Network Pharmacology and Molecular Docking Analysis of the Mechanism Underlying Yikunyin’s Therapeutic Effect on Menopausal Syndrome

Tan, Xin; Du, Yan-Ping; Luo, Qian; Zhan, Xue-Bing; Kuang, Yun-Shu; Liang, Xiao; Zhang, Yun; Wang, Lin; Chen, Bing; Wen, Ming

doi:https://doi.org/10.1155/2022/7302419

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 7302419 | https://doi.org/10.1155/2022/7302419

Network Pharmacology and Molecular Docking Analysis of the Mechanism Underlying Yikunyin’s Therapeutic Effect on Menopausal Syndrome

Xin Tan,¹Yan-Ping Du,¹Qian Luo,¹Xue-Bing Zhan,¹Yun-Shu Kuang,¹Xiao Liang,¹Yun Zhang,²Lin Wang,²Bing Chen,¹and Ming Wen²

Academic Editor: Talha Bin Emran

Received16 Feb 2022

Revised07 Apr 2022

Accepted08 Apr 2022

Published06 Jun 2022

Abstract

Objective. Yikunyin is an empirical prescription that exhibits good efficacy in the clinical treatment of menopausal syndrome; however, its underlying mechanism remains unclear. This study investigates the mechanism implicated in the therapeutic effect of Yikunyin by identifying its hub genes, central pathways, and key active ingredients. Method. The active ingredients and targets of Yikunyin were obtained from the Traditional Chinese Medicine Systems Pharmacology database, whereas the targets related to menopausal syndrome were obtained from GeneCards, PharmGKB, Therapeutic Target Database (TTD), and Comparative Toxicogenomics Database (CTD). To reveal the pharmacological mechanism, the component-target and the intersecting protein-protein interaction (PPI) networks were constructed, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Finally, molecular docking was carried out to assess the strength of binding between the key active ingredients and key targets. Results. A total of 418 targets and 121 active ingredients were identified in Yikunyin. The intersection of Yikunyin’s 418 targets with the 2822 targets related to menopausal syndrome shows that there are 247 common targets that can be considered potential targets of Yikunyin in the treatment of menopausal syndrome. The topology analysis of the constructed PPI network conducted using the Cytoscape software shows that there are 15 hub genes implicated in the therapeutic effect of Yikunyin: AKT1, PRKCA, TLR9, CXCL10, PRKCD, PARP1, ABCB1, TP53, CAV1, MAPK8, PPARA, GRB2, EGFR, IL-6, and JAK2. Moreover, the key active components acting on these genes are paeoniflorin, luteolin, quercetin, beta-sitosterol, and kaempferol. GO and KEGG analyses indicate that Yikunyin can treat menopausal syndrome by regulating cellular response to chemical stress (GO:0062197), cellular response to oxidative stress (GO:0034599), phosphatase binding (GO:0019902), cytokine receptor binding (GO:0005126), PI3K-Akt signaling (hsa04151), lipid and atherosclerosis (hsa05417), and hepatitis B (hsa05161). Finally, the results of molecular docking suggest that the key active ingredients and key targets can bind well, with binding energies of less than −5 kJ/mol. Conclusion. The research conducted herein reveals that Yikunyin treats menopausal syndrome by targeting AKT1 and IL-6 and by regulating the PI3K-Akt signaling pathway. Moreover, it provides a new idea for understanding the therapeutic effects of traditional Chinese medicines.

1. Introduction

Menopause is a physiological stage characterized by the permanent cessation of a woman’s menstrual period due to the loss of ovarian follicle function. According to a global project comprising 36 international studies conducted in 24 countries across 6 continents, the mean age of natural menopause onset ranges between 46 and 52 years, with an overall average of 48.8 years [1]. During the menopausal transition, women experience a variety of physical, psychological, and social symptoms, such as depression, insomnia, osteoarthritis, and atherosclerosis. These symptoms occur frequently and are usually associated with a worsening quality of life, both at home and at work [2–4]. As one of the most important therapies in the field of complementary and alternative medicine, traditional Chinese medicine (TCM) makes use of naturally occurring, plant-based ingredients that are not industrially processed. Consequently, it has become increasingly popular in the treatment of diseases, including menopausal and perimenopausal syndrome, in China and around the world [5]. In Western countries, hormone therapy is most commonly used to treat menopausal symptoms, with an incidence of 19.5%. However, this therapy can cause many adverse reactions, such as increased risk of breast cancer, dyslipidemia, and osteoporosis, and thus, it must be used with great caution [6]. Considering that the side effects of TCM are few and that its efficiency is high, this natural medicine constitutes a feasible candidate for the treatment of menopausal syndrome.

Yikunyin is a Gui Shen Pill-based formula composed of nine herbs, namely Gou Qi Zi (Lycii Fructus), Fu Ling (Poria), Bai Shao (Paeoniae Radix Alba), Xian Ling Pi (Epimedii Folium), Huang Qi (Astragali Radix), Shu Di Huang (Rehmanniae Radix Praeparata), Gou Teng (Ramulus Uncariae Cum Uncis), Muli (oysters), and He Huan Pi (Albizia Peel). These herbs are rich in flavonoids, which are known for their anti-inflammatory, antioxidant, anticancer, and antidiabetic effects [7, 8]. Clinical studies have shown that Yikunyin can significantly alleviate the symptoms of menopausal syndrome in patients, with a total effective rate of 81.25% after treatment [9]. However, the underlying mechanism remains unclear. Therefore, to promote the clinical application of Yikunyin, its therapeutic mechanism must be elucidated.

