key: cord-0775637-3cwm4y19
authors: Fan, Qiaowei; Guo, Lin; Guan, Jingming; Chen, Jing; Fan, Yujing; Chen, Zhendong; Li, Hulun
title: Network Pharmacology-Based Study on the Mechanism of Gegen Qinlian Decoction against Colorectal Cancer
date: 2020-11-26
journal: Evid Based Complement Alternat Med
DOI: 10.1155/2020/8897879
sha: b0331fe78b3edc727aa10a4f39604cbdc4fab69f
doc_id: 775637
cord_uid: 3cwm4y19

PURPOSE: Gegen Qinlian decoction (GQD) has been used to treat gastrointestinal diseases, such as diarrhea and ulcerative colitis (UC). A recent study demonstrated that GQD enhanced the effect of PD-1 blockade in colorectal cancer (CRC). This study used network pharmacology analysis to investigate the mechanisms of GQD as a potential therapeutic approach against CRC. MATERIALS AND METHODS: Bioactive chemical ingredients (BCIs) of GQD were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. CRC-specific genes were obtained using the gene expression profile GSE110224 from the Gene Expression Omnibus (GEO) database. Target genes related to BCIs of GQD were then screened out. The GQD-CRC ingredient-target pharmacology network was constructed and visualized using Cytoscape software. A protein-protein interaction (PPI) network was subsequently constructed and analyzed with BisoGenet and CytoNCA plug-in in Cytoscape. Gene Ontology (GO) functional and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis for target genes were then performed using the R package of clusterProfiler. RESULTS: One hundred and eighteen BCIs were determined to be effective on CRC, including quercetin, wogonin, and baicalein. Twenty corresponding target genes were screened out including PTGS2, CCNB1, and SPP1. Among these genes, CCNB1 and SPP1 were identified as crucial to the PPI network. A total of 212 GO terms and 6 KEGG pathways were enriched for target genes. Functional analysis indicated that these targets were closely related to pathophysiological processes and pathways such as biosynthetic and metabolic processes of prostaglandins and prostanoids, cytokine and chemokine activities, and the IL-17, TNF, Toll-like receptor, and nuclear factor-kappa B (NF-κB) signaling pathways. CONCLUSION: The study elucidated the “multiingredient, multitarget, and multipathway” mechanisms of GQD against CRC from a systemic perspective, indicating GQD to be a candidate therapy for CRC treatment.

Colorectal cancer (CRC) is a global health burden and is the third most commonly diagnosed malignancy and the second leading cause of cancer deaths worldwide [1] . It constituted approximately 1.8 million new cases and 900,000 deaths annually, according to estimates from the International Agency for Research on Cancer in 2018 [2] . Despite the progress in the treatment of CRC, effects of current therapies including surgery, radiotherapy, chemotherapy, and targeted therapy are still unsatisfactory, especially for patients with metastatic lesions. erefore, innovative therapeutic agents are needed, which are more effective and less toxic.

Traditional Chinese medicine (TCM) has been widely used in China for millenniums. It has been proved highly effective for a wide range of diseases. During the fight against infectious pneumonia caused by the 2019 novel coronavirus (2019-nCoV), TCM has made vast contributions to the prevention, treatment, and rehabilitation of coronavirus disease 2019 (COVID-19) among Chinese population [3] .

is highlights the great value of TCM in the treatment of complicated diseases, especially those with poor response to Western medicine alone. Gegen Qinlian decoction (GQD) is a well-known TCM formula originally described in the "Treatise on Exogenous Febrile Disease ("Shang Han Lun" in Chinese)." GQD had been used in China for approximately 2,000 years, most commonly for the treatment of gastrointestinal diseases, such as infectious diarrhea. GQD is composed of four herbal components, Radix Puerariae ("Gegen" in Chinese), Scutellariae Radix ("Huangqin" in Chinese), Coptidis Rhizoma ("Huanglian" in Chinese), and licorice ("Gancao" in Chinese). In recent years, studies have shown promising therapeutic effects of GQD in various diseases. A metaanalysis showed that GQD used alone or in combination with Western medicine might have potential benefits in curing ulcerative colitis (UC). UC can lead to the accumulation of high levels of proinflammatory cytokines within the colonic mucosa, resulting in dysplastic lesions and CRC [4, 5] . GQD has been shown to maintain colonic mucosal homeostasis in ulcerative colitis via bidirectionally regulating Notch signaling [6] . GQD also attenuated high-fat diet-induced steatohepatitis via modulation of the gut microbiome and reduced nonalcoholic steatohepatitis-associated liver injuries [7, 8] . In the field of cancer research, GQD has been proved to inhibit the expansion and neoangiogenesis of renal carcinoma by suppressing matrix metalloproteinase-2 [9] . Moreover, GQD was found to enhance the effect of PD-1 blockade in CRC by remodeling the gut microbiota and the tumor microenvironment [10] .

