Yuan-Yuan Ding, Dong-Hua Zhang, Rong-Sheng Zhang, Li-Yuan Zhang, Cheng-Hao Guo, Xuan Wang

Affiliated 81st Hospital of University of Chinese Medicine,Nanjing 210002,China

Keywords:Network pharmacology Aidi injection Hepatocellular carcinoma Mechanism of action

ABSTRACT Objective: To explore the active ingredients and potential mechanism of Aidi injection in the treatment of hepatocellular carcinoma by network pharmacology. Methods: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicines Integrated Database (TCMID) were used to screen the active ingredients of four traditional Chinese medicines of Renshen, Huangqi, Ciwujia, and Banmao and corresponding potential targets. Screening of hepatocellular carcinoma-related targets through the Online Mendelian Inheritance in Man (OMIM) and GeneCards Suite (The Human Gene Database) database platforms. The drug and disease targets are merged to obtain the intersection, and the information is imported into Cytoscape 3.7.2 to construct a network diagram of the active ingredients of Aidi injection and related targets of hepatocellular carcinoma, and the topology analysis is performed. A protein-protein interaction (PPI) network was constructed and analyzed using the STRING online analysis platform. Uses the Database for Annotation, Visualization and Integrated Discovery (DAVID) to perform GO function enrichment analysis of targets and enrichment of KEGG pathways analysis. Results: A total of 33 potential active ingredients were screened from Aidi injection for treating hepatocellular carcinoma, including quercetin, kaempferol, beta-sitosterol, isorhamnetin and other important active ingredients. There are 106 potential targets for active ingredient action, 6,677 diseaserelated targets, and 89 drug-disease common targets. Through the network diagram, it was found that the highest degree of target is PTGS1.In the PPI graph, a total of 87 nodes. Among them, the higher degree values include IL6, CASP3, VEGFA, MAPK8, JUN, EGFR, MYC, PTGS2 and FOS.A total of 60 related signal pathways were obtained by GO enrichment analysis. It mainly involves biological processes such as inhibiting abnormal proliferation and differentiation of hepatocellular carcinoma cells, inhibiting angiogenesis of hepatocellular carcinoma, regulating cell cycle and promoting apoptosis. KEGG pathway enrichment analysis screened a total of eight significantly different signal pathways. Among them, P53, VEGF, MAPK, Toll-like receptor, ErbB signaling pathways play an important role in treatment. Conclusion: This study initially revealed the potential mechanism of multi-component, multi-target and multi-pathway treatment of Aidi injection for hepatocellular carcinoma, and provided ideas for the subsequent verification of the molecular mechanism of Aidi injection for hepatocellular carcinoma.?Corresponding author: Wang Xuan, Professor, chief physician.

1. Introduction

Hepatocellular carcinoma accounts for 85% -90% of primary liver cancer [1]. At present, the early detection rate of HCC screening in China is not high, the diagnosis is often advanced [2] and the opportunity for surgery is lost. Although chemical drugs have a positive role in the treatment of hepatocellular carcinoma, their strong toxic side effects and drug resistance cannot be ignored

[3]. Traditional Chinese medicine is a feature of comprehensive treatment of tumors in China, and it plays a unique role in improving symptoms, treating adverse reactions, delaying disease progression, preventing recurrence and metastasis, prolonging survival, and improving quality of life [4]. The clinical application of traditional Chinese medicine to treat hepatocellular carcinoma based on the existing treatment methods has achieved good results [5], and has been widely recognized at present. Traditional Chinese medicine has the advantages of multiple orientations, multiple targets, and less prone to drug resistance [6]. Therefore, it is of great significance to actively explore the mechanism of Chinese medicine in the prevention and treatment of this disease.