Unlike Western medicines, TCM prescriptions, such as Yikunyin, comprise a variety of herbs. Considering their complex composition, the effects of these prescriptions cannot be readily studied using simple methods. In 2008, Hopkins proposed network pharmacology as an effective method that can be used to study the molecular mechanisms of complex systems (e. g., natural herbs and traditional Chinese medicines) by establishing the relationships between multiple compounds and targets [10]. In this study, we explore the effect of Yikunyin in treating menopausal syndrome, and we systematically evaluate its therapeutic targets and mechanisms using network pharmacology and molecular docking.

2. Materials and Methods

2.1. Screening for the Active Chemical Constituents in Yikunyin

The chemical composition of Yikunyin was obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://tcmspw.com/tcmsp.php) [11], the Shanghai Institute of Organic Chemistry of Chinese Academy of Science, the Chemistry (CASC) database (https://202.127.145.134/scdb/), and a high-throughput experiment- and reference-guided database of traditional Chinese medicine (HERB) database (http://herb.ac.cn/). The oral bioavailability (OB) and drug-likeness (DL) of each component were obtained from the TCMSP database. Since Yikunyin is administered orally, OB, a measure of the drug’s ability to enter systemic circulation, is an important indicator [12]. Drug-likeness is also important, as it helps reduce the cost of drug discovery. Of the numerous tangible small molecules in herbs, only those that meet the drug-likeness criterion are considered for further research [13]. In this study, the criteria of OB ≥ 30% and DL ≥ 0.18 were used to screen the active ingredients in Yikunyin [14].

The Swiss Absorption, Distribution, Metabolism, and Elimination (ADME) system (https://www.swissadme.ch/) provides pharmacokinetic and drug-likeness data of compounds not included in the TCMSP database. In this research, Swiss ADME was used to identify the active compounds in Yikunyin based on GI absorption and drug-likeness. In particular, the compounds with “High” GI absorption and more than three “Yes” drug-likeness criteria were considered to be active.

2.2. Determining the Targets of Yikunyin in the Treatment of Menopausal Syndrome

The TCMSP database and SwissTargetPrediction (https://www.swisstargetprediction.ch/) were used to predict the relevant targets of Yikunyin. The compound SDF files required for SwissTargetPrediction were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The UniPort database (https://www.uniprot.org/) was used to convert protein targets into genetic targets, species restricted to humans, and reviewed targets.

As for the targets of menopausal syndrome, they were collected from four databases, namely GeneCards (https://www.genecards.org/), PharmGKB (https://www.pharmgkb.org/), the Therapeutic Target Database(TTD) (https://db.idrblab.net/ttd/), and Comparative Toxicogenomics Database(CTD) (https://ctdbase.org/), using the keyword “menopausal syndrome.” The identified menopausal syndrome targets were intersected with the drug targets using the online website https://bioinformatics.psb.ugent.be/webtools/Venn/. The resulting Venn diagram was visually processed, and the intersection targets were considered to be the targets of Yikunyin in the treatment of menopausal syndrome.

2.3. Construction and Analysis of the Network

2.3.1. PPI Network

To construct the intersecting protein-protein interaction (PPI) network, the identified targets were entered into the STRING database (https://string-db.org). The organism criterion was set to “Homo sapiens,” and the minimum required interaction score was fixed at 0.7. For visualization and topology analysis, the resulting TSV files were downloaded from the STRING database and uploaded into Cytoscape 3.7.2. The important targets in the network were identified based on their degree value (large degree) [15].

2.3.2. Identification of Hub Genes

CytoHubba is a Cytoscape plug-in that uses 12 algorithms (betweenness, bottleneck, closeness, clustering coefficient, degree, DMNC, eccentricity, EPC, MCC, MNC, radiality, and stress) to characterize the nodes in the PPI network and to identify the central elements based on their importance [16]. After constructing the PPI network of common targets, the 12 algorithms in CytoHubba were used for topology analysis. The top-ranking targets under each algorithm were selected as hub genes, and the active compounds corresponding to these genes were identified as the key active compounds in Yikunyin [17].

2.3.3. GO and KEGG Pathway Enrichment Analyses

Gene Ontology (GO) analysis was used to identify the genes implicated in biological process (BP), cell composition (CC), and molecular function (MF) related to menopausal syndrome, whereas the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify the corresponding pathways. The analyses were carried out using the clusterProfiler R package. values below 0.01 were considered statistically significant [18].

2.3.4. Verification by Molecular Docking

To predict whether the key active compounds can bind well to the key protein targets, molecular docking analysis was carried out using AutoDockTools 1.5.6. The molecular structures of the key active compounds were taken from PubChem (https://pubchem.ncbi.nlm.nih.gov/), whereas the structures of proteins were obtained from the Protein Data Bank (PDB, https://www.rcsb.org/). The PDB files of proteins and drugs were uploaded into AutoDockTools 1.5.6 for pretreatment (e.g., deleting water and hydrogenation), and then, molecular docking was conducted to determine the binding energy. The obtained PDBQT files were converted to PDB files and imported into PyMOL 2.2.0 for visual processing.

3. Results

3.1. Screening for the Active Chemical Constituents in Yikunyin

Based on ADME screening and searches conducted in TCMSP, CASC, and HERB databases, the nine herbs in Yikunyin comprise 121 active components (common active ingredients in two or more Chinese medicines are counted only once), eight of which are in Bai Shao, six in Fu Ling, 32 in Gou Teng, 35 in Gou Qi Zi, 16 in Huang Qi, two in Shu Di, 21 in Xian Ling Pi, 10 in He Huang Pi, and seven in Muli. These components are listed in Supplementary Table 1.

3.2. Determining the Targets of Yikunyin in the Treatment of Menopausal Syndrome

In total, 418 targets corresponding to the 121 active ingredients in Yikunyin were identified using the TCMSP database and SwissTargetPrediction. Based on searches conducted in the GeneCards, CTD, PharmGKB, and TTD databases, a total of 2,822 targets are associated with menopausal syndrome. Of these targets, 247 are also associated with Yikunyin, as shown in the Venn diagram presented in Figure 1.

