A network pharmacology approach to investigate the mechanisms of Si-Jun-Zi decoction in the treatment of gastric precancerous lesions

Liang-Jun Yang, Dao-Rui Hou, Ya Li, Zhi-Peng Hu, Yong Zhang*

1Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China. 2The First People's Hospital of Xiangtan City, Xiangtan, Hunan, China. 3Lin’an Hospital of Traditional Chinese Medicine, Lin’an, Zhejiang, China. 4Chengdu University of Traditional Chinese Medicine, Chengdu, China. 5Sichuan Second Hospital of Traditional Chinese Medicine,Chengdu, Sichuan China.

Background

Gastric cancer is one of the most common and aggressive malignant tumors around the world, with a 5-year overall survival rate less than 25% [1, 2]. And it is still the most prevalent cancer in Eastern Asia, such as China, Japan,and Korea [3]. Because of the high mortality, early detection and treatment is an important way to reduce the risk of death from gastric cancer. It is well known that gastric cancer is a continuous process starting from chronic superficial gastritis, atrophic gastritis, intestinal metaplasia, and ultimately to dysplasia and adenocarcinoma [4]. There are pieces of evidence that gastric precancerous lesions (GPL), which mainly includes intestinal metaplasia and dysplasia [5], plays a crucial role in the progression of gastric cancer. A cohort study showed that nearly 25% of patients with high-grade dysplasia received a diagnosis of gastric cancer within 1 year and 7% of patients with low-grade dysplasia progressed to invasive carcinoma [6]. Therefore, further researches on GPL is imperative to prevent the formation and development of gastric carcinoma.

Traditional Chinese medicine (TCM), which characterized by multiple compounds, has been used for preventing and treating cancers for centuries [7] and shows great potential in complex disease for its multi-targets manner [8]. Evidence has shown that TCM therapy offered a better survival benefit for patients with stage IV gastric adenocarcinoma compared with patients without any TCM treatment [9]. Si-Jun-Zi (SJZ)decoction, originated from Taiping Huimin Heji Jufang compiled by the bureau of peaceful benevolent dispensary in 1078 A.D. - 1085 A.D. (Song Dynasty of China), is a famous herbal formula for treating digestive diseases. The formula is composed of four herbs:Dang-Shen (Codonopsis Radix, DS), Fu-Ling (Poria Cocos Sc hw. W olf, FL), Bai-Zhu (Atractylodes Macrocephala Koidz, BZ) and Gan-Cao (licorice, GC).According to TCM theory, SJZ decoction is wildly used in spleen-deficiency syndrome for its efficacy in invigorating spleen and replenishing Qi. And pharmacological researches have demonstrated that SJZ decoction could promote the restoration of intestinal function [10, 11], modulate the immunity [12], alleviate the intestinal inflammatory response and improve the nutrition absorption [13]. Clinical study shows that SJZ decoction can reverse gastric pathological changes including chronic atrophic gastritis, intestinal metaplasia and gastric epithelial dysplasia, and favorably relieve clinical symptoms [14-16]. Although the therapeutic efficacy of SJZ decoction in the treatment of GPL has been verified, the active ingredients, cellular targets, and the molecular mechanisms of action are still unknown.

In this paper, we explore the underlying mechanisms of SJZ decoction by a network pharmacology approach,which is a powerful discipline incorporating systems biology, bioinformatics, and pharmacology [17]. As a new advanced analytical technique, network pharmacology analysis has been applied to TCM research to identify the effect and mechanism of action of medications used to treat complicated diseases [18, 19].The protocol of this work is presented above. Firstly, the active compounds of SJZ were selected via oral bioavailability (OB), drug-likeness (DL), Caco-2 cell permeability evaluation, and literature mining. Secondly,the targets were obtained by integrating the drug and the GPL 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 for further analysis. Finally, an integrated “GPL pathway” was established to illuminating the molecular pathogenesis of SJZ on GPL, which provided a new approach for TCM modernization.

Material and methods

Active compounds screening

The compounds of SJZ were obtained from the database TCMSP (Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform,http://lsp.nwsuaf.edu.cn/tcmsp.php), which is a platform of Chinese herbal medicines that captures the relationships between drugs, targets, and diseases [20].This database has collected 499 herbs with a total of 12144 chemicals based on precisely examined pharmacology and clinical knowledge. Simultaneously,literature search was conducted to find ingredients that have functions on humans. In this work, the ingredients of SJZ were put into TCMSP, a total of 491 molecules with 134 in DS, 34 in FL, 55 in BZ, 280 in GC were obtained. In order to get the ADME (absorption,distribution, metabolism, and excretion) properties of compounds, the molecular pharmacokinetic parameters including OB, DL, and caco-2 permeability were calculated to screen the potential chemical components.

