Pharmacology of Active Components of Glechomae Herba in the Treatment of Bladder Cancer

Chunfeng WU, Damei NONG, Huajiang ZUO

1. Liuzhou People’s Hospital Affiliated to Guangxi Medical University, Liuzhou 545006, China; 2. Guangxi University of Science and Technology, Liuzhou 545006, China

Abstract [Objectives] To explore the action mechanism of Glechomae Herba in the treatment of bladder cancer through network pharmacology. [Methods] TCMSP database retrieval and literature text mining were used to find out the main chemical active components and their targets, and the "component-target" network map was constructed by Cytoscape 3.7.0 software. The CTD database was used to select targets related to bladder cancer, and the interaction between the targets of the main active components of Glechomae Herba and the targets related to bladder cancer was taken. A total of 87 targets were obtained, then imported into the STRING database to obtain PPI network, and the topological analysis of the network was conducted to find out the key targets. The key targets were introduced into the DAVID database, and the biological process and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of the key targets in the treatment of bladder cancer were obtained. [Results] Active compound components interfere with the occurrence and development of bladder cancer possibly through bladder cancer signaling pathway, proteoglycan in cancer signaling pathway, HIF-1 signaling pathway, tumor necrosis factor signaling pathway, PI3K-Akt signaling pathway, toll-like receptor signaling pathway, node-like receptor signaling pathway, and MAPK signaling pathway, etc. [Conclusions] The pharmacological mechanism of Glechomae Herba in the treatment of bladder cancer may be realized through multiple components, multiple targets and multiple pathways. In conclusion, Glechomae Herba has certain medicinal value.

Key words Glechomae Herba, Bladder Cancer, Network pharmacology

1 Introduction

In 2014, the incidence of bladder cancer in China ranked 13thin the incidence of malignant tumors, and the mortality rate ranked 12thin the deaths of malignant tumors, and both incidence and mortality of bladder cancer in men were much higher than that in women[1]. The pathogenesis of bladder cancer is complicated, and it is clinically divided into muscle-invasive and non-muscle-invasive bladder cancer[2]. In recent years, the surgical techniques are constantly advancing. At present, the bladder cancer is mainly treated by surgical operation, supplemented by chemotherapy drugs after surgery, which significantly improves the prognosis of patients with early bladder cancer, but the recurrence rate and metastasis rate of mid-term patients still exceed 60%[3]. Therefore, exploring new treatment methods and improving the treatment effect of bladder cancer will have great clinical significance.

Prevention and postoperative conditioning of patients are generally inseparable from traditional Chinese medicines, and traditional Chinese medicine have always been considered an important source of drug discovery. Glechomae Herba, as a kind of Chinese herbal medicine, is used in the prevention and treatment of human diseases, but its related reports are few. Glechomae Herba is the dry above-ground part ofGlechomalongituba(Nakai) Kupr., a perennial herb in the Lamiaceae family. It belongs to liver, kidney, and bladder medians. Clinically, it is mainly used for facilitating urination, treating bladder stones, kidney stones, ureteral stones,etc.[4-6]. Modern pharmacological studies have proved that Glechomae Herba has the diuretic and choleretic effects, can regulate blood lipid, dissolve stone, and has hypoglycemic, anti-inflammatory, antibacterial, anti-cancer effects[7]. The main chemical components of Glechomae Herba are flavonoids, glycosides, and terpenoids. In this study, we used the method of network pharmacology to make a systematic evaluation of the chemical components of Glechomae Herba and its mechanism of treatment of bladder cancer.

2 Materials and methods

2.1 Screening of active components, related diseases and targetsFrom the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), we obtained 44 chemical components of Glechomae Herba. Taking oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18 as the conditions for screening the active components of Glechomae Herba, and combining relevant literature, we obtained a total of 12 active components. We imported the target proteins of the compounds into String and UniProt database (https://www.uniprot.org/) to search to eliminate duplicate and non-human targets, and convert them into the gene symbols of target proteins of active compounds. Finally, we obtained 213 corresponding 213 targets and 301 related diseases, to provide basic data for constructing a "component-target" network diagram.

2.2 Obtaining bladder cancer related targetsIn the CTD search box of the database, we entered "bladder cancer" to search, and obtained 28 405 bladder cancer-related genes. We selected the first 300 genes according to the "Inference Score" order as targets for subsequent analysis.

2.3 Screening common targets and key targetsWe imported target genes and disease target genes related to the chemical components of the drug into the analysis platform of Veneney 2.1.0, and plotted the Venn diagram to obtain the potential targets of the active components of Glechomae Herba in the treatment of bladder cancer. We imported the screened common targets into the String platform (http://string-db.org), set the Organism to "homo aspiens", and set the minimum interaction score to 0.4 to obtain the Protein-Protein Interaction (PPI) network model of target interaction. Since the STRING database cannot visually show the degree change of the target protein,we saved the protein interaction information of STRING in TSV format and imported into Cytoscape 3.7.0 software for visual analysis to screen out key targets.