3.3. Common Target-Component Network Construction

To better understand the relationship between TCM components and targets, a common target-component network was constructed, as shown in Figure 2. The circular and triangular nodes in this network represent the active ingredients of Yikunyin and the common targets, respectively, whereas the lines signify relationships between nodes. Different colors are used for ingredients belonging to different herbs. The molecular IDs corresponding to signs in circular nodes are given in Supplementary Table 2. In total, the network comprises 368 nodes, including 247 nodes of common targets and 121 nodes of active ingredients. Using the CytoHubba plug-in in Cytoscape, the degree value of each node in the network was analyzed and ranked. The top five active ingredients were found to be quercetin (degree = 420), kaempferol (degree = 160), beta-sitosterol (degree = 51), luteolin (degree = 44), and norarmepavine (degree = 43).

3.4. In-Depth Analysis of Common Targets

3.4.1. PPI Network of Common Targets and Identification of Hub Genes

The PPI network was constructed by inputting all 247 common targets into the STRING database, and it was visualized using Cytoscape (Figure 3(a)). Topology analysis of the constructed network was carried out using the CytoHubba plug-in, and 12 subnetworks were obtained using 12 algorithms (betweenness, bottleneck, closeness, clustering coefficient, degree, density of maximum neighborhood component (DMNC), eccentricity, edge percolated component (EPC), maximal clique centrality (MCC), maximal clique centrality (MNC), radiality, and stress). Each subnetwork contains the top ten targets under the corresponding algorithm, as shown in Figure 3(b) (deeper red color signifies higher score). The ranking of targets under different algorithms is provided in Supplementary File 3. The top-ranking targets in the 12 subnetworks (in eccentricity, 10 genes rank first, so we take these 10 genes into consideration, that is why there are 15 top-ranking genes in 12 subnetworks) were considered hub genes that play an important role in Yikunyin treatment of menopausal syndrome. These genes are AKT1, PRKCA, TLR9, CXCL10, PRKCD, PARP1, ABCB1, TP53, CAV1, MAPK8, PPARA, GRB2, EGFR, IL-6, and JAK2 (Figure 4(a)). Figure 4(b) shows the two functional modules obtained by analyzing the hub genes using the MCODE plug-in. Clearly, AKT1 and IL-6 are the seeds of the two functional modules, which suggest that these two targets may play a pivotal role in Yikunyin’s therapeutic effect.

(a)

(b)

3.4.2. Screening for Key Active Compounds and Construction of the Hub Gene-Component Network

The key compounds corresponding to the 15 hub genes identified herein were determined by a reverse search, and they are paeoniflorin, luteolin, octadecatrienoic acid, quercetin, beta-sitosterol, kaempferol, acacic acid lactone, julibroside j24, julibrotriterpenoidal lactone A, norarmepavine, S-(2-carboxyethyl)-L-cysteine, and machaerinic acid lactone. Figure 5 presents the hub gene-component network consisting of 27 nodes (15 hub genes and 12 key compounds).

3.4.3. GO and KEGG Pathway Enrichment Analyses of Hub Genes

Since the 15 hub genes play important roles in the treatment of menopausal syndrome by Yikunyin, it is necessary to identify the pathways in which these genes are enriched. Based on GO and KEGG analyses, the hub genes are implicated in 644 GOs (624 BPs and 20 MFs) and 91 KEGGs. Figure 6 shows the top 10 most statistically significant BPs and MFs, as well as the top 20 most statistically significant KEGG pathways (sorted based on value). The obtained results indicate that the mechanism of menopausal syndrome treatment by Yikunyin is mainly related to cellular response to chemical stress (GO:0062197), cellular response to oxidative stress (GO:0034599), phosphatase binding (GO:0019902), cytokine receptor binding (GO:0005126), PI3K-Akt signaling (hsa04151), lipid and atherosclerosis (hsa05417), and hepatitis B (hsa05161).

(a)

(b)

3.4.4. Molecular Docking Validation

The strength of binding between hub genes and key active compounds was assessed by molecular docking. With binding energies less than −5 kJ/mol, the proteins in the hub genes are well connected to the key compounds. The 3D representation and binding energies of AKT1 and IL-6 docking are shown in Figure 7.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

Figure 7

“Molecular docking 3D diagram. Small drug molecules are represented by sky blue sticks, hydrogen bonds by yellow dotted lines, and amino acid residues bound to small drug molecules by green sticks. (a) Paeoniflorin-AKT1 (binding energy = −8.35 kcal·mol-1). (b) Luteolin-AKT1 (binding energy = −8.61 kcal·mol-1). (c) Beta-sitosterol-AKT1 (binding energy = −10.09 kcal·mol-1). (d) Kaempferol-AKT1 (binding energy = −7.38 kcal·mol-1). (e) Quercetin-AKT1 (binding energy = −7.24 kcal·mol-1). (f) Beta-sitosterol-IL-6 (binding energy = −8.9 kcal·mol-1). (g) Kaempferol-IL-6 (binding energy = −8.17 kcal·mol-1). (h) Luteolin-IL-6 (binding energy = −7.34 kcal·mol-1). (i) Paeoniflorin-IL-6 (binding energy = −14.76 kcal·mol-1). (j) Quercetin-IL-6 (binding energy = −8.0 kcal·mol-1)”.