ese studies suggest potential for the use of GQD in the treatment of CRC. However, more preclinical evidence is needed. It is difficult to illustrate the complex anticancer mechanisms of GQD due to its "multi-"component and "multi-"target characteristics.

Network pharmacology, first proposed by Andrew L Hopkins, integrates a series of disciplines including pharmacology, bioinformatics, chemoinformatics, and systems biology. It offers a new framework for drug design and drug-target relationship prediction and enables unknown mechanisms of drug action to be inferred [11] . e history of "TCM network pharmacology" dates back to 1999, when Li proposed a possible relationship between TCM syndrome and molecular networks [12] . Since then, numerous studies have been conducted to support the concept and practice of TCM network pharmacology [13] . e TCM network pharmacology approach provides a new research paradigm for the discovery of bioactive compounds and elucidation of the mechanisms of herbal formulas [13] . TCM has also been successfully used to identify active compounds and elucidate mechanisms of GQD in the treatment of diseases such as type 2 diabetes and rotavirus enteritis [14, 15] . In the present study, we used a network pharmacology-based approach to investigate the potential mechanisms of how GQD exerts its anticancer effects on CRC. First, CRC-specific genes and bioactive chemical ingredients (BCIs) of GQD were obtained from the Gene Expression Omnibus (GEO) and the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), respectively. en, CRC-specific genes related to the BCIs of GQD were screened by chemical-target interaction analysis. A pharmacological network and a protein-protein interaction (PPI) network were subsequently constructed to provide a comprehensive overview of the anti-CRC pharmacological action of GQD. Gene Ontology (GO) functional and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses were finally conducted for target genes in the pharmacological network to reveal their functional implications during the anticancer process. e flowchart of the analysis procedures of our study is shown in Figure 1 . [16] . en, the data were converted into expression measures, and background correction, quartile data normalization, and probe summarization were performed using the robust multiarray average (RMA) algorithm in R [17] . e paired ttest based on the Linear Models for Microarray data (LIMMA) package in R was used to identify differentially expressed genes (DEGs) between CRC and normal samples [18] . e DEGs with an adjusted P value < 0.05 and a |log 2 fold-change (log 2 FC)|≥1 were considered significant and defined as CRC-specific genes.

Chemical compounds were obtained from the TCMSP database (https://tcmspw.com/) [19] . BCIs were screened out according to predicted oral bioavailability (OB) and drug-likeness (DL) values and reserved if OB ≥ 30% and DL ≥ 0.18, which were the suggested drug screening criteria by the TCMSP database.

To retrieve all the possible interactions among target genes in the pharmacological network, a PPI network was constructed using the BisoGenet plug-in in Cytoscape [21] . Subsequently, CytoNCA, a Cytoscape plugin for network centrality analysis, was used to identify crucial genes in the network [22] . Genes with the top 30% highest degree centrality (DC) values were first selected for subnetwork construction using CytoNCA. en, genes with the top 30% highest betweenness centrality (BC) values in the subnetwork were identified as crucial genes and formed the core network.

Network Construction. GO functional enrichment analysis was performed for target genes in three categories: biological process (BP), cellular component (CC), and molecular function (MF) [23] . Both GO functional and KEGG pathway enrichment analysis for target genes were performed using the R package of clusterProfiler [24, 25] . Benjamini-Hochberg correction was performed for multiple testing, and adjusted P value ＜0.05 was set as the threshold. A targetpathway network was then constructed in Cytoscape to visualize the relationships between target genes and KEGG pathways.

Genes. Based on the cutoff criteria, a total of 533 DEGs (including 235 upregulated and 298 downregulated genes) were identified between CRC tissues and normal tissues. e top 10 significantly upregulated and downregulated DEGs are listed in Table 1 .

Using the TCMSP database, 489 compounds were retrieved: 18 in Gegen, 143 in Huangqin, 48 in Huanglian, and 280 in Gancao. After filtering with the criteria of OB ≥ 30% and DL ≥ 0.18, 146 bioactive components from GQD were collected. ese included 4, 36, 14, and 92 from Gegen, Huangqin, Huanglian, and Gancao, respectively. After excluding duplicates, 140 BCIs were selected for further analysis (as shown in Supplementary Table  S1 ).