Aidi injection is a Chinese medicine injection independently researched and invented in China with intellectual property rights in the treatment of hepatocellular carcinoma. Consisting of four Chinese herbal extracts of Banmao, Renshen, Ciwujia and Huangqi. With Banmao as the king's medicine, Renshen as the minister's medicine, Ciwujia and Huangqi as adjuvant medicine, it has the functions of clearing away heat and detoxifying, eliminating stasis and dispersing stagnation, and replenishing Qi [7]. Among them, Banmao, also known as Huabanmao has the effects of breaking blood, removing blood stasis, eliminating stasis, attacking toxicity and dispersing stasis; Renshen has always been regarded as the king of herbs. It has the effects of nourishing vitality, nourishing the spleen and lungs, stabilizing the nerves and strengthening the body's immunity; Ciwujia can nourish the body, enhance body resistance and tolerance; Huangqi supplements qi and yang, strengthens body surface, diuresis swelling, clears store and grows muscle, stimulates bone marrow cell proliferation and enhances the phagocytosis of macrophages. Four flavors of drugs have been used in combination to treat hepatocellular carcinoma with satisfactory clinical results. At present, its active ingredients and molecular mechanism of action for the treatment of hepatocellular carcinoma have not been fully clarified, and further investigation is needed.

Network pharmacology is an emerging product of multidisciplinary integration proposed in recent years [8], using computer simulation, data analysis, multiple database retrieval, and other methods to clarify the material basis of drugs and its mechanism of action through a multilevel network [9], providing new ideas for the modern study of the interaction between drugs and the body [10]. In this study, we searched public databases such as TCMSP, TCMID, OMIM, and GeneCard Suite, explored the relationship between drugs and diseases, and constructed a "drug-target-disease" network to provide scientific evidence for the clarification of the mechanism of Aidi injection in treating hepatocellular carcinoma.

2.Materials and methods

2.1 Database

Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php); Traditional Chinese Medicines Integrated Database (TCMID, http://119.3.41.228:8000/tcmid/); UniProt database (Universal Protein, http://www.uniprot.org/); OMIM database (Online Mendelian Inheritance in Man, https://omim.org); GeneCards Suite database (The Human Gene Database，https://www.genecards.org)；STRING database (https://string-db.org); Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/).

2.2Obtaining Active Ingredients and Potential Targets of Drugs

Retrieve the active ingredients of each traditional Chinese medicine through the TCMSP database with the keywords "Banmao", "Huangqi" and "Renshen". The oral bioavailability (OB) of the drug (≥30%) and the drug-likeness (DL) (≥0.18%) were selected as the criteria for active ingredients. Using "CI WU JIA" as the key word, search the active ingredients of Ciwujia in the TCMID database, and input the results back to TCMSP to screen out active ingredients with OB ≥ 30% and DL ≥ 0.18%, and obtain potential target information for candidate compounds of four Chinese medicine. And through the RSQLite data packet to search UniProt database to add gene abbreviation to each target online.

2.3 Obtaining the disease-related targets

In the OMIM and GeneCards Suite databases, using "Hepatocellular carcinoma" as the search term, the two database results are combined to eliminate duplication to obtain potential targets related to hepatocellular carcinoma disease.

2.4 Obtaining the intersecting targets of drug and disease

Use the R language (Version 3.6.1) to install the Venn Diagram data package, map the potential target of Aidi injection to the target of hepatocellular carcinoma, and make a Venn diagram to obtain the intersection of them.

2.5 Making a network graph

Run the script to fuse the selected intersecting targets and the active ingredients and their corresponding potential target information selected from the four traditional Chinese medicines to generate a network diagram preparation file, which mainly includes the relationship files between nodes and attribute files of each node. The file was imported into the Cytoscape software, and a network diagram of the interaction between the active ingredient and the target was generated, and the topology analysis was performed.

2.6 Making a PPI Graph

Enter the intersection of the drug target and the disease target into the STRING database, select "Homo sapiens" as the species, obtain the interaction network diagram (PPI) of the common target, and perform topology analysis to export the string_interactions.tsv. The number of links of each target is detected by R language to make a histogram.