4. Discussion

Gou Qi Zi (Lycii Fructus), Fu Ling (Poria), Bai Shao (Paeoniae Radix Alba), Xian Ling Pi (Epimedii Folium), Huang Qi (Astragali Radix), Shu Di Huang (Rehmanniae Radix Praeparata), Gou Teng (Ramulus Uncariae cum Uncis), Muli (oysters), and He Huan Pi (Albizia Peel) are the nine TCM components of Yikunyin. According to Jae Hyun Kim et al., the water extract of Gou Qi Zi can inhibit RANKL-induced osteoclast differentiation and improve menopause osteoporosis [19]. Similarly, the combination of Rubus coreanus and Huang Qi can improve osteoporosis in ovariectomized mice [20]. Gou Teng alleviates menopause-induced depression in mice by activating the 5-HT1A receptor [21], and flavonoid-rich He Huan Pi suppresses oxidative stress in postmenopausal women [8, 22]. Finally, the Muli extract prevents bone loss due to ovariectomy [23]. Despite the established effects of different herbs, the mechanism of Yikunyin in treating menopausal syndrome remains unclear. In this study, the hub genes, key active compounds, and pathways implicated in the therapeutic activity of Yikunyin against menopausal syndrome are identified for the first time, using network pharmacology. The underlying mechanism of Yikunyin’s therapeutic activity is also revealed.

The key active ingredients identified herein are paeoniflorin, luteolin, octadecatrienoic acid, beta-sitosterol, kaempferol, quercetin, acacic acid lactone, julibroside J24, julibrotriterpenoidal lactone A, norarmepavine, S-(2-carboxyethyl)-L-cysteine, and machaerinic acid lactone. Among these ingredients, paeoniflorin, luteolin, kaempferol, beta-sitosterol, and quercetin are noteworthy, as they can provide a reference for the future development of new drugs. Based on previous studies, luteolin inhibits insulin resistance, a condition caused by diseases (diabetes and metabolic syndrome) associated with the onset of menopause, by promoting the PI3K-Akt signaling pathway [24, 25]. Meanwhile, kaempferol reduces inflammation and lipid peroxidation, and thus, it may have a therapeutic effect on menopausal atherosclerosis [26, 27]. According to Yue-Hua Jiang et al., beta-sitosterol plays an important role in regulating anti-LDL and anti-atherosclerosis processes via PI3K-Akt signaling [28]. As regards paeoniflorin, it prevents atherosclerosis by inhibiting oxidized LDL [29] and alleviates atherosclerotic inflammation [30]. It also protects nerves by promoting Akt phosphorylation and exerting antidepression effects [31], and thus, it may have a positive therapeutic impact on menopausal depression. Finally, quercetin inhibits inflammation and slows atherosclerosis by regulating the PI3K-Akt signaling pathway [32]. To the best of our knowledge, no other active ingredients in Yikunyin have been reported in the literature. In conclusion, the effect of Yikunyin in treating menopausal syndrome is related to paeoniflorin, luteolin, kaempferol, beta-sitosterol, and quercetin key active ingredients.

The 15 hub genes identified herein are as follows: AKT1, PRKCA, TLR9, CXCL10, PRKCD, PARP1, ABCB1, TP53, CAV1, MAPK8, PPARA, GRB2, EGFR, IL-6, and JAK2. MCODE analysis shows that AKT1 and IL-6 played the most important roles. IL-6 was used to develop tocilizumab, a drug that clinically treats arthritis and alleviates the symptoms of COVID-19 by targeting interleukin-6 receptors [33]. According to Reeta Kangas and colleagues, the amount of AKT1 in the subcutaneous fat of postmenopausal women is lower than that in premenopausal women [34]. Considering that AKT1-deficient adipocytes are less sensitive to insulin [35], menopause may thus interfere with lipid storage, leading to lipid metabolism disorders. Studies conducted on beta-sitosterol show that it improves lipid metabolism in mice [36] and that it may antagonize oxidized LDL by targeting the AKT1 implicating PI3K-Akt signaling pathway [28]. In a study [37] involving 281 middle-aged (45–60 years old) healthy women, Huang et al. showed that poor sleep efficiency is associated with elevated levels of IL-6 and that menopausal animal models exhibit increased inflammatory response and elevated levels of IL-6 [38]. Paeoniflorin reduces IL-6 levels [39], and thus, it can improve sleep efficiency in menopausal women. Among the key active ingredients, quercetin and luteolin suppress CXCL10 expression at protein and mRNA levels [40], thereby inhibiting osteoclast differentiation and protecting against ovariectomy-induced bone loss [41], which is of great significance in the treatment of menopausal osteoporosis. The remaining core targets identified in our research have not yet been developed into drugs, and more research is needed to elucidate their therapeutic effects.

To further assess the complex relationships existing between different component, targets, and pathways implicated in the treatment of menopausal syndrome by Yikunyin, GO and KEGG analyses of the 15 key targets were conducted. The results of GO analysis show that the 15 targets are mainly enriched in phosphatase binding, cytokine receptor binding, cellular response to oxidative stress, and cellular response to chemical stress, with the former three biological processes being the most important. After entering menopause, the level of alkaline phosphatase in women increases significantly, and it keeps on increasing with time. This indicates that the bone turnover level, a marker of menopausal osteoporosis, also increases [42]. As mentioned above, Huang et al. report that menopausal animal models exhibit increased inflammatory responses and elevated levels of IL-6 [37, 38]. In general, high levels of IL-6 cytokine are associated with increased inflammatory reaction, which in turn is closely related to the occurrence and development of cancer [43]. Therefore, the regulation of IL-6 plays a key role in suppressing chronic inflammation in menopausal women, which ultimately reduces the incidence of cancer and slows down its progression. We speculate that the key active ingredients in Yikunyin (such as paeoniflorin) may inhibit inflammation in menopausal women by regulating cytokine (IL-6) receptor binding. In addition to inflammation, menopause is associated with lower estrogen levels. Therefore, menopause diminishes or eliminates the protective effects of estrogen against drug- or poison-induced damage. For example, estrogen protects the heart against the toxic effects of anthracyclines by regulating oxidative stress [44], an indicator that is also closely related to the development of cancer [45], and the advent of menopause inhibits this protective effect. Based on the available data, it may be hypothesized that Yikunyin can treat menopausal syndrome by regulating phosphatase binding, cytokine receptor binding, and oxidative stress responses.

