersection data between the two were obtained, and these data were the effective targets of QiXiong Fang in the treatment of oligoasthenospermia. These targets were imported into the STRING database to construct protein-protein interaction (PPIs), and their topological features were analyzed using MCODE and entiscape plug-ins of Cytoscape. The median degree, betweenness, closeness, and k-core were used as the screening values to screen the core targets of QXF in the treatment of oligoasthenozoospermia. These targets were imported into the STRING database to construct PPIs, and their topological features were analyzed using MCODE and entiscape plug-ins of Cytoscape.

Construction of the visual network of disease-drug-active ingredient-target

Cytoscape (version 3.7.1) is an open source platform based on network visualization and high-throughput data analysis. Its powerful plug-in function can help multiple functions to realize visualization, such as protein interactions, gene function annotation, and pathway analysis.[18] Based on the results of the collection of active ingredients and targets, Cytoscape software was used to build a disease-medicine-active ingredient-target network for the treatment of oligozoospermia with QXF. In this network, each data point is a node, and the degree of interaction between each node is the degree of nodes. In the Layout tab, the degree sorted circle layout (ALL NODES) was selected to extract the degree for each datum.

Gene ontology and Kyoto encyclopedia of genes and genomes analysis

Gene ontology analysis is an international standard classification system for gene functions. It aims to establish a language and vocabulary standard that is suitable for various species to limit and describe the function of genes and proteins, and can be updated as research continues, annotating gene product functions from high-throughput.[19] KEGG is an integrated database resource for linking sequences to molecular functions from higher levels.[20] It is divided into three parts: Molecular function, biological process, and cellular component. DAVID (version 6.8) is a biological information database that provides systematic comprehensive biological function annotation information. It is mainly used to analyze the function and pathway enrichment of differential genes. The core target genes were imported into the DAVID database, the functional annotation was selected, “Homo sapiens” was selected in species selection, GO and KEGG enrichment analyses were performed, and GOTERM_BP_DIRECT was selected under Gene_Ontology and CC, MP, CF and KEGG_PATHWAY under Pathways. The module parameters were set to; GO analysis: P < 0.01, Pathway analysis: P < 0.05, the signal pathway enriched by KEGG was screened. Finally, Cytoscape software was used to draw a visual network of gene pathways, and the degree of each gene and pathway was extracted. The overall steps of the study are shown in [Figure 1].
Figure 1: Research steps

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  Results Top


Main active ingredients and related targets of Qixiong formula

Through the mining of TCMSP and BATMAN-TCM databases and the collation of relevant studies, after removing unclear options and duplicates, a total of 536 active ingredients and 1765 related targets were obtained: 719 targets for A. membranaceus, 1097 for fruit of Chinese wolfberry, 268 for Cuscutae Semen, 255 for Fructus Amomi, 341 for C. spatholobi, 664 for C. officinalis, 289 for A. bidentata, 309 for C. chinensis, 653 for L. wallichii, 236 for Epimedium Herb, 354 for R. Rehmanniae Praeparata, and 575 for Chinese yam.

Prediction of core treatment targets for oligoasthenospermia

Through GeneCards database mining, 5008 genes were obtained, of which 1801 were “deficiency of sperm motility,” 46 were “asthenospermia,” 188 were “oligospermia,” 353 were “spermatogenic dysfunction,” and 2620 were “low sperm motility.” According to the feedback from the OMIM database, a total of 497 genes were obtained, of which 98 were “spermatogenic dysfunction,” 200 were “deficiency of sperm motility,” and 199 were “low sperm motility.” In the DisGeNET database, 109 genes of “oligospermia” were obtained. In the DrugBank database, 18 genes of “spermatogenic dysfunction” were obtained. In the GAD, two genes of “asthenospermia” and 88 of “oligospermia” were obtained. Removing duplicate values from these data yielded a total of 3222 genes for participation in oligoasthenozoospermia treatment. Mapping these targets with therapeutic targets of Qixiong prescription, a total of 677 genes, in which QXF could be used as a core target for the treatment of oligoasthenospermia, were obtained.

Production of protein-protein interaction

Through PPI analysis and data screening of 677 targets, a protein interaction relationship including 40 nodes and 748 edges was obtained. The screening process and results are shown in [Figure 2]. The target represented by these 40 nodes is the core target of QXF in the treatment of oligozoospermia and asthenospermia.
Figure 2: Protein-protein interaction network of the core target of Qixiong formula

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Visualization of the disease-drug-active ingredient-target network