(1) OB screening. OB reflects the capability of the orally administered drug be delivered to the circulatory system. It is one of the most important pharmacokinetics features in drug screening [21]. Compounds with a high OB values often have the potential to become therapeutic drugs [22]. In this work, the OB evaluation was calculated by a robust in silico model OBioavail 1.1 [21],which has advantages in accelerating the prediction of OB. Molecules with OB ≥ 30% were selected as the candidate compounds for further analysis [23-25].

(2) DL evaluation. DL is a qualitative concept used in drug design for estimating how “drug-like” a prospective compound is in line with the majority of known drugs[26]. It has been widely used to screen out chemicals with desired properties in pharmaceutical research [27]. Based on the molecular descriptors and Tanimoto coefficient[28], a database-dependent model is applied to calculate the DL index of each compound in SJZ. The DL calculation approach is shown as follows:

where A is the molecular properties of herbal ingredients,and B represents the average molecular properties of all molecules in the DrugBank database (http://www.drugbank.ca/) based on Dragon soft descriptors [29]. In this work, the ingredients with DL ≥ 0.18 were selected as candidate compounds for further research.

(3) Caco-2 cell evaluation. Caco-2 cell permeability is a valuable index for assessing oral absorption and is usually used as a model to evaluate the absorption of drugs from the intestinal epithelium barrier [30, 31]. In this work, a Caco-2 cell permeability prediction work platform preCaco2 was applied to calculate the drug absorption rate [32]. The threshold of Caco-2 cell permeability was set to 0 to screen possible candidate drugs presently.

Finally, the threshold values for the integrative screening system were OB ≥ 30%, DL ≥ 0.18, Caco-2 ≥ 0,and the ingredients meeting the conditions were selected as potential active compounds for further analysis.

Compound targets of SJZ

Target identification is crucial to illuminate the therapeutic mechanism of SJZ because of the multiple active components. The targets for these compounds were collected from TCMSP database, which used the SysDT model and the HIT (Herb ingredients’ targets) database[20]. Based on two powerful methods named Random Forest and SVM (Support Vector Machine), the SysDT shows outstanding in predicting drug-target interactions,with a concordance of 82.83%, a sensitivity of 81.33%,and a specificity of 93.62% [33].

The compounds with unknown targets were subjected to Swiss Target Prediction (http://www.swisstargetprediction.ch/), which is a database for target prediction of bioactive small molecules based on a combination of 2D and 3D similarity measures with known ligands [34]. The potential targets were carried out using the SMILES format of each constructed structure,and Homo sapiens was chosen as the source of the target.

Owing to the non-canonical description of the compound targets, the UniProt Knowledgebase(www.uniprot.org/) was used. The protein names were retrieved with the species limited to ?Homo sapiens? to obtain official symbols. Finally, the ingredient targets with gene symbol were obtained.

Disease targets of GPL

Information on GPL related target genes was screened by The Human Gene Database named Genecards(http://www.genecards.org/), which is a database containing information on all annotated and predicted human genes, proteins, and diseases [35]. Sources of Genecards come from 125 databases including genomic,transcriptomic, proteomic, genetic, clinical and functional information, and the information is considered reliable[36]. The database was searched using the keywords?gastric precancerous lesions? or ? precancerous lesion of gastric cancer?, and the genes which quality score ≥5 were selected as the GPL related genes.

GO and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment

In order to explore the underlying mechanisms of SJZ decoction that provides therapeutic effects in GPL, the GO and KEGG pathway enrichment were conducted. The GO defines the concepts relating to gene functions from three aspects: molecular function, cellular component,and biological process [37]. In this research, we performed GO analysis only for biological process.KEGG pathway database is a collection of comprehensive inferences for pathway mapping and is useful in predicting gene functions [38]. The enrichment analyses were performed by linking the targets to Enrichr(http://amp.pharm.mssm.edu/Enrichr/), which allows for the identification of associated pathways and ontology tools [39]. Enriched GO terms and pathways with adjusted P < 0.01 were obtained and analyzed for further research. And the terms which have no connection with the disease were detached.

Network construction

To decipher the complex therapeutic mechanisms of SJZ decoction in treating GPL, a compound-target network(C-T network) and target-pathway (T-P network) were constructed to uncover the relationship between candidate compounds and potential targets. (1) C-T network. A C-T network was constructed to illustrate the drug-target interactions of active ingredients in SJZ and their potential targets. (2) T-P network. Based on the previous work, a T-P network was built by mapping the target proteins to the enriched pathways.