2.4 GO analysis and KEGG pathway analysisWe imported the key targets screened in Section2.3into the DAVID (https://david.ncifcrf.gov/) database for GO analysis and KEGG analysis.

3 Results and analysis

3.1 Screening and collection of active components of Glechomae HerbaThrough the TCMSP platform database withOB≥30%,DL≥0.18 as the conditions, we obtained a total of 7 active components of Glechomae Herba that meet the conditions, and checked relevant literature to include components with larger content in Glechomae Herba, and determined a total of 12 compounds as the main active components of Glechomae Herba for subsequent analysis (Table 1).

Table 1 Active components of Glechomae Herba

3.2 "Component-Target Network" of traditional Chinese medicineWe imported the components and targets into Cytoscape software to construct a component-target network with 237 nodes and 427 edges (Fig.1). The top five of Degree were quercetin, kaempferol, luteolin, ursolic acid, and rosmarinic acid; and the top six targets for Degree were PTGS2 (prostaglandin G/H synthetase 2), CASP3 (cysteine-aspartic acid protease 3), PTGS1 (Prostaglandin G/H synthase 1), PPARG (peroxisome proliferator activated receptor γ), CD40LG (tumor necrosis factor), and HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A Member 1).

3.3 Collection of bladder cancer-related disease targets and treatment targets of Glechomae Herba for bladder cancerIn the CTD search box of the database, we entered "bladder cancer" to search, and obtained 28 405 bladder targets. According to the default score value of the database system, we selected the first 300 related genes. Through the Venny 2.1.0 database platform, we plotted a Venn diagram of the traditional Chinese medicine and disease targets, and collected a total of 87 targets. It is believed that these targets are the potential targets for the treatment of bladder cancer by the main chemical components of Glechomae Herba (Fig.2).

3.4 Interaction between targets and proteinsIn order to better explore the action mechanism of action of the active components in Glechomae Herba against bladder cancer, we input the targets of Glechomae Herba for the treatment of bladder cancer into the String platform to obtain the target network interaction diagram. Through visualized analysis with the aid of Cytoscape 3.7.0 software (Fig.3), we observed the size and color of the ellipse, from pink ellipse to blue ellipse, the shape is getting bigger and bigger, and the color is getting darker, indicating the Degree value is higher. We selected the lowest interaction threshold "highest confidence" to be 0.4, and the number of interaction edges was 1 758. Through calculation, the average Degree value was 40.4, the average shortest path length was about 1.541, and the average betweenness was about 0.006 36, 43 targets exceeded the average degree, including TP53, AKT1, CASP3, JUN , VEGFA, IL6, MYC, EGFR,etc.These targets have a high degree value and can be considered as key targets, they have a relatively large relationship with the disease, and play an important role in the treatment of bladder cancer (Table 2).

Note: blue diamonds represent the active components of traditional Chinese medicine, and the pink ellipses represent the targets.

Fig.2 Venny diagram for active component targets of Glechomae Herba and bladder cancer-related disease targets

Table 2 Key targets and their topological characteristics

Fig.3 Protein interaction of potential therapeutic targets

3.5 Related pathway information and interpretation analysisWe imported the key targets into the David 6.8 database for GO analysis and KEGG analysis, and obtained 363 biological processes (BP) and 94 signal pathways. Taking the correctedPvalue of Bonferroni <0.01, we obtained 187 biological processes, including drug reaction, positive regulation of RNA polymerase II promoter transcription, positive regulation of transcription, DNA templating, negative regulation of apoptosis process, aging, positive regulation of smooth muscle cells Proliferation, positive regulation of nitric oxide biosynthesis process, response to estradiol, cell response to organic ring compounds,etc., which are closely related to bladder cancer and play an important role in the treatment of bladder cancer by Glechomae Herba. These key therapeutic targets involve a variety of signal pathways. TakingP<0.01, we obtained 82 signal pathways, cancer pathways, hepatitis B, pancreatic cancer, bladder cancer, Chagas disease (American trypanosomiasis), HIF-1 signaling pathway, proteoglycans in cancer, tumor necrosis factor signaling pathway, PI3K-Akt signaling pathway, toxoplasma Body diseases, colorectal cancer, in-flammatory bowel disease (IBD), leishmaniasis, toll-like receptor signaling pathway, prostate cancer,etc.The smaller thePvalue, the higher the correlation with bladder cancer, and the more relevant genes related to the enrichment (Fig.4-5).

Note: only the first 20 biological processes according to the P value were shown.

Note: only the first 20 KEGGs according to the P value were shown.

