Ze-Yun Li, Zhi-Qiang Chen, Cheng-Xin Liu,Yang Cao

1.The First Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510405, China

2.Department of Oncology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China

Keywords:

ABSTRACT

1. Introduction

Ranunculus ternatus Thunb is an annual herb belonging to the Ranunculaceae family. It is mainly distributed in the Guangxi、Henan and Anhui region of China. Ranunculi Ternati Radix, the root of Ranunculus ternatus Thunb, is a common Chinese medicine, which is accepted in the Chinese pharmacopoeia since 1977. In terms of traditional Chinese medicine (TCM) theory, Ranunculi Ternati Radix has the function of resolving phlegm, resolving toxin and swelling. In recent decades, Ranunculi Ternati Radix has become a research hotspot due to its anticancer effect. Ranunculi Ternati Radix has been used in traditional Chinese medicine for the treatment of various cancer, tuberculosis, Chronic Hepatitis B, faucitis, neck scrofula, etc[1]. Previous investigations of Ranunculi Ternati Radix have revealed the presence of lactones, biflavonoids, triterpenoids, glycosides, alkaloids[2]. In recent years, several studies have demonstrated that the extract in Ranunculi Ternati Radix has clearly exhibited antitumor and antituberculosis activities. In addition, the extracts of the plant strengthens the nonspecial immune function of the normal mice[3] and exerts significant hepatoprotective effect[4]. Although well-practiced in clinical medicine, the mechanism by which these effects are exerted has yet to be fully elucidated.

TCM is a comprehensive medicinal system that plays an important role in health maintenance for Asian people. It offers bright prospects for the prevention and treatment of complex diseases such as cancer. Herbal medicine, is multicomponent and multitarget agents, achieving the therapeutic effectiveness through regulation of the molecular network of body systems utilizing its active components[5]. However, because of the complex chemical composition, it's difficult to conduct a comprehensive study of herbal medicine in the body to reveal the TCM mechanism. As an emerging discipline, network pharmacology provides new ideas and perspectives to solve the complex pharmacological problems and to discover the molecular mechanisms related to the therapeutic efficacy of TCM[6-8]. Thus, we select the network pharmacology approach to investigate the mechanisms of how Ranunculi Ternati Radix exerts the therapeutic effects on different diseases to clarify its medical value. The flowchart of the experimental procedures of our study was shown in Figure 1.

Figure 1: The flowchart of the network pharmacological study of Ranunculi Ternati Radix

2. Materials and Methods

2.1 Chemical database construction of Ranunculi Ternati Radix

To collect the compounds of Ranunculi Ternati Radix, we used the Traditional Chinese Medicine Systems Pharmacology Database[9](TCMSP, http://lsp.nwu.edu.cn/tcmsp.php, updated on February 27, 2019), a unique system pharmacology platform designed for Chinese herbal medicines. TCMSP includes information of all 500 Chinese herbal medicines with a total of 30,069 ingredients. In this study, the compounds meeting the criteria of oral bioavailability[10] (OB) ≥≥ 30% and drug-likeness[11] (DL) ≥≥ 0.18 are selected as potential active compounds and used for further research.

2.2 Identification of Associated Proteins and Gene Names

The protein targets of the compounds were retrieved from the TCMSP database[12]. Because of the nonstandard naming, we input the protein names with the species limited as “Homo sapiens” to the UniProt Knowledgebase (UniProtKB, https://www.uniprot.org/) to receive their official symbol. Because the active compounds without corresponding targets or the targets of the compounds without corresponding gene, we decided to remove these compounds after deleting the replicate data. After these operations, the main active substance of Ranunculi Ternati Radix and proteins information of compound targets were finally obtained.

2.3 Protein-Protein Interaction Construction

Protein-Protein interaction (PPI) data come from String[13](http://string-db.org/, ver. 10) with the species limited to “Homo sapiens”. The graphical interactions in this network was visualized using Cytoscape software[14] (version 3.6.0). In a network, degree is the basic network topological properties which was utilized to evaluate the characterization of different drug treatments from a network perspective. Degree stands for the number of neighbors of a node in undirected graph[15]. Nodes with higher degree can be considered as the key nodes, which would be highly influential in the whole networks.