After treatment with Cytoscape software, QXF was used to treat the disease-medicine-active ingredient-target network of oligoasthenospermia [Figure 3]. For each node, the higher the degree of the node, the higher the degree of enrichment, indicating that the participation in the network is higher, and the effect is greater. According to the degree value, the components with higher node degrees were quercetin, kaempferol, beta-sitosterol, formononetin, stigmasterol, luteolin, wogonin, isorhamnetin, calycosin, and licochalcone A. Nine targets; MAPK1, AKT1, MAPK8, MAPK14, TNF, JUN, EGFR, CCND1, and NOS3, had a node degree of more than 10, which suggests that QXF may play an important role in the treatment of oligoasthenospermia.
Figure 3: Disease-drug-active ingredient-target network

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Function and pathway enrichment analysis of active ingredients of Qixiong formula

With the help of the Metascape database, GO and KEGG analyses were performed on the target genes acting on the active ingredients of QXF. Histograms were developed based on-logP [Figure 4], [Figure 5], [Figure 6]. The biological processes of these genes are mainly enriched in cell migration, cell motility, cellular component movement, and response to growth factors. The main cell components of the target were the vesicle lumen, cytoplasmic vesicle lumen, secretory granule lumen, etc. Molecular functions mainly included phosphatase binding, receptor activity, growth factor receptor binding, kinase binding, etc., [Table 1].
Figure 4: Molecular function results of gene ontology analysis

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Figure 5: Cell composition results of gene ontology analysis

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Figure 6: Biological process results of gene ontology analysis

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Table 1: Gene ontology analysis results of the core target of Qixiong formula

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As shown in [Table 2], the signaling pathways involved included the AGE/RAGE signaling pathway, PI3K-Akt signaling pathway in diabetic complications, TNF signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, FoxO signaling pathway, and Ras signaling pathway. According to related studies, PI3K/Akt, MAPK, mammalian target of rapamycin (mTOR), and other signaling pathways are closely related to oligoasthenospermia. Among them, PI3K/Akt, Foxo, and other signaling pathways have high node degrees, suggesting that these signaling pathways may be the key pathways of QXF in the treatment of oligospermia and asthenospermia [Figure 7].
Table 2: Main signal pathways of the core target of Qixiong formula

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Figure 7: Gene-pathway network diagram

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  Discussion Top


QXF is effective in invigorating the kidneys and spleen, promoting blood circulation and spermatogenesis, and has an obvious effect on enhancing semen viability. A. membranaceus and L. wallichii are the monarch herb of this prescription. The combination of the two drugs promotes blood circulation and invigorates the spleen and kidneys. It also removes blood stasis and invigorates Qi. A. membranaceus is the most common prescription for treating Qi stagnation and blood stasis syndrome. L. wallichii is characterized by its pungent-warm dispersion, good channeling, and blood circulation. R. Rehmanniae Praeparata nourishes kidney water, and nourishing essence and blood as well as dredging blood vessels can supplement the deficiency of the five zang organs. C. officinalis supplements the liver and kidneys, Chinese yam can supplement the spleen, lung, and kidney Qi and yin. The combination of the three drugs has the same function of tonifying the kidneys and nourishing yin. Epimedium herbs strengthen essence and yang, invigorate the kidneys, and strengthen bones, especially for impotence, sequestration of essence, deficiency, and cold infertility. A. bidentata is more effective than tonifying the liver and kidneys and strengthening the waist and knees. The Compendium of Materia Medica states, “The merit of nourishing is like the strength of cattle.” The combination of these six drugs serves as a minister to assist the monarch in tonifying the kidneys, activating blood circulation, and promoting spermatogenesis. With the adjuvant use of Semen Cuscutae and fruit of Chinese wolfberry, there is an endless source of medicines to help the monarch generate essence. C. chinensis is bitter and descending. It contains the essence of nourishing. The effect of Fructus Amomi is to dissipate dampness and promote Qi. It warms and invigorates the spleen. The two drugs are used together with the tonic to prevent it from being greasy and hindering Qi. At the same time, it can invigorate the spleen and stomach. Qi and blood are biochemically active. All drugs are used together to invigorate the kidneys and spleen, activate the blood, and generate essence.[12]

In this study, 536 active ingredients of QXF and 1765 therapeutic targets were distributed in 12 drugs, including A. membranaceus and L. wallichii. Among them, the same component existed in different drugs and acted on different targets, and acted on the same target. This reflects the characteristics of QXF of being multi-component and-target. After treatment, 40 core targets were obtained from 677 targets intersecting with oligoasthenozoospermia targets. This indicates that the treatment with QXF results from a large and complex protein interaction.