4 Discussion

Bladder cancer is the most common malignant tumor in urology, and its incidence rate and mortality are increasing year by year, and the trend is not optimistic. There are many methods for the treatment of bladder cancer, such as surgery, chemotherapy, and radiotherapy, but the effect is still not satisfactory. Thus, it is necessary to explore new treatment methods. The rich resources of Chinese medicine and traditional Chinese medicines provide a broad range of ideas and space for the research on the prevention and treatment of bladder cancer. New and effective methods are needed to treat bladder cancer.

In this study, using the method of network pharmacology, we conducted a "component-target" network analysis and found that the key components may be quercetin, kaempferol, luteolin, acacetin, ursolic acid,etc.The action mechanism of Glechomae Herba in treating the bladder cancer is possibly achieved through these main chemical components. Studies have shown that the mechanism of quercetin may be through inhibiting tumor cell signal transduction, inhibiting cell cycle arrest, and inducing apoptosis of bladder cancer cells; the main mechanism of inhibiting cell proliferation may be achieved through up-regulating the expression of tumor suppressor genes such asp53[8]. Besides, quercetin can inhibit tumor angiogenesis and metastasis to exert its anti-tumor effect. Acacetin is a natural flavonoid compound and it is widely found in nature. Acacetin inhibits the expression of related receptors and transcription factors, inhibits the secretion of carcinogenic metabolites, inhibits tumor cell proliferation, promotes tumor cell apoptosis, regulates signal pathways, resists tumor invasion and migration, prevents the expression of VGEF in cancer cells and inhibits angiogenesisinvivoandinvitro[9]. Kaempferol has physiological activities such as anti-inflammatory, antibacterial, anti-cancer effects. Luteolin has multi-target anti-tumor ability and is a potential anti-tumor drug. According to the literature[10], luteolin acts on bladder cancer cells BIU-87 culturedinvitro, and can effectively inhibit the proliferation of BIU-87 cells, block the cell cycle, and induce cell apoptosis. Ursolic acid can significantly inhibit the proliferation and induce apoptosis of human bladder cancer 5637 culturedinvitro, but the exact action mechanism is still not clear. In 1990, Japan listed it as one of the most promising anti-cancer drugs in the 21stcentury. The results of variousinvitroexperiments have shown that ursolic acid can significantly inhibit the growth and reproduction of a variety of human tumor cells[11].

From the PPI data network, according to the Degree size, average shortest path length, and betweenness centrality, it could be determined that TP53, AKT1, CASP3, JUN, VEGFA, IL6, MYC, EGFR, STAT3, CCND1, MAPK8, ESR1, EGF,etc.are key targets and these targets are involved in multiple signaling pathways. TP53 is the main driving gene of most cancers and a common mutation gene in human cancers. Large-scale whole-genome screening of human tumor samples confirmed the important role of TP53 in tumor suppression[12]. JUN is widely involved in cell regulation of cell life processes. The overexpression of MYC is an important feature of human cancer. It is believed that MYC oncogene releases the regulation of the expression of a large number of specific target proteins through transcription factors, accordingly exerting the function of treating bladder cancer. Studies have shown[13]that VEGF and TGF-β detected by ELISA in tumor tissues of patients with bladder cancer are significantly higher than those of normal bladder tissues. Matrix metalloproteinases (MMPs) can decompose extracellular matrix and play an important role in promoting tumor infiltration and metastasis. MMP-2 of MMPs participates in the invasion and metastasis of bladder cancer[14], and can be used as a detection indicator for clinical diagnosis and prognosis. For high-risk patients who are prone to metastasis, early treatment can improve their survival rate. MMP-9 can also be used as a crucial point for follow-up diagnosis and testing.

Through key targets, we obtained bladder cancer related signal pathways, including cancer pathways, hepatitis B, pancreatic cancer, bladder cancer, Chagas disease (American trypanosomiasis), HIF-1 signaling pathway, proteoglycans in cancer, tumor necrosis factor signaling pathway, PI3K-Akt signaling pathway, toxoplasma diseases, colon and colorectal cancer, inflammatory bowel disease (IBD), leishmaniasis, toll-like receptor signaling pathway, prostate cancer signaling pathway,etc.According to the literature[15], baicalein induces bladder cancer cell apoptosis by inhibiting the PI3K/AKT/mTOR pathway, and it can be inferred that the PI3K-Akt signaling pathway related to bladder cancer may also have this function; the toll-like receptors (TLRs) of TLRs signaling pathway are type I transmembrane protein receptors, they are widely distributed on the surface of immune cells and epithelial cells. The TLRs/NF-κB signaling pathway in the signal transduction pathway of TRLs-mediated immunity plays an important role in immune regulation. TNF-α in the tumor necrosis factor signaling pathway induces synovial cells to produce IL2, IL4, IL6 and other cytokines and the expression of matrix metalloproteins (MMPs).

Network pharmacology is a method of traditional Chinese medicine research. Using this method, we determined the potential active components of Glechomae Herba and elaborated the active components and target mechanism. It is expected to provide important reference value for the treatment of the bladder cancer by Glechomae Herba.