2.4 Gene Ontology (GO) and KEGG Pathway Enrichment Analysis

In this study, to detect and analyze the specific biological properties of the target genes, the Gene Ontology biological process (GOBP) were introduced. We used the Database for Annotation, Visualization and Integrated Discovery[16] (DAVID, http://david.abcc.ncifcrf.gov/, ver. 6.7) for GOBP analysis. In GO enrichment analysis, the bubble chart was plotted by using the OmicShare tools (http://www.omicshare.com/tools), a free online platform for data analysis. DAVID was also applied to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Threshold count ≥≥ 6 and EASE scores ≤≤0.1 were selected in functional annotation clustering.

2.5 “Compound-target-pathway” network construction

Based on the pathway enrichment analysis, to further elucidate the target pathway mechanisms, we picking out the closely linked pathways related with Ranunculi Ternati Radix through consulting a large number of literature material. Then, a “compound-targetpathway” network was constructed on the basis of the significant signaling pathways and their corresponding targets and compound. The graphical interactions in this network was visualized using Cytoscape software[14] (version 3.6.0).

3. Results

3.1 Identification of Active Compounds

A total of 79 compounds of Ranunculi Ternati Radix were retrieved from the TCMSP database, then we employed OB and DL to screen out the potential active components. As a result, a total of 10 active compounds pass through the criteria of OB ≥ 30% and DL ≥≥0.18. The majority of these potential bioactive compounds with favorable OB values are phytosterol, such as beta-sitosterol、Stigmasterol、campesterol、CLR. The details of the 10 compounds are presented in Table 1.

Table 1 Active ingredients and parameters of Ranunculi Ternati Radix

3.2 Identification of Targets

For the 10 compounds, 91 targets were obtained from the TCMSP, and 52 targets were finally obtained after removal of the duplicates. Input the Protein targets with the species limited as “Homo sapiens” to UniProtKB, and 52 predicted target genes were obtained. The details of the 52 Potential targets are presented in Table 2.

3.3 Protein-Protein Interaction network

In total, there are 185 interactions between 52 proteins, the interactions among which are shown in Figure 2. In this network, Relatively red nodes (JUN、MAOA、SLC6A4、CASP3、HSP90AA1、ADRA1B、ADRA2A、MAOB、HTR2A、CHRM1、CHRM2、PTGS2) have higher degrees. The number of edges of each node is quite large (14 in JUN、MAOA and SLC6A4, 13 in CASP3, 12 in HSP90AA1、ADRA1B and ADRA2A, 11 in MAOB、HTR2A、CHRM1、CHRM2 and PTGS2). This demonstrates that these proteins are closely related to other proteins in the network, suggesting that these proteins may play an important role in Ranunculi Ternati Radix.

Figure 2: Protein-Protein Interaction network of Ranunculi Ternati Radix. The circle nodes represent proteins and the node size is proportional to their degree. The edges represent the interaction between them.

3.4 GO enrichment analysis

To clarify the multiple mechanisms of Ranunculi Ternati Radix from a systematic level, we performed a GO enrichment analysis for the biological process of the 52 protein targets. Figure 3 listed the top 20 significantly enriched GO terms of these targets. The results suggested that the major biological processes involved response to estradiol, positive regulation of vasoconstriction, positive regulationof ERK1 and ERK2 cascade, and so on. These annotations are all related with the processes of different molecular signal transmissions, indicating that Ranunculi Ternati Radix may intervene in these pathological progresses.

Table 2 The information of the Potential targets of Ranunculi Ternati Radix

Figure 3: The GO pathway enrichment analysis of potential targets in Ranunculi Ternati Radix

3.5 KEGG Pathway Enrichment Analysis

Input all of the genes into DAVID to do pathway enrichment analysis. As shown in Table 3, Neuroactive ligand-receptor interaction should be the crucial pathway and is regulated by 17 genes (OPRM1, DRD1, GABRA2, GABRA1, GABRA3, GABRA5, ADRB2, CHRM4, CHRM3, CHRM2, CHRM1, ADRA1B, ADRA2A, ADRA1A, CHRNA7, CHRNA2 and HTR2A), which is the most. The second-most frequent associations were Pathways in cancer includes 13 genes, followed by Calcium signaling pathway includes 12 genes, Morphine addiction includes 9 genes, and so on. The details are described in Table S5. Through our research, we can find that the aforementioned pathway may be the focus of the future study of the effect of Ranunculi Ternati Radix and its compounds.