A total of 240 BCI-related targets were screened out. After intersecting the 240 BCI-related targets with 533 CRC-specific genes, 20 genes were collected as CRC-specific GQD-target genes. After excluding BCIs whose targets were not CRC-specific, 118 effective BCIs were finally used for pharmacological network construction.

e Pharmacological Network. One hundred and eighteen BCIs of GQD together with 20 CRC-specific GQDtarget genes were introduced into Cytoscape to create the pharmacological network ( Figure 2 ). BCIs are displayed as ellipse nodes in the network, and BCIs from different herbs are painted in different colors. BCIs from Gancao, Gegen, Huangqin, and Huanglian are painted in green, brown, purple, and yellow, respectively. Shared BCIs of multiple medicines are painted in red. One hundred and three BCIs with only one target are distributed in two larger circles in the upper half of the figure Evidence-Based Complementary and Alternative Medicine 3 (formononetin), and MOL000422 (kaempferol). Information from 15 BCIs with multiple targets is listed in Table 2 . Twenty CRC-specific GQD-target genes are displayed as Vshaped polygons in blue, including PTGS2, OLR1, NR3C2, HSD3B2, TNFSF15, MMP1, MMP3, MMP9, AKR1C3, CA2, PLAU, IL1B, DUOX2, CCNB1, ABCG2, CXCL11, CXCL10, SPP1, ADH1C, and MAOA. Information from 20 CRCspecific GQD-target genes, including full name, log 2 FC, adjusted P value, and aliases, is shown in Table 3 . e regulation relationships between BCIs and their targets are displayed as lines in figure. As the most important gene, PTGS2 is targeted by 116 BCIs and is emphasized centrally in the upper half of the figure.

A PPI network comprising 446 nodes and 3,518 edges was generated using BisoGenet (Figure 3(a) ). After DC calculation, 130 nodes together with 1,619 edges were selected to form the subnetwork (Figure 3(b) ). Ten target genes including CCNB1, SPP1, MMP9, NR3C2, PLAU, MMP3, PTGS2, CA2, MMP1, and IL1B ranked as the top 30% after DC evaluation and were integrated into the subnetwork, with the degree of 140, 90, 38, 32, 26, 26, 25, 19, 18 , and 17, respectively. After BC calculation, 41 nodes and 379 edges were further selected for core network construction (Figure 3(c) ). Two target genes, CCNB1 and SPP1, gained the top 30% highest BC values and were finally identified as crucial genes. Target Figure 4 represents a GO term, plotted by the number of genes enriched in the term on the horizontal axis. e color of each term represents its adjusted P value. e more red the color of the term, the smaller its adjusted P value. Six KEGG pathways were enriched for target genes, and these are listed in Table 4 . e bubble graph of KEGG pathways is shown in Figure 5 , with gene ratio on the horizontal axis. e size of each bubble indicates the number of genes enriched in each KEGG pathway. e larger the bubble, the greater the number of genes involved in the pathway. As in the GO barplot, the color of each bubble in Figure 5 represents the adjusted P value of each KEGG pathway. e more red the color of the bubble, the smaller its adjusted P value. e target-pathway network is displayed in Figure 6 . Target genes and KEGG pathways are visualized as V-shaped polygons and parallelograms, respectively. e larger the size of the V polygon, the more KEGG pathways the target gene is involved in. Similarly, the larger the size of the parallelogram, the greater the number of target genes the KEGG pathway contains.

Colorectal carcinogenesis is a complex and consecutive progression. It involves a multiscale and systemic framework integrating genetic, proteomic, and metabolic networks from responses to DNA damage, gene mutations, population dynamics, inflammation, and metabolism-immune balance [26] [27] [28] . e evolution of the genomic landscape through novel sequencing techniques has uncovered major clues into the key mechanisms of CRC. Medicines have been designed to target specific genetic keypoints to block the progression of the disease [29] . However, survival of CRC patients remains unsatisfactory due to the complex crosstalk among these alterations. TCM has unique benefits such as being naturally sourced, multitargeted, and a holistic concept. It has advantages in treating complicated diseases, especially those with a poor response to Western medicine alone. In the present study, we used a network pharmacology-based approach to reveal the pharmacological effects of GQD on CRC, which might provide novel therapeutic strategies for better treatment of CRC.

