Zi-Yang Chen, Zhao-Da Ye, Li-Jin You, Yan-Hong Hu

Analysis of the action mechanism of Si-Jun-Zi decoction in cataract by network pharmacology

Zi-Yang Chen1#, Zhao-Da Ye2#, Li-Jin You2, Yan-Hong Hu3*

1Comprehensive Clinic of Fujian Academy of Traditional Chinese Medicine, Fujian 350001, China;2Fujian University of Traditional Chinese Medicine, Fujian 350122, China;3Second People’s Hospital of Fujian University of Traditional Chinese Medicine, Fujian 350001, China.

To explore the action mechanism of the classic ancient prescription Si-Jun-Zi decoction in slowing downcataract process by network pharmacology.In Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform Database, screening the chemical components and functional targets of the classic ancient prescription Si-Jun-Zi decoction, and the targets of cataract disease were obtainded through GeneCards and the Online Mendelian Inheritance in Man Database. In the STRING database, the protein-protein interaction network was built. And network visualization were created in Cytoscape. The genetic ontology and Kyoto Gene and Genome Encyclopedia pathways of key proteins were enriched via the DAVID data platform.Ninety-six active components and 33 key targets were obtained in this work. The protein-protein interaction network consisted of 33 proteins. There were 33 genetic ontology entries and 72 Kyoto Gene and Genome Encyclopedia pathway enrichment analysis.This work provided a new means to understand the pathogenesis of cataract and revealed the action mechanisms of the classic ancient prescription Si-Jun-Zi decoction.

Network pharmacology, Cataract, Si-Jun-Zi decoction, Action target, Genetic ontoloy

In this work, the pharmacological action mechanism of the classic ancient prescription Si-Jun-Zi decoction in slowing down cataract process by network pharmacology was explored. the classic ancient prescription Si-Jun-Zi decoction slow down cataract process by antioxidant activity, cell apoptosis, and proliferation, which had been identified by early studys. And we predicted the classic ancient prescription Si-Jun-Zi decoction mainly acted on the prostaglandin G/H synthase 2. And this target is relevant with ferroptosis. This study provide an idea that cataract may be linked with len ferroptosis, and the classic ancient prescription Si-Jun-Zi decoction can regulate the ferrpotosis. This is worth to be verified by experiment in future.

Background

Cataract refers to a blinding eye disease characterized by intraocular lens opacity, ranking first in the world [1]. Most senile cataracts develop slowly in stages, and low vision can cause great trouble to patients’ life and work. For early senile cataract patients, surgical treatment is not recommended. Traditional Chinese medicine (TCM), which characterized by multiple compounds, has been used for slowing the process of cataract.

The classic ancient prescription Si-Jun-Zi decoction (SJZ) originates from(compiled by the bureau of peaceful benevolent dispensary in Song Dynasty of China). The formula consists of Renshen (, RS), Fuling (, FL), Baizhu (BZ) and Gancao (, GC). SJZ is wildly used in “Spleen-Deficiency Syndrome” for its efficacy in “Invigorating Spleen” and “Replenishing Qi”. In early studys, it was found that SJZ could delay the development of senile cataract. Meanwhile, SJZ antioxidation effect on lens fiber [2, 3]. However, the active components, specific targets and the molecular mechanisms of action are still unknow.

Network pharmacology provides an ideal paradigm to dispose of multiple component drugs [4] and has been applied to study TCM prescriptions along with their active compounds and therapeutic targets [5, 6]. By this means, the underlying mechanisms underlying TCM formulations were illuminated. In this paper, we explore the underlying mechanisms of SJZ by a network pharmacology approach. Firstly, the active compounds of SJZ were selected via oral bioavailability (OB), drug-likeness (DL). Secondly, the targets were obtained by integrating the drug and the cataract targets. These predicted targets were mapped into a drug-target interaction and validated by GO (gene ontology) and pathway enrichment. Since then, the targets were used to construct an integrated pathway. Finally, an integrated pathway was established to illuminating the molecular pathogenesis of SJZ on cataract, which provided a new approach for TCM modernization.

Material and methods

Screening the active ingredients and targets of SJZ

The chemical constituents of RS, FL, BZ and GC were obtained from the Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform (TCMSP, http://lsp.nwu.edu.cn/tcmspsearch. php), and a total of 96 compounds were collected. Among them, 11 were from RS, 3 from BZ, 1 from FL, and 81 from GC. Due to limitations and high cost of biological experiments, the use of computer simulation tools to identify the pharmacokinetics of drugs (absorption, distribution, metabolism and excretion) has become one of the important methods in drug research [7]. In this study, 2 important in silico absorption, distribution, metabolism, and excretion indexes including OB (OB ≥ 30%) and DL (DL ≥ 0. 18) were employed to screen the candidate active ingredients[8]. And 96 compounds were selected for follow-up studies. Then, target protein of each active ingredient was subsequently obtained from the TCMSP database. The gene name of protein was gained from the UniProtKB web platform (http://www.uniprot.org) andtheGenecards(http://lsp.nwu.edu.cn/tcmspsearch.php) database.