3.6 “Compound-Target-Pathway” Network

According to Pathway Enrichment Analysis and a large number of literature material, we picking out 9 closely linked pathways related with Ranunculi Ternati Radix. There are three pathways related to tumor diseases, including pathways in cancer, colorectal cancer and PI3K Akt signaling pathway; three pathways related to other diseases, including Hepatitis B, Non-alcoholic fatty liver disease and tuberculosis, and two pathways related to endocrine, including estrogen signaling pathway and thyroid hormone signaling pathway. In addition, cAMP signaling pathway, a signal transduction pathway. These 9 pathways are mainly involved in tumor cell proliferation, metabolism, nerve conduction and other important biological processes. As shown in Figure 4, “compound-target-pathway” network was constructed on the basis of the significant signaling pathways and their corresponding targets and compound. As we can see, the number of nodes of beta-sitosterol and Stigmasterol is the largest, which indicates the two compounds play a major role in thetreatment. In addition, we can find that these pathways have several of the same genes. For instance, Pathways in cancer、PI3K-Akt signaling pathway and Colorectal cancer have PIK3CG, HSP90AA1, BAX and BCL2, which are closely associated with cancer. These may be the key targets for the anti-tumor effect of Ranunculi Ternati Radix. From this we learn that different signal pathways are closely related and have great complexity. This fact implied that the therapeutic effects of Ranunculi Ternati Radix may be based on the synergistic interactions of different components by different pathways, which virtually showed the properties of multicomponent, multitarget, and multidisease of the herbal medicine.

Table 3 The KEGG pathway enrichment analysis of potential targets in Ranunculi Ternati Radix

Figure 4: “compound-target-pathway” network of Ranunculi Ternati Radix. The yellow squares represent significant pathways, the green circles represent potential targets, brown octagon represents Ranunculi Ternati Radix and the fuchsia triangles represent compounds. The edges represent the interaction between them.

4. Discussion

In recent decades, Ranunculi Ternati Radix has become a research hotspot due to its anticancer effect. In this study, we know the majorities of bioactive compounds of Ranunculi Ternati Radix are phytosterol. Phytosterols are a kind of plant steroid substances whose structures are similar to that of zoosterol, but their branched car- bon chains are different. Phytosterol has a variety of biological activities. Poriferasterol monoglucoside_qt （CAS Number:19716-26-8）belongs to steroidal saponin, also known as stigmasterol-3-O-β-D-glucopyranoside. Zhang and colleagues showed that stigmasterol-3-O-β-D-glucopyranoside has an certain inhibitory effect on human hepatocarcinoma cells[17]. Βeta-sitosterol has also been considered as a potent anticancer agent with anticancer properties against breast cancer, prostate cancer, colon cancer, lung cancer, stomach cancer, ovarian cancer, and leukemia[18; 19]. Zhang found that β- sitosterol exerted antiproliferative effects and induced HepG2 cells apoptosis via mitochondrial pathway and membrane death receptor pathway[20]. In this work, we know BAX、BCL2 and HSP90AA1 may be the key targets for the antitumor effect of Ranunculi Ternati Radix. Bax and BCL2 are both apoptosis related proteins. β-Sitosterol could induce apoptotic cell death by up-regulation the expression levels of Bax, downregulation the expression levels of BCL2, and this is one of the mechanisms by which β-Sitosterol possesses anti-proliferation and apoptosis inductive effect in lung cancer cell[21]. Heat shock proteins (HSPs) were significantly associated with cancers. It has been reported that HSP90 is overexpression in many solid tumors and haematological malignancies, and it plays an important role on tumor invasion, metastasis[22]. Many studies have demonstrated that the overexpression of HSPs is associated with poor prognosis[23; 24]. So it could act as a promising target for prognostic prediction. For instance, the detection of HSP90α is of high diagnostic value for breast cance[25]. GO enrichment analysis suggested that the major biological processes involved response to estradiol, which indicates that Ranunculi Ternati Radix may intervene in these pathological progresses. Nieminen et al have observed through experiments that phytosterol had different enhancement effects on plasma estradiol and TH levels in female polecat[26]. Modern pharmacological studies shows that estrogen and estrogen signaling pathway play an important role in the development of hormone-dependent tumors such as breast endometrial cancer, prostate cancer, ovarian cancer, thyroid cancer[27-29]. As shown in Figure 4, PI3K-Akt signaling pathway should be the crucial pathway. Aberrant PI3K-AKT pathway activation is found in many types of human cancers. It is the most significant pathways that resulting in the development and progression of these malignant tumors[30-32]. It has been proved that the PI3K pathway is often dysregulated in endometrial and ovarian cancer and other gynaecological cancers[33; 34]. Patients with advanced gynaecological malignancy, refractory to standard therapies, achieved meaningful clinical efficacy from PI3K pathway inhibitors[35]. In additon, the PI3K-AKT pathway appear to have a complex role in Hepatocellular carcinoma (HCC)[38]. Inhibiting PI3K/AKT signaling markedly suppresses HCC cell proliferation[36]. Ranunculus ternatus Thunb is also widely used in the clinic to treat Other diseases, such as tuberculosis、Chronic Hepatitis B, and it can also stimulate the immunologic function.