Network pharmacology analysis showed that 20 target genes were regulated by GQD in CRC, including PTGS2, CCNB1, SPP1, PLAU, MAOA, OLR1, NR3C2, HSD3B2, TNFSF15, AKR1C3, CA2, MMP1, MMP3, MMP9, IL1B, DUOX2, ABCG2, CXCL11, CXCL10, and ADH1C. Most of these have been reportedly associated with CRC.

PTGS2 (prostaglandin G/H synthase 2, also known as PGHS-2; COX-2) is one of the most important genes in the pharmacological network. Many studies have demonstrated that CRC is closely related to PTGS2. Studies have found PTGS2 to be overexpressed in CRC tissues [30] , which is consistent with results in the present study (Table 3 ). e elevation of PTGS2 predicts poor prognosis in colon cancer [31] . PTGS2 produces the inflammatory mediator prostaglandin E2 (PGE2), which is suggested to promote the development and progression of CRC [32] [33] [34] . Furthermore, epidemiological evidence indicates that the regular use of aspirin (a PTGS2 inhibitor) reduces the risk of CRC [35] . In the present study, PTGS2 was targeted by as many as 116 BCIs in GQD, indicating it may possess a potentially important PTGS2-related anti-CRC mechanism.

Reduction of NR3C2 (also known as MR) expression was found to be a potential early event involved in CRC progression [36] . Expression of AKR1C3 may be used for the prediction of lymph node metastasis in CRC [37] . High mRNA expression of DUOX2 was significantly associated with better overall survival of CRC patients [38] . ABCG2 was shown to play a potential protective role in CRC by inhibiting the NF-κB signaling pathway to relieve oxidative stress and decrease the inflammatory response [39] . Downregulation of CXCL11 inhibited cell growth and epithelial-mesenchymal transition in CRC [40] . e crucial MMP1, MMP3, and MMP9 genes in the pharmacology network are all matrix metalloproteinase family members. Increasing evidence has demonstrated their oncogenic significance in CRC carcinogenesis [41] [42] [43] .

After PPI network analysis, CCNB1 and SPP1 were identified as crucial genes of the highest degree. Cyclin B1 (CCNB1) is a well-known gene involved in mitosis. It produces a complex with cyclin-dependent kinase 1 (CDK1), which is necessary for proper control of the G2/M transition phase of the cell cycle [44, 45] . Previous study indicated that CCNB1 is overexpressed in CRC tissues, and inhibition of CCNB1 suppressed the proliferation of CRC cells in vitro and tumorigenicity in vivo [46] . SPP1, also known as OPN, has been shown to regulate multiple functions contributing to CRC progression [47] . Upregulation of SPP1 promoted CRC cell proliferation in vitro and tumor growth in vivo [47] . It also promoted metastasis in CRC by activating the EMT pathway and was associated with poor survival outcomes in CRC [48, 49] .

Fifteen BCIs were correlated with multiple target genes in the pharmacological network. Some of these have already been shown to exert anti-CRC properties. Quercetin, a dietary flavonoid, was reported to induce human colon cancer cell apoptosis by inhibiting the NF-κB pathway and inducing apoptosis in KRAS-mutant CRC cells via JNK signaling pathways [50, 51] . Wogonin, a naturally occurring monoflavonoid, induced antiproliferation and G1 arrest via the Wnt/β-catenin signaling pathway in CRC cells [52] . Baicalein was shown to inhibit the proliferation and migration of CRC cells [53] [54] [55] . Oroxylin A was reported to suppress the growth and development of CRC via reprogramming of HIF1α-modulated fatty acid metabolism [56] . ese findings all suggest the promising potential of GQD in the treatment of CRC.