Screening the targets of cataract

Information on cataract related target genes was screened by the Human Gene Database named Genecards (http://www.genecards.org/) and the Online Mendelian Inheritance in Man Database named OMIM (http://www.omim.org/). The database was searched using the keywords “cataract”, and the genes which quality score ≥ 5 were selected as the cataract related genes [9].

Constructing active ingredient-target network

Cytoscape is an open source bioinformatics software platform, whose core architecture is the network. Each node is a gene, protein, or molecule, and the junction between nodes represents the interaction between these biomolecules [10]. The chemical compositions of the pre-organized RS, FL, BZ and GC and the corresponding targets were input into Cytoscape 3.5.1 (http://www.cytoscape.org/) software to create the compound-target network. Subsequently, the network analyzer plug-in network characteristic analysis was used to identify important ingredients and targets in the decoction, and simultaneously, track the literature to analyze the interaction.

Protein-protein interaction network construction

To illustrate the role of SJZ in the treatment of cataract, target protein at the system level, the gene for drug component, and disease intersection target were uploaded to the online STRING 10.5 (https://string-db.org/) database to obtain protein interaction information. The STRING database is a database that stores known and predicted protein interactions, including direct and indirect interactions between proteins. It has a score for each protein interaction. The higher the score, the more mutual the interaction between proteins and the higher the confidence level [11]. Therefore, the high-confidence protein interaction data with a score of > 0. 4 were selected and imported into Cytoscape 3.5.1 software to construct the protein-protein interaction (PPI) network. Further, the key genes based on the degree of value were screened.

Genetic ontology Kyoto Gene and Genome Encyclopedia pathway enrichment analyses

The DAVID 6.7 data platform (https://david.ncifcrf.gov/) was used for the genetic ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) enrichment analyses of proteins in the PPI network. The DAVID database is featured with integrating multiple categories of database resources and using the improved Fisher’s exact test algorithm to execute enrichment analysis on gene sets, then provides-value and false discovery rate (FDR) that enrich the results of the analysis [12].

Results

Component-target network construction and analysis of SJZ

In this study, all compounds were obtained from the TCMSP database. After screening by OB and DL values, 96 components for cataract targets were retained, including 11 were from RS, 3 from BZ, 1 from FL, and 81 from GC. Table 1 provides the information of top 5 active ingredients. As shown in Figure 1, the compound-target network included 129 nodes and 151 edges. The blue node represents the Mol ID of drug component, and green represents target. The edge represents interaction between nodes. In this network, different compounds act on the same target, which embodies the mechanism of interaction between multi-component and multi-target of SJZ. Using the degree value, the representative components in the network and nodes of the target were effectively screened to obtain nodes with high correlation. With respect to compounds, there were approximately nine compounds with targets > 5, of which the representative component nodes were quercetin (MOL000098), kaempferol (MOL000422), naringenin (MOL004328).

With respect to targets, the targets with highest degree of value included prostaglandin G/H synthase 2 (PTGS2), which combined with 96, respectively. After data mining and molecular docking, the drug targets were input into the STRING database for analysis. As shown in Figure 2, the PPI network had a total of 33 nodes, and the interactions between the nodes were as many as 226. The nodes are proteins, and edges reveal theinteractionbetween proteins.Accordingto the network topology, the highest value was for interleukin 6, which can interact with 28 proteins, followed by VEGFA and CASP3, which can interact with 26 and 25 proteins, respectively.

Table 1 Active ingredients information statistics

OB, oral bioavailability; DL, drug-likeness.

GO enrichment and KEGG analysis

The GO and KEGG enrichment analyses were performed using the DAVID data analysis platform. In the GO analysis, a total of 33 entries (FDR < 0. 05) were determined according to the false discovery rate (FDR). Figure 3 lists the top 20 biofunctional items of the crossover target genes, mainly related to growth factor activity, antioxidant activity, cytokine receptor, and so on. In the KEGG pathway enrichment analysis, we obtained a total of 72 pathways, including pathways in fluid shear stress and atherosclerosis, mitogen-activated protein kinase (MAPK) signaling pathway, and IL-17 signaling pathway. Figure 4 lists the top 20 KEGG pathways based on FDR values. The results indicate that the active ingredients of SJZ might be used to treat cataract, and they act on these signaling pathways.