The KEGG analysis results demonstrate that most of the 15 hub genes are involved in PI3K-Akt signaling, lipid and atherosclerosis, and hepatitis B, and the former seems to be the most critical pathway implicated in the therapeutic effect of Yikunyin on menopausal syndrome. Unlike Lee et al. [46], we believe that Yikunyin can alleviate the symptoms of menopause by upgrading the phosphorylation level of Akt. As mentioned above, a pronounced increase in alkaline phosphatase is observed in women after menopause [42]. Considering that phosphatase is a major antagonist of P13 K [47], the high levels of this enzyme in menopausal women may lead to excessive inhibition of the PI3K-Akt signaling pathway, resulting in a series of menopausal symptoms. A study conducted by Meng et al. suggests that the upregulation of PI3K-Akt signaling can reduce cholesterol accumulation, inhibit inflammatory signaling, and alleviate postmenopausal dyslipidemia [48]. Osteoarthritis, a disease occurring in women over the age of 50, often in menopause [4], may also be prevented upon the activation of the PI3K-Akt signaling pathway, which plays an important role in the development of postmenopausal osteoarthritis [49]. Based on previous reports, menopause is an additional risk factor for atherosclerosis [3]. Therefore, the prevention and improvement of atherosclerosis have a positive impact on the treatment of menopausal syndrome. Moreover, Lee et al. demonstrated that the phosphorylation of eNOS may be increased by regulating the PI3K-Akt signaling pathway, which ultimately results in the treatment of cardiovascular disease caused by vascular endothelial cell dysfunction [50]. The PI3K-Akt signaling pathway is also involved in the alleviation of depression, a condition that is more prevalent among postmenopausal women than among premenopausal ones [51, 52]. Luteolin and quercetin, two key active flavonoid compounds in Yikunyin, have antidepressant activity [53, 54], and thus, the natural Yikunyin prescription can potentially improve depression, one of the symptoms of menopausal syndrome. Previous studies that demonstrate the effect of menopause in increasing inflammation and IL-6 levels [38]suggest that the alleviation of excessive inflammatory response in menopausal women may have a positive effect on the treatment of menopausal atherosclerosis, a disease instigated by chronic inflammation [27]. They also highlight the importance of regulating cytokine levels, such as IL-6 levels. With structures similar to curcumin (phenolic O–H, unsaturated carbonyl, and C–H groups), flavonoids such as quercetin and luteolin are expected to have the same biological activity as curcumin, including antioxidant and anti-inflammatory activities [55]. Indeed, the anti-inflammation effect of these two compounds has been established in previous studies [56, 57]. Interestingly, the 15 hub genes identified herein are also significantly enriched in the hepatitis B pathway. Based on the available literature [58], the antiviral effect of hepatitis B in premenopausal women is better than that in postmenopausal women (), as manifested by the recovery of liver fibrosis. Moreover, curcumin, a compound whose structure is similar to those of quercetin and luteolin, has a hepatoprotective effect [55], and quercetin and kaempferol have been shown to inhibit the activity of the hepatitis B virus, which may interfere with hepatitis B virus proteins [59]. Therefore, we speculate that the key compounds in Yikunyin may have anti-hepatitis effects or can promote the antiviral treatment of hepatitis B, to eliminate the difference between the effects of this treatment before and after menopause. In summary, Yikunyin alleviates the symptoms of menopause mainly by regulating the PI3K-Akt signaling pathway, lipid and atherosclerosis, and hepatitis B.

5. Conclusion

This study highlights the importance of Yikunyin in improving menopausal syndrome by upregulating the PI3K-Akt signaling pathway, improving inflammatory response, promoting antioxidant, and adjusting lipid metabolism to prevent atherosclerosis. The most important active ingredients in Yikunyin are paeoniflorin, luteolin, kaempferol, beta-sitosterol, and quercetin. In addition, our research provides some new targets for the future development of new drugs.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure

The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing of the manuscript.

Conflicts of Interest

The authors declare that they have no known conflicts of interests or personal relationships that could have appeared to influence the work reported in this study.

Authors’ Contributions

XT and YP-D conceived the research. QL, XB-Z, YS-K, XL, YZ, and LW designed the methodology. MW and BC reviewed and edited the manuscript. MW and BC supervised the study. Xin Tan and Yan-Ping-Du contributed equally to this work.

Acknowledgments

The authors thank LetPub (https//www.letpub.com) for its linguistic assistance during the preparation of this manuscript. This research was funded by the Scientific and Technological Achievements Transformation plan of WUHU (grant no. 2019cg18), the Scientific Research Project of Teaching Hospital of Wannan Medical College (grant no. XJ2021001101), and the Scientific Research Project of Anhui Provincial Health Commission (grant no. AHWJ2021b059).

Supplementary Materials

Supplementary File 1: 121 potential active ingredients in Yikunyin screened by ADME. Supplementary File 2: molecular IDs corresponding to signs in circles. Supplementary File 3: ranking of 15 hub genes by 12 CytoHubba algorithms. (Supplementary Materials)