The graphs were visualized and analyzed by Cytoscape version 3.2.1 [40], a powerful bioinformatics software for data visualization and integration. In the graphical networks, the compounds, targets, and pathways were represented by nodes, and the interaction between these nodes was represented by edges. Meanwhile, a vital topological parameter called degree was analyzed by the plugin Network Analyzer of Cytoscape [41]. The degree of a node is the number of edges associated with it and the nodes with a high degree can be regarded as the key nodes in the network.

GPL pathway construction and analysis

To explore the systematic mechanisms of SJZ decoction on GPL treatment, an incorporated “GPL pathway” was integrated based on the T-P network. Pathways related to GPL pathogenesis were picked out and assembled according to the pathological and clinical data.

Results

Active compounds identification

In total, 491 compounds were collected from 4 herbs of SJZ decoction. By screening three classical ADME parameters including OB, DL and Caco-2 permeability,123 potentially active compounds with 19 in DS, 13 in FL, 7 in BZ, 84 in GC were obtained. Additionally, 12 typical components of SJZ decoction were also reserved as the active components even though they do not meet the parameter. For instance, atractylenolide Ⅲ, a common ingredient in DS and BZ, has anti-inflammatory effects by suppressing the release of nitric oxide, prostaglandin E2, TNF-α (tumor necrosis factor-α) and IL-6(interleukin-6) related to the NF-κB and MAPK-signaling pathways [42]. Thus, a total of 135 active herbal ingredients were obtained in this work for further target prediction and network analysis (as showed in Table S1)

Target identification

Based on the target fishing approach introduced above, a total of 286 targets for the 135 compounds with 2170 connection between them were obtained. While, targets involving the pathogenesis of GPL, as well as the interaction mechanism, remained unknown. In order to find out the GPL related genes, the target genes related to GPL were searched in the GeneCards, which covers nearly 90% of human protein-coding genes in GeneCards[43]. Since then, 350 GPL related genes which known to be expressed were collected. Through a combination of the compound targets of SJZ decoction and the disease targets, 90 overlapping genes were selected as the key targets in the GPL treatment. And 111 compounds were saved as the active ingredients after deleting 24 candidate compounds which had no related targets. Finally, these 90 targets were considered to have effective therapeutic actions in the pathogenesis of GPL (Table S2).

To get the logical structure of the biological functions,GO enrichment and analysis were conducted. According to the results, these targets were significantly associated with 654 GO biological process terms, like the cellular response to cytokine stimulus (ID: 0071345, Count = 36),cytokine-mediated signaling pathway (ID: 0019221,Count = 37), regulation of apoptotic process (ID:0042981, Count = 37), negative regulation of apoptotic process (ID: 0043066, Count = 29), positive regulation of gene expression (ID: 0010628, Count = 33), etc. The top 20 significantly enriched terms were shown in Figure 1.

Figure 1 GO analysis of target genes

Network Construction

C-T network and analysis.TCM decoction exerts multiple biological and pharmacological effects through multiple compounds and targets. To illustrate the interrelationship between the key targets and their correlated herbal ingredients, a C-T network was built.The active compounds, targets, and interactions between them were showed in Figure 2, which have 201 nodes(111 compounds and 90 targets) and 944 edges. The C-T network analysis displayed that the average degree (the number of edges connecting to the nodes) per compound is 10.49, indicating that most compounds regulated multiple targets to exert therapeutic effects and the compounds with high degree may play crucial roles in the treatment of GPL. Typically, three compounds with degrees more than 20, such as quercetin, luteolin, and kaempferol become the critical ingredients for the SJZ because of their important positions in this network.

Additionally, lots of targets were connected with multiple compounds of different herbs, which exhibit an integrative function of SJZ decoction in the treatment of GPL. Among the candidate targets, COX-2(Cyclooxygenase-2), estrogen receptor 1, PPARG(peroxisome proliferative activated receptor) are targeted by 90, 79 and 66 compounds, which demonstrated the potential therapeutic targets of SJZ decoction.

T-P Network and analysis.To examine the signaling pathways of these target genes, KEGG pathway enrichment was conducted. After enrichment, 90 targets were mapped to 130 pathways according to the adjusted P-value. According to the pathogenesis of GPL and the gene counts related to these pathways, like hepatitis B(hsa05161, degree = 31), bladder cancer (hsa05219,degree = 19) which had no relationship with the GPL and degree less than 10 were detached. Finally, 21 remarkably pathways are likely to be the major pathways in the treatment of GPL (shown in Table 1). Taken together, 76 target genes were involved in these pathways, resulting in a T-P network (shown in Figure 3). It can be seen from the figure below that these targets were closely interact with pathways in cancer (hsa05200, degree = 46),PI3K-Akt signaling pathway (hsa04151, degree = 28),apoptosis (hsa04210, degree = 19), etc. These pathways could be regarded as the key pathways in the treatment of GPL.