The disease-drug-active component-target network showed that all drugs in QXF had a high node degree (≥15), which indicated that the QXF for the treatment of oligoasthenospermia was reasonable, targeted, and effective. Studies have shown that the main active extract from A. membranaceus and Astragalus polysaccharides can effectively reduce the levels of the sperm protein tyrosine phosphatase 1B B, improving the acrosome reaction of sperm.[21] A. membranaceus and L. wallichii both have good oxidation resistance and can remove oxygen free radicals.[22] Relevant pharmacological studies have found that C. officinalis has a great anti-aging effect, protects the liver, and regulates bone metabolism, and its specific component C. officinalis polysaccharides prefrontal cortex has a good antioxidant function,[23] the mechanism of which may be related to the decreased expression of related genes by the polysaccharides from the leaves of dogwood. Modern pharmacological studies have shown that Epimedium herbs can regulate the transcription of target genes and protein expression in the nucleus by regulating the neuroendocrine immune network, thus warming the kidneys and nourishing the essence.[24] Relevant studies have shown that high-dose icariin can promote sperm capacitation and increase the content of TNBP, thus significantly improving the sperm viability and PR value in the rat model of human disease and effectively improving the reproductive function of male rats.[25] C. spatholobi dispels wind and promotes blood circulation, and its main components, flavonoids, contain a variety of pharmacological activities, including antioxidants, which affect the circulation of the blood system, etc.[26] Dodder flavonoids can regulate the apoptosis process of sperm cells, repair the sperm cells S period, and protect living sperm cell proliferation.[27]

The GO analysis results showed that the treatment of QXF on oligoasthenospermia involved biological processes such as cell migration, cell motility, cellular component movement, and response to growth factors, and involves cell components, such as vesicle lumen, cytoplasmic vesicle lumen, and secretory granule lumen, in a complex regulatory process. KEGG analysis showed that the target of QXF in treating oligoasthenospermia involves PI3K/Akt, MAPK, mTOR, AGE/RAGE, FoxO, and Ras. Among these, PI3K/Akt and mTOR have been used in recent years to study the mechanisms underlying oligoasthenospermia.[28],[29] In addition, these pathways involve genes such as MAPK1, AKT1, MAPK8, and MAPK14, which are consistent with the above visual processing results of Cytoscape, which proves the reliability of network pharmacological research methods. This also provides a new basis for the continued study of the mechanism of QXF in treating oligoasthenospermia.

Spermatogonial proliferation and spermatogenesis can be regulated by the regulation of p70S6K and 4EBP1 expression in the testis tissue by PI3K/mTOR signaling molecules. By activating the PI3K/AKT/mTOR signaling pathway, Kit protein synthesis during spermatogonia differentiation can be increased,[30] and spermatogenesis may be regulated by feedback from Kit proteins and PI3K/AKT/mTOR signaling pathways. Spermatogenesis includes the proliferation and differentiation of spermatogonial stem cells, and meiosis of spermatocytes, and spermatogenesis. Related studies have found that the mTOR pathway is involved in regulating the formation of the blood-testis barrier, and during spermatogenesis, it likely regulates the meiosis of spermatids by regulating the proliferation and differentiation of spermatogonia stem cells. The level of DNA methylation[31] affects spermatogenesis and maturation. The PI3K/Akt signaling pathway regulates DNA methylation by regulating the level of DNA methyltransferase phosphorylation,[32] thereby affecting the process of spermatogenesis. The PI3K/Akt, mTOR, and other signaling pathways are important signaling pathways involved in spermatogenesis. Although research on these pathways is still in its infancy, based on the results of this network pharmacological study, it can be presumed that QXF can treat oligoasthenospermia by regulating PI3K/Akt, mTOR, and other pathways. APK1, AKT1, MAPK8, MAPK14, TNF, JUN, EGFR, CCND1, NOS3, and other targets have higher node degrees. These genes may play a key role in the treatment of oligoasthenospermia through the PI3K/Akt and mTOR pathways. The study of these potential targets and pathways will provide a new reference for the treatment of oligoasthenospermia by QXF at the molecular level.


  Conclusion Top


To sum up, the network pharmacology of QXF showed that 536 active ingredients act on different targets and involve multiple biological processes and signaling pathways, reflecting QXF being “multi-component, multi-target, multi-path.” These results are consistent with the research direction of the treatment of oligoasthenospermia in recent years,[28] and a systematic analysis of the role and mechanism of QXF in the treatment of oligoasthenospermia at the molecular level is suggested. The PI3K/Akt and mTOR signaling pathways may be important pathways that affect spermatogenesis and development, laying a foundation for further exploration of the mechanism of QXF in treating oligoasthenospermia, and also providing new ideas for related research of other TCM compounds. However, the results of this part of the study require further investigation in in vitro cells, animal experiments, and other means to directly verify and explain the molecular mechanism of QXF in treating oligoasthenospermia.

Financial support and sponsorship

This work was supported by the Youth Program of the National Natural Science Foundation of China (81703929); the Fundamental Research Funds for Central Public Welfare research institutes (ZZ070855, ZZ11-062); and Beijing Traditional Chinese Medicine Science and Technology Development Fund Project (JJ-2020-76).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

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