References

S. R. El Khoudary, “Age at menopause onset and risk of cardiovascular disease around the world,” Maturitas, vol. 141, pp. 33–38, 2020.
View at: Publisher Site | Google Scholar
G. Nie, H. Yang, J. Liu, C. Zhao, and X. Wang, “Psychometric properties of the Chinese version of the Menopause-Specific Quality-of-Life questionnaire,” Menopause, vol. 24, no. 5, pp. 546–554, 2017.
View at: Publisher Site | Google Scholar
M. Gerhard, P. Baum, and K. E. Raby, “Peripheral arterial-vascular disease in women: prevalence, prognosis, and treatment,” Cardiology, vol. 86, no. 4, pp. 349–355, 1995.
View at: Publisher Site | Google Scholar
F. E. Watt, “Hand osteoarthritis, menopause and menopausal hormone therapy,” Maturitas, vol. 83, pp. 13–18, 2016.
View at: Publisher Site | Google Scholar
Y. Wang, X.-T. Lou, Y.-H. Shi, Q. Tong, and G.-Q. Zheng, “Erxian decoction A Chinese herbal formula, for menopausal syndrome: an updated systematic review,” Journal of Ethnopharmacology, vol. 234, pp. 8–20, 2019.
View at: Publisher Site | Google Scholar
P. Pacello, L. F. Baccaro, A. O. Pedro, and L. Costa-Paiva, “Prevalence of hormone therapy factors associated with its use, and knowledge about menopause: A population-based household survey,” Menopause, vol. 25, no. 6, pp. 683–690, 2018.
View at: Publisher Site | Google Scholar
M. S. Bari, L. Khandokar, E. Haque et al., “Ethnomedicinal uses, phytochemistry and biological activities of plants of the genus Gynura,” Journal of Ethnopharmacology, vol. 271, Article ID 113834, 2021.
View at: Publisher Site | Google Scholar
D. Ahmed, V. Kumar, M. Sharma, and A. Verma, “Target guided isolation in-vitro antidiabetic, antioxidant activity and molecular docking studies of some flavonoids from Albizzia Lebbeck Benth. bark,” BMC Complementary and Alternative Medicine, vol. 14, no. 1, p. 155, 2014.
View at: Publisher Site | Google Scholar
C. X. Wang Jun, “Clinical observation of 64 cases about the treatment of Yikunyin for the patients with postmenopausal syndrome,” Practical Geriatrics, vol. 25, 2011, (in Chinese).
View at: Google Scholar
H.-X. Yan, C.-F. Xu, H. Yang et al., “Network pharmacology-based analysis on the curative effect of kunxian capsules against rheumatoid arthritis,” Evidence-based Complementary and Alternative Medicine, vol. 2021, pp. 1–11, 2021.
View at: Publisher Site | Google Scholar
J. Ru, P. Li, J. Wang et al., “TCMSP: a database of systems pharmacology for drug discovery from herbal medicines,” Journal of Cheminformatics, vol. 6, no. 1, p. 13, 2014.
View at: Publisher Site | Google Scholar
S. Tian, Y. Li, J. Wang, J. Zhang, and T. Hou, “ADME evaluation in drug discovery 9 Prediction of oral bioavailability in humans based on molecular properties and structural fingerprints,” Molecular Pharmaceutics, vol. 8, no. 3, pp. 841–851, 2011.
View at: Publisher Site | Google Scholar
S. Tian, J. Wang, Y. Li, D. Li, L. Xu, and T. Hou, “The application of in silico drug-likeness predictions in pharmaceutical research,” Advanced Drug Delivery Reviews, vol. 86, pp. 2–10, 2015.
View at: Publisher Site | Google Scholar
S. He, T. Wang, C. Shi, Z. Wang, and X. Fu, “Network pharmacology-based approach to understand the effect and mechanism of Danshen against anemia,” Journal of Ethnopharmacology, vol. 282, Article ID 114615, 2022.
View at: Publisher Site | Google Scholar
Y. Cao, Y Chen, P. Wang, J. Lu, X. Han, and J. She, “Network pharmacology and experimental validation to explore the molecular mechanisms of Bushen Huoxue for the treatment of premature ovarian insufficiency,” Bioengineered, vol. 12, no. 2, pp. 10345–10362, 2021.
View at: Publisher Site | Google Scholar
C.-H. Chin, S. H. Chen, H. H. Wu, C. W. Ho, M. T. Ko, and C. Y. Lin, “CytoHubba: identifying hub objects and sub-networks from complex interactome,” BMC Systems Biology, vol. 8, no. S4, p. S11, 2014.
View at: Publisher Site | Google Scholar
T. Liu, T. Li, X. Chen et al., “A network-based analysis and experimental validation of traditional Chinese medicine Yuanhu Zhitong Formula in treating neuropathic pain,” Journal of Ethnopharmacology, vol. 274, Article ID 114037, 2021.
View at: Publisher Site | Google Scholar
G. Yu, L.-G. Wang, Y. Han, and Q.-Y. He, “clusterProfiler: an R package for comparing biological themes among gene clusters,” OMICS: A Journal of Integrative Biology, vol. 16, no. 5, pp. 284–287, 2012.
View at: Publisher Site | Google Scholar
J.-H. Kim, E.-Y Kim, B. Lee et al., “The effects of Lycii Radicis Cortex on RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells,” International Journal of Molecular Medicine, vol. 37, no. 3, pp. 649–658, 2016.
View at: Publisher Site | Google Scholar
H. Jung Koo, E.-H. Sohn, Y. J. Kim, S. A. Jang, S. Namkoong, and S. C. Kang, “Effect of the combinatory mixture of Rubus coreanus Miquel and Astragalus membranaceus Bunge extracts on ovariectomy-induced osteoporosis in mice and anti-RANK signaling effect,” Journal of Ethnopharmacology, vol. 151, no. 2, pp. 951–959, 2014.
View at: Publisher Site | Google Scholar
Y.-L. Qiao, J.-J. Zhou, J. H. Liang, X. P. Deng, Z. J. Zhang, and H. L. Haung, “Uncaria rhynchophylla ameliorates unpredictable chronic mild stress-induced depression in mice via activating 5-HT1A receptor: insights from transcriptomics,” Phytomedicine, vol. 81, Article ID 153436, 2021.