Figure 2 Compound-target network

Table 1 The KEGG pathways of therapy target genes

Figure 3 Target-pathway network

GPL pathway.In order to explore the integral regulation of SJZ for the treatment of GPL, an integrated GPL pathway was constructed through the T-P network analysis above. As showed in Figure 4, this GPL associated pathway can be separated into three therapeutic modules, including inflammation module,apoptosis module, and proliferation module, which reveal the underlying therapeutic effects of SJZ decoction.

Inflammation module.It is well known that gastric cancer is one of the best examples of inflammation-associated cancer in human beings. As a vital step in the gastric pathogenesis, gastric precancerous lesions triggered by environmental stimuli is closely connected with the deleterious effects of inflammation[44]. And chronic mucosal inflammation is associated with a great risk in the progression from chronic gastritis to gastric cancer [45]. From the figure above we can see that the inflammatory cytokines play crucial roles in the activation of the PI3K-Akt signaling pathway, the FoxO signaling pathway, and the NF-κB signaling pathway. For instance, H. pylori infection often induces the expression of the pro-inflammatory cytokine IL-8 (interleukin-8)through the activation of the NF-κB pathway [46]. And the modulation of these pathways is considered a useful approach to inhibit proinflammatory gene expression[47-49].

Apoptosis module.Apoptosis is the programmed cell death which keeps the survival and death balance in metazoan cells. Deficiency in cell apoptosis is related to the formation of some gastrointestinal malignancies [50],and exerts a positive influence on the morphogenesis of GPL [51]. As can be seen from the Figure 4, pathways including the PI3K-Akt signaling pathway, the p53 signaling pathway, the TNF signaling pathway, the FoxO signaling pathway, and the Ras signaling pathway are involving in the disorder of apoptosis. For example, p53,which participates in the PI3K-AKT signaling pathway and p53 signaling pathway, is playing a vital role in anti-tumor by inducing cell apoptosis [52]. And the C-T network shows that the p53 could be regulated by luteolin from DS and quercetin from GC. The results of the correlational analysis suggest that SJZ decoction may treat GPL by inducing cell apoptosis in gastric mucosa.

Proliferation module.It is well known that tumor cell growth is closely associated with the imbalance between cell proliferation and apoptosis [53]. And the acquisition of cell proliferation allows the progression of gastric precancerous lesions, invasion, and metastasis [54]. The figure above indicates that the PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and FoxO signaling pathway have a tight relationship with the disorder of cell proliferation. For instance, the PI3K-Akt pathway is an important signaling pathway that is activated in a large variety of tumors and the phosphorylation of AKT (Protein kinase B) could promote cell cycle progression and cell proliferation [55].As a vital molecule, some ingredients like luteolin,kaempferol, naringenin, and quercetin which connected to the Akt, may exert some influences on this target. It has been reported that kaempferol could induce G2/M cell cycle arrest by suppressing AKT activation, which suggested that kaempferol may be useful in cancer prevention [56]. Thus, this work showed that SJZ decoction might integrate diverse signaling pathways to modulate cell proliferation to prevent cancer.

Figure 4 Gastric precancerous lesions associated pathway

Discussion

Gastric cancer is one of the most common types of cancer and it remains the second leading cause of cancer death worldwide [57]. Although the incidence of gastric cancer has decreased, the mortality rate has markedly improved[58] and there is still a long way to go before complete conquest of this disease [59]. Therefore, early diagnosis and treatment of GPL are crucial to reduce the morbidity and mortality of gastric cancer [59, 60]. As a useful alternative medicine with a long history, TCM is attracting more and more attention across the world for its remarkable effect in clinical practice. It is well acknowledged that spleen deficiency is the key pathogenesis of GPL [61]. SJZ decoction has been applied to treat GPL owing to its ability to fortify the spleen and replenish the Qi. Thus, it is imperative to use the network pharmacology approach to explore the therapeutic mechanism of SJZ decoction in the treatment of GPL.