Based on GO enrichment analysis, BP terms enriched by target genes were mainly concentrated in response to various materials and biochemical processes of different substances. Response to inorganic substance (GO: 0071241), nutrient (GO: 0007584), vitamin D (GO: 0033280), and metal ion (GO: 0010038) are all pivotal in cellular metabolism. Biosynthetic and metabolic processes of prostaglandins and prostanoids (including GO:0006693, GO:0046457, GO:0006692, and GO: 0001516) are also important in colorectal tumorigenesis, since prostaglandins and prostanoids have been implicated in various pathological processes in CRC [57] [58] [59] [60] . Some enriched MF terms are associated with inflammation, such as GO:0005125: cytokine activity, GO:0005126: cytokine receptor binding, GO:0042379: chemokine receptor binding, GO:0008009: chemokine activity, and GO:0045236: CXCR chemokine receptor binding. Inflammation, a hallmark of hsa04620 hsa04657 hsa01523 hsa04668 hsa04064 hsa04913 CRC, has been discussed in many publications. Cytokines such as interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10 (IL-10), and interleukin-17 (IL-17) have been shown to be involved in the development of CRC [61] [62] [63] [64] . Chemokines direct leukocyte recruitment and migration under inflammatory conditions [65, 66] . e expression of many chemokines, such as CCL2-4, CXCL1, CXCL5, and CXCL8-10, was reportedly elevated in the CRC microenvironment compared to normal tissues [67, 68] .

KEGG enrichment analysis showed that the pharmacological effects of GQD on CRC are closely related to wellknown tumor-associated pathways, including the IL-17, tumor necrosis factor (TNF), Toll-like receptor, and NF-κB signaling pathways. Numerous studies have highlighted the important role of the IL-17 signaling pathway in the tumorigenesis, angiogenesis, and metastasis of CRC. Interleukin-17 (IL-17), a proinflammatory cytokine, was significantly upregulated in CRC tissues [69] . IL-17 can promote CRC tumorigenesis by stimulating the production and recruitment of myeloid-derived suppressor cells (MDSCs) [70] . It promotes angiogenesis via stimulating VEGF production in CRC cells [71] . Additionally, IL-17 stimulates the production of PGE2, MMP9, and MMP13, which are involved in the migration of CRC cells [72] [73] [74] .

TNF-α is one of the most important cell signaling proteins involved in cell growth, differentiation, and apoptosis [75, 76] . It plays a pivotal role in proliferation, angiogenesis, and metastasis in CRC [77] . Serum TNF-α was demonstrated to contribute to CRC susceptibility, and anti-TNF therapy has been considered for CRC treatment [78] .

A large body of data demonstrates an important relationship between the Toll-like receptor (TLR) signaling pathway and CRC. e TLR signaling pathway exerts a fundamental role in colorectal epithelium hemostasis and in activating the innate and adaptive immune responses [79] . Activation of the TLR signaling pathway leads to activation of downstream signaling pathways and recruitment of transcription factors such as NF-κB, interferon regulatory factor-(IRF-) 3, AP-1, PI3K/Akt kinases, the mitogen-activated protein kinase (MAPK), and the subsequent generation of cytokines and chemokines [80, 81] . e TLR2 and TLR4 agonists HMGB1 and S100A9 have been proposed as potential biomarkers for CRC [82, 83] . Several TLR-based therapeutic agents have been developed for targeting this pathway and are currently used in clinical trials in patients with CRC [84] [85] [86] .

e NF-κB signaling pathway is a key regulator of CRC cell proliferation, apoptosis, inflammation, angiogenesis metastasis, and drug resistance [87] . Constitutive NF-κB activation was observed in CRC cell lines and human CRCs [88, 89] . It promotes the proliferation of cancer cells and rescues cancer cells from cell death [90] . Studies suggest that inhibition of the NF-κB pathway can sensitize CRC cells to chemotherapy and radiotherapy, providing more effective strategies for cancer treatment [91, 92] . ese findings suggest that the molecular mechanisms of GQD against CRC are closely related to these key target genes, biochemical processes, and important signaling pathways. However, detailed experiments are still needed to confirm these findings.

In conclusion, this study is the first to reveal the pharmacological effects of GQD against CRC via network pharmacology analysis. A total of 118 BCIs from GQD were identified, and 20 corresponding genes including PTGS2, NR3C2, CXCL11, CCNB1, and SPP1 were demonstrated to be key targets for GQD in CRC. GO functional and KEGG pathway enrichment analysis indicated that the molecular mechanisms of GQD in CRC were closely related to important biochemical processes and signaling pathways, such as biosynthetic and metabolic processes of prostaglandins and prostanoids, cytokine and chemokine activities, the IL-17 signaling pathway, the TNF signaling pathway, the Tolllike receptor signaling pathway, and the NF-κB signaling pathway.

e study provides a research basis for further studies of GQD in the treatment of CRC.

e data used to support the findings of this study are included within the article and supplementary information files.

e authors declare that there are no conflicts of interest regarding the publication of this paper.

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Acknowledgments e authors greatly appreciate the financial support from Harbin Medical University, which ensured the accomplishment of the work.

Supplementary