Discussion

The diverity of TCM ingredients and the complexity of interaction in human body, it is difficulty to figure the potential mechanism. To uncover the mechanism of action of SJZ, this study relied on the TCMSP to study the active constituents of SJZ. The Genecards and the Online Mendelian Inheritance in Man Database queried the target of cataract and constructed a compound-target network.

The direct interaction between the compound and the target was analyzed, which provided a reference for the treatment mechanism of multi-component, multi-target, and multi-channel.

The main compounds of SJZ are quercetin and kaempferol in GC, and their degree value was much higher than that of other compounds. It is indicated that GC might play a major role in the decoction in delay process of cataract. Studies have shown that quercetin not only has antioxidant, inhibiting apoptosis, vasodilating, and antihypertensive effects [13–15], but also mediate the antidiabetic cataract effects [16]. And kaempferol also intervene oxidative and inflammatory stress induced apoptosis [17].

The key target of SJZ is PTGS2 in the compound-target network. It was found that PTGS2 mediates anti-apoptosis in many disease [18]. As a potential target genes for cataract, PTGS2 plays a vital role in MAPK, microRNA and NF-κB signaling pathways in process of cataract [19–22]. And PTGS2 is an important gene in ferroptosis. In future, more works, experimental verification, are required to be done to find this key mechanisms.

According to the GO analysis, the results demonstrated that SJZ may slow down cataract process by antioxidant activity, cell apoptosis, and proliferation. For example, antioxidant activity involves in antioxidant activity, cell apoptosis involve in cell apoptosis, and growth factor receptor binding and growth factor activity involves in cell proliferation, respectively. Our study had indentifided that SJZ can regulate the lens cells apoptosis induced by H2O2. This means that network analysis can gain results, which are similar to experiments.

Figure 1 Compound-target network. Yellow nodes represent disease, red nodes represent SJZ compounds, blue nodes on behalf of herbal ingredients, green nodes represent potential targets, and the gray lines shows the interactions. Node size means its degree. SJZ, the classic ancient prescription Si-Jun-Zi decoction.

Figure 2 PPI network.The nodes represent proteins and each lines represents the interaction between proteins. PPI, protein-protein interaction.

Figure 3 GO analysis of genes. The X-axis means the enrichment counts, and the Y-axis represents the GO biological process terms (adjusted-value < 0. 01). GO, gene ontology.

Figure 4 KEGG analysis of genes. The X-axis means the gene radio of these terms, and the Y-axis represents the KEGG biological process terms of the target genes (adjusted-value < 0. 01). KEGG, Kyoto Gene and Genome Encyclopedia.

KEGG pathway analysis indicated that SJZ could target 72 pathways which involved in these biological processes to intervene in the pathogenesis of cataract. For instance, Fluid shear stress and atherosclerosis, MAPK, and IL-17 signaling pathway, etc. These pathways involve in the oxidative stress process of age-related cataract[23–24].

Although studies have manifested that TCM formulations are effective and safe in slowing down the process of cataract [2, 25], the mechanisms of action are still unknown. The network pharmacology method provided a novel mean to explore the molecular mechanisms of SJZ and improved our understanding of herbal medicines.

Conclusion

In this study, we utilized a network pharmacology approach with integrating active compounds screening, targets prediction, network and pathway analysis to explore the therapeutic mechanism of SJZ on cataract. The result revealed that SJZ exerts a therapeutic effect via the antioxidant activity, cell apoptotic process, and cell proliferation. Meanwhile, the approach applied in this work offers a new research orientation for SJZ mechanism in cataract.

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10.12032/MDM2020123004

#Authors contributed equally to this article.

Yan-Hong Hu. Second People’s Hospital of Fujian University of Traditional Chinese Medicine, 282 Wusi Road, Fuzhou City, Fujian 350001, China. Email: 46665871@qq. com.

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SJZ, the classic ancient prescription Si-Jun-Zi decoction; TCM, traditional Chinese medicine; PPI, protein-protein interaction; GO, genetic ontology; KEGG, Kyoto Gene and Genome Encyclopedia; TCMSP, Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform; RS, Renshen; FL, Fuling; BZ, Baizhu; GC, Gancao; OB, oral bioavailability; DL, drug-likeness; FDR, false discovery rate; PTGS2, prostaglandin G/H synthase 2; MAPK, mitogen-activated protein kinase.

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The authors declare that they have no conflict of interest.

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Zi-Yang Chen, Zhao-Da Ye, Li-Jin You, et al. Analysis of the action mechanism of Si-Jun-Zi decoction in cataract by network pharmacology. Medical Data Mining 2020, 3 (4): 177–184.

:Yu-Ping Shi.

:24 September 2020,

: 10 December 2020,

:30 December 2020