In this study, there are 111 compounds collected from SJZ decoction and 90 targets considered to have therapeutic effects in treating GPL. Among the 111 compounds linked to the C-T network, quercetin, luteolin,kaempferol, stigmasterol, atractylenolide I are known bioactive compounds and have been shown to have anti-cancer effects [62-66]. For instance, quercetin, a vital member belonging to the flavonoid family, is one of the most prominent dietary antioxidants and famous for its anticarcinogenic potential. It can regulate the balance of gastric cell proliferation and apoptosis to protect against gastritis and induce the apoptosis of human gastric carcinoma cells by stimulating the activity of caspase-3 and decreasing the rate of Bcl-2/Bax [67, 68]. According to a clinical research in Sweden, high dietary quercetin intake is inversely related to the risk of gastric cancer,especially for women exposed to oxidative stress [69].Luteolin is a natural flavonoid present in several herbs.Researches indicated that luteolin can prevent cancer by arresting the cell cycle, inducing apoptosis [70, 71]. And it exerts a significant anti-tumor effect in gastric cancer through decreasing the expression and phosphorylation of c-Met (tyrosine-protein kinase Met), and downstream phosphorylation of AKT and ERK (extracellular signal-regulated kinase) [72]. Kaempferol is a natural flavonoid found in many herbs [73]. Epidemiologic studies have demonstrated that dietary Kaempferol intake could reduce the risk of cancer, especially gastric cancer[74], and induce apoptosis in human stomach carcinoma cells [75]. All in all, these findings suggest that the active components in SJZ decoction are effective in treating the disease.

On the other hand, lots of targets were found to be linked to multiple compounds in the C-T network. Among these targets, COX-2, TNF, and NOS2 (Nitric Oxide Synthase 2) are inflammation-associated enzymes with oncogenic function in tumor progression [76, 77]. Such as COX-2, evidence shows that COX-2 over expression is related to gastric premalignant changes and subsequent tumor occurrence [78], and the decrease of COX-2 is related to the regression of precancerous lesions [79].CDK2 (Cyclin-dependent kinase 2), GSK3B (Glycogen synthase kinase 3 beta), Bax, and Bcl-2, which involved in cell proliferation or apoptosis processes, are playing important roles in the tumorigenesis [80-83]. Certain compounds in SJZ decoction like quercetin, whose targets are Bcl-2 and Bax in the C-T network, have been shown to be effective in deregulating the expression of Bcl-2 and Bax [84]. By analyzing the targets, the work revealed that this formula may treat GPL at numerous levels.

According to the GO analysis and the KEGG pathway enrichment, the results demonstrated that SJZ decoction may treat GPL by modulating gastric mucosal inflammation, cell apoptosis, and proliferation. For example, cellular response to cytokine stimulus (GO:0071345) involves in inflammation, regulation of apoptotic process (GO: 0042981) and negative regulation of apoptotic process (GO: 0043066) involve in cell apoptosis, and regulation of cell proliferation (GO:0042127) involves in cell proliferation, respectively. It is well known that chronic inflammation is an established risk factor during the progression from GPL to gastric cancer [45]. And the disruption in the balance between gastric epithelial cell proliferation and cell death is linked to the progression from chronic gastritis, atrophy,intestinal metaplasia, dysplasia and ultimately to cancer[85]. KEGG pathway analysis indicated that SJZ decoction could target 21 pathways which involved in these biological processes to intervene in the pathogenesis of GPL. For instance, BZ, a widely used traditional herbal medicine for gastrointestinal system diseases, has been shown to be effective in down regulating the production and mRNA expression of inflammatory cytokines like TNF-α, IL-6, and IL-8 through inhibition of the NF-κB signal pathway [86].Studies have demonstrated that Atractylenolide III,obtained from DS and BZ, is able to decrease the TNF level in a dose-dependent manner [87]. All these data indicate that these pathways might interact to exert their combined effects against GPL, which could reveal the potential effects of SJZ decoction.

Although numerous studies have demonstrated that TCM formulations are effective and safe in treating GPL[88], and could inhibit the carcinogenesis of gastric mucosa [89], the mechanisms of action are still unknown.The network pharmacology approach applied in this work provided a novel approach for exploring the molecular mechanisms underlying the therapeutic effects of SJZ decoction and improved our understanding of herbal medicines. The results indicated that the pharmacological mechanisms of SJZ decoction on GPL might be strongly associated with its synergic modulation on inflammation,cell apoptosis, and proliferation. However, there was no experimental verification in this work, such as quantitative real-time PCR or western blot analysis to estimate the predictions. More works are required to be done to verify the result and to find the key mechanisms.

Conclusion

In this study, we utilized a network pharmacology approach by integrating active compounds screening,targets prediction, network analysis, and pathway analysis to explore the therapeutic mechanism of SJZ decoction on GPL. The result revealed that SJZ decoction exerts a therapeutic effect by intervening in the mucosal inflammation, cell apoptotic process, and cell proliferation. The approach applied in this work offers a new approach to explore TCM formulations and promotes the therapeutic application of traditional medicines in modern medicine.

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