View at: Publisher Site | Google Scholar
S. S. Signorelli, S. Neri, S. Sciacchitano et al., “Behaviour of some indicators of oxidative stress in postmenopausal and fertile women,” Maturitas, vol. 53, no. 1, pp. 77–82, 2006.
View at: Publisher Site | Google Scholar
H. J. Ihn, J. A. Kim, S. Lim et al., “Fermented oyster extract prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis,” Nutrients, vol. 11, no. 6, p. 1392, 2019.
View at: Publisher Site | Google Scholar
H. L. Brooks, D. P. Pollow, and P. B. Hoyer, “The VCD mouse model of menopause and perimenopause for the study of sex differences in cardiovascular disease and the metabolic syndrome,” Physiology, vol. 31, no. 4, pp. 250–257, 2016.
View at: Publisher Site | Google Scholar
Y. Huang and X. Zhang, “Luteolin alleviates polycystic ovary syndrome in rats by resolving insulin resistance and oxidative stress,” American Journal of Physiology Endocrinology and Metabolism, vol. 320, no. 6, pp. E1085–e1092, 2021.
View at: Publisher Site | Google Scholar
P. Rajendran, R. B. Ammar, F. J. Al-Saeedi et al., “Kaempferol inhibits zearalenone-induced oxidative stress and apoptosis via the PI3K/Akt-Mediated Nrf2 signaling pathway: in vitro and in vivo studies,” International Journal of Molecular Sciences, vol. 22, no. 1, p. 217, 2020.
View at: Publisher Site | Google Scholar
T. Kipari, P. W. Hadoke, J. Iqbal et al., “11β‐hydroxysteroid dehydrogenase type 1 deficiency in bone marrow‐derived cells reduces atherosclerosis,” The FASEB Journal, vol. 27, no. 4, pp. 1519–1531, 2013.
View at: Publisher Site | Google Scholar
Y. H. Jiang, X. Li, W. Niu, D. Wang, B. Wu, and C. H. Yang, “β-Sitosterol regulated microRNAs in endothelial cells against an oxidized low-density lipoprotein,” Food and Function, vol. 11, no. 2, pp. 1881–1890, 2020.
View at: Publisher Site | Google Scholar
Y. Wang, J. Che, H. Zhao, J. Tang, and G. Shi, “Paeoniflorin attenuates oxidized low‐density lipoprotein‐induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells,” Journal of Cellular Biochemistry, vol. 120, no. 6, pp. 9291–9299, 2019.
View at: Publisher Site | Google Scholar
H. Li, Y. Jiao, and M. Xie, “Paeoniflorin ameliorates atherosclerosis by suppressing TLR4-mediated NF-κB activation,” Inflammation, vol. 40, no. 6, pp. 2042–2051, 2017.
View at: Publisher Site | Google Scholar
N. Yuan, L. Gong, K. Tang et al., “An integrated pharmacology-based analysis for antidepressant mechanism of Chinese herbal formula xiao-yao-san,” Frontiers in Pharmacology, vol. 11, p. 284, 2020.
View at: Publisher Site | Google Scholar
X.-L. Lu, C.-H. Zhao, X.-L. Yao, and H. Zhang, “Quercetin attenuates high fructose feeding-induced atherosclerosis by suppressing inflammation and apoptosis via ROS-regulated PI3K/AKT signaling pathway,” Biomedicine and Pharmacotherapy, vol. 85, pp. 658–671, 2017.
View at: Publisher Site | Google Scholar
F. Islam, S. Bibi, A. F. K. Meem et al., “Natural bioactive molecules: an alternative approach to the treatment and control of COVID-19,” International Journal of Molecular Sciences, vol. 22, no. 23, Article ID 12638, 2021.
View at: Publisher Site | Google Scholar
R. Kangas, C. Morsiani, G. Pizza et al., “Menopause and adipose tissue: miR-19a-3p is sensitive to hormonal replacement,” Oncotarget, vol. 9, no. 2, pp. 2279–2294, 2018.
View at: Publisher Site | Google Scholar
P. Fischer-Posovszky, D. Tews, S. Horenburg, K.-M. Debatin, and M. Wabitsch, “Differential function of Akt1 and Akt2 in human adipocytes,” Molecular and Cellular Endocrinology, vol. 358, no. 1, pp. 135–143, 2012.
View at: Publisher Site | Google Scholar
S. Feng, Z. Dai, A. B. Liu et al., “Intake of stigmasterol and β-sitosterol alters lipid metabolism and alleviates NAFLD in mice fed a high-fat western-style diet,” Biochimica et Biophysica Acta (BBA)—Molecular and Cell Biology of Lipids, vol. 1863, no. 10, pp. 1274–1284, 2018.
View at: Publisher Site | Google Scholar
W.-Y. Huang, C. C. Huang, C. C. Chang, C. T. Kor, T. Y. Chen, and H. M. Wu, “Associations of self-reported sleep quality with circulating interferon gamma-inducible protein 10, interleukin 6, and high-sensitivity C-reactive protein in healthy menopausal women,” PLoS One, vol. 12, no. 1, Article ID e0169216, 2017.
View at: Publisher Site | Google Scholar
T. Iwasa, T. Matsuzaki, R. Kinouchi et al., “Changes in central and peripheral inflammatory responses to lipopolysaccharide in ovariectomized female rats,” Cytokine, vol. 65, no. 1, pp. 65–73, 2014.
View at: Publisher Site | Google Scholar
Y. Yoshihisa, M. Furuichi, M. Ur Rehman, C. Ueda, T. Makino, and T. Shimizu, “The traditional Japanese formula keishibukuryogan inhibits the production of inflammatory cytokines by dermal endothelial cells,” Mediators of Inflammation, vol. 2010, Article ID 804298, 8 pages, 2010.
View at: Publisher Site | Google Scholar
X.-F. Huang, W.-B. Cheng, Y. Jiang et al., “A network pharmacology-based strategy for predicting anti-inflammatory targets of ephedra in treating asthma,” International Immunopharmacology, vol. 83, Article ID 106423, 2020.