Pathway Enrichment Analysis suggested that Tuberculosis and cAMP signaling pathway should be the crucial pathway for the treatment of diseases. Chinese traditional herbal medicine has been a good resource for new bioactive chemical entities. The identification of potential therapeutic agents to prevent and treat multiple drugresistant tuberculosis infection by natural drug screening is important. Ranunculus ternatus Thunb is a traditional Chinese antituberculosis medicine with an undefined mechanism[37]. Feng et al. extracted five new heterocyclic compounds from the roots of the root of Ranunculus ternatus Thunb. The structures of these compounds are similar to some anti-tuberculosis drugs[38]. Zhou et al. found Radix Ranuncoli Ternati may increase the expression of GLS and enhance the body's killing effect on pathogenic bacteria to achieve the therapeutic effect[39]. GO enrichment analysis suggested that the major biological processes involved positive regulation of ERK1 and ERK2 cascade. Other research has found that macrophages infected with mycobacterium tuberculosis that carries cAMP-induced gene Rv1265 increased the production of pro-inflammatory cytokines IL-1β, IL-6 and IL-12 P40 through activation of ERKl/2 pathway[40]. The results of KEGG pathway enrichment analysis demonstrate that Hepatitis B is one of the main enrichment pathways for the candidate targets from active components of Ranunculi Ternati Radix. Ranunculi Ternati Radix also has been used as an herbal drug for treatment of Hepatitis B in China. Ganoderma lucidum and Ranunculi Ternati Radix were used as monarch medicine to make "Lingmao Formula", which was used as hospital preparation to treat Chronic Hepatitis B and proved to be very effective[41]. Crude polysaccharides from the root of Ranunculus ternatus significantly inhibited the secretion of HBsAg and HBeAg in HepG2.2.15 cells[4]. Further research found that the Lingmao Formula Suppresses the Replication of Hepatitis B Virus by Enhancing Innate Immune Response[42].

The active part of Radix Ranunculi Ternati can stimulate the immunologic function of immune suppression mouse models. The extracts of Radix Ranunculi Ternati were found to be capable of stimulating the expression of IFN-γ, IL-12 and GLS mRNA and downregulating the expression of IL-4 and IL-10[43]. Experiments also find Radix Ranunculi have the effect of immune excitation. It can improve the phagocytic function of macrophages in immunosuppressive mice, and increase the number of T lymphocytes in peripheral blood obviously[44].

Phytosterols have received much attention from the medical and biochemical community in recent years because of their cholesterollowering properties[45]. A large number of studies have shown that phytosterols can regulate blood lipid and lower total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C)[46; 47]. Phytsoterols decrease the absorption of lipid from diet by decreasing the intestinal bile acid and increasing the exertion of lipid into feces[48]. Zhou et al. discovered that Stigmasterol and β-sitosterol could effectively improve the situation of non-alcoholic fatty liver disease through relieving steatosis and oxidative stress[49]. The results of KEGG pathway enrichment analysis demonstrate that Non-alcoholic fatty liver disease (NAFLD) is one of the main enrichment pathways for the candidate targets from active components of Ranunculi Ternati Radix. Therefore, we predict that Ranunculi Ternati Radix had the potential to treat non-alcoholic fatty liver disease. These also provide new ideas and new directions for the research and development of hypolipidemic drugs.

In this study, the complex relationship between the multicomponent, multi-target and a variety of diseases of Ranunculi Ternati Radix was studied by using the method of network pharmacology. The results of the study preliminarily verified the main target and pathways of Ranunculi Ternati Radix in the treatment of cancer and other different diseases, and laid a foundation for further study on its mechanism.