View at: Publisher Site | Google Scholar
Y. Dong, C. Song, Y. Wang et al., “Inhibition of PRMT5 suppresses osteoclast differentiation and partially protects against ovariectomy-induced bone loss through downregulation of CXCL10 and RSAD2,” Cellular Signalling, vol. 34, pp. 55–65, 2017.
View at: Publisher Site | Google Scholar
R. L. Prince, I. Dick, A. Devine et al., “The effects of menopause and age on calcitropic hormones: a cross-sectional study of 655 healthy women aged 35 to 90,” Journal of Bone and Mineral Research, vol. 10, no. 6, pp. 835–842, 2009.
View at: Publisher Site | Google Scholar
A. J. Chakraborty, S. Mitra, T. E. Tallei et al., “Bromelain a potential bioactive compound: A comprehensive overview from a pharmacological perspective,” Life, vol. 11, 2021.
View at: Publisher Site | Google Scholar
C. Cadeddu Dessalvi, A. Pepe, C. Penna et al., “Sex differences in anthracycline-induced cardiotoxicity: the benefits of estrogens,” Heart Failure Reviews, vol. 24, no. 6, pp. 915–925, 2019.
View at: Publisher Site | Google Scholar
S. Ahmed, H. M. H. Khan, and E. R. Küpeli Akkol, “Anticancer potential of furanocoumarins: mechanistic and therapeutic aspects,” International Journal of Molecular Sciences, vol. 21, no. 16, p. 5622, 2020.
View at: Publisher Site | Google Scholar
E. Lee, M.T Jang, T. G. Lim et al., “Selective activation of the estrogen receptor-β by the polysaccharide from Cynanchum wilfordii alleviates menopausal syndrome in ovariectomized mice,” International Journal of Biological Macromolecules, vol. 165, pp. 1029–1037, 2020.
View at: Publisher Site | Google Scholar
N. Haddadi, Y. Lin, G. Travis, A. M. Simpson, N. T. Nassif, and E. M. McGowan, “PTEN/PTENP1: “Regulating the regulator of RTK-dependent PI3K/Akt signalling” new targets for cancer therapy,” Molecular Cancer, vol. 17, no. 1, p. 37, 2018.
View at: Publisher Site | Google Scholar
Q. Meng, J. Li, Y. Chao et al., “β-estradiol adjusts intestinal function via ERβ and GPR30 mediated PI3K/AKT signaling activation to alleviate postmenopausal dyslipidemia,” Biochemical Pharmacology, vol. 180, Article ID 114134, 2020.
View at: Publisher Site | Google Scholar
L.-W. Huang, T.-C. Huang, Y. C. Hu et al., “S-equol protects chondrocytes against sodium nitroprusside-caused matrix loss and apoptosis through activating PI3K/Akt pathway,” International Journal of Molecular Sciences, vol. 22, no. 13, p. 7054, 2021.
View at: Publisher Site | Google Scholar
J. H. Lee, A. Parveen, M. H. Do, Y. Lim, S. H. Shim, and S. Y. Kim, “Lespedeza cuneata protects the endothelial dysfunction via eNOS phosphorylation of PI3K/Akt signaling pathway in HUVECs,” Phytomedicine, vol. 48, pp. 1–9, 2018.
View at: Publisher Site | Google Scholar
N. Santoro, C. N. Epperson, and S. B. Mathews, “Menopausal symptoms and their management,” Endocrinology and Metabolism Clinics of North America, vol. 44, no. 3, pp. 497–515, 2015.
View at: Publisher Site | Google Scholar
F. K. Ludka, L. C. Constantino, T. Dal-Cim et al., “Involvement of PI3K/Akt/GSK-3β and mTOR in the antidepressant-like effect of atorvastatin in mice,” Journal of Psychiatric Research, vol. 82, pp. 50–57, 2016.
View at: Publisher Site | Google Scholar
A. Akter, F. Islam, S. Bepary et al., “CNS depressant activities of Averrhoa carambola leaves extract in thiopental-sodium model of Swiss albino mice: implication for neuro-modulatory properties,” Biologia, vol. 10, 2022.
View at: Google Scholar
M. M. Rahman, F. Islam, A. Parvez et al., “Citrus limon L. (lemon) seed extract shows neuro-modulatory activity in an in vivo thiopental-sodium sleep model by reducing the sleep onset and enhancing the sleep duration,” Journal of Integrative Neuroscience, vol. 21, no. 1, p. 042, 2022.
View at: Publisher Site | Google Scholar
P. Tagde, P. Tagde, F. Islam et al., “The multifaceted role of curcumin in advanced nanocurcumin form in the treatment and management of chronic disorders,” Molecules, vol. 26, 2021.
View at: Publisher Site | Google Scholar
M. F. Khan, F. B. Kader, M. Arman et al., “Pharmacological insights and prediction of lead bioactive isolates of Dita bark through experimental and computer-aided mechanism,” Biomedicine and Pharmacotherapy, vol. 131, Article ID 110774, 2020.
View at: Publisher Site | Google Scholar
M. A. Freitas, A. Vasconcelos, E. C. D. Gonçalves et al., “Involvement of opioid system and TRPM8/TRPA1 channels in the antinociceptive effect of spirulina platensis,” Biomolecules, vol. 11, no. 4, p. 592, 2021.
View at: Publisher Site | Google Scholar
M. Xiong, J. Li, S. Yang et al., “Influence of gender and reproductive factors on liver fibrosis in patients with chronic hepatitis B infection,” Clinical and Translational Gastroenterology, vol. 10, no. 10, Article ID e00085, 2019.
View at: Publisher Site | Google Scholar
M. K. Parvez, S. Ahmed, M. S. Al Dosari et al., “Novel anti-hepatitis B virus activity of Euphorbia schimperi and its quercetin and kaempferol derivatives,” ACS Omega, vol. 6, no. 43, pp. 29100–29110, 2021.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2022 Xin Tan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

558

Downloads

609

Citations