2.7 GO function enrichment and KEGG pathway enrichment analysis

In order to describe and annotate the function of the differential genes, the differential genes screened out were subjected to GO enrichment analysis through the DAVID database. The species selection was "Homo sapiens". The analysis includes Molecular Function (MF), Biological Process (BP), and Cellular Components (CC). Through these three functional categories, a gene is defined and described in many aspects. Then KEGG pathway enrichment analysis is performed using the R language through the ClusterProfiler. With P ＜0.05 as the screening condition, the pathways with significant differences in the main biological processes and drug action mechanisms of Aidi injection for treating hepatocellular carcinoma were obtained.

3.Results

3.1 Active ingredients and potential targets of Aidi injection

Under the limited conditions of OB ≥ 30% and DL ≥ 0.18%, a total of 36 active ingredients can be obtained by searching the TCMSP database for the three Chinese medicines of Banmao, Huangqi and Renshen, and the TCMID database for Ciwujia(Table 1). Of which Banmao is 1, Huangqi are 14, Renshen are 16, and Ciwujia are 5. Quercetin is shared by Huangqi and Ciwujia, betasitosterol is shared by Ciwujia and Renshen, and kaempferol is shared by Huangqi and Renshen. The highest bioavailability was 7-O-methylisomucronulatol from Huangqi. The highest druglikeness is 9,10-dimethoxypterocarpan-3-O-β-D-glucoside from Huangqi. A total of 106 potential targets were screened for the active ingredients (Figures 1 and 2A), and gene abbreviations were added for each target.

3.2 The intersection targets of drug and disease

A total of 6,677 disease-related targets were retrieved through the OMIM and GeneCards Suite databases. The potential targets of Aidi injection were mapped to the obtained disease-related targets, and a total of 89 intersecting targets were obtained to make a Venn diagram, as shown in Figure 1. It can be seen that the proportion of all targets of Aidi injection related to hepatocellular carcinoma is as high as 84%, which proves the scientificity and effectiveness of Aidi injection in treating this disease from the side. We will analyze it further in the discussion part.

Fig. 1: The intersection of drug and disease targets

Table 1 Basic information of active ingredients of Aidi injection

3.3Network graph

A total of 122 nodes can be seen in Fig. 2A, including 31 active ingredients, 89 targets, and 467 lines of action, including 347 lines of action of active ingredients and targets. The number of nodes connected represents the level of the node degree, and the BetweennessCentrality value represents the core degree of the node in the network graph. Both are important indicators for describing the node. It can be seen from the figure that the highest active ingredient value is quercetin (MOL000098), followed by kaempferol (MOL000422), beta-sitosterol (MOL000358), isorhamnetin (MOL000354), 7-0-methylisomucronulatol (MOL000378). Each active ingredient acts an average of 11.19 (347/31) targets, and each target connects an average of 3.90 (347/89) active ingredients. The above can also explain that Aidi injection achieves the purpose of treating hepatocellular carcinoma through the combined action of multiple components and multiple targets. The topology analysis of the network shows that the network density is 0.048, the network concentration is 0.699, the network heterogeneity is 1.897, the shortest path is 14762 (100%), the average degree value of the nodes is 7.66, and the mean value of the BetweennessCentrality is 0.011. Analyze a total of 9 nodes with the degree value greater than 7.66 and the BetweennessCentrality greater than 0.011. (see Figure 2B), including 4 active ingredients and 5 targets. The highest active ingredient is quercetin, and the highest target is PTGS1.

Fig. 2: Network graph and target topology analysis

3.4 Building a PPI network

89 target proteins were entered into the STRING database for analysing, and the species "Homo sapiens" was selected. At the same time, 2 unlinked free target proteins were eliminated to obtain a PPI network diagram (see Figure 3). The network has 87 nodes and 928 interaction lines with an average degree of 10.67. Nodes with a degree value greater than 30 are calculated by R language and made into a histogram (see Figure 4). These targets work most closely with other nodes. For example, IL6, CASP3, VEGFA, MAPK8, JUN, EGFR, MYC, PTGS2 and FOS are target proteins clearly related to hepatocellular carcinoma.

Fig. 3: Interaction network target protein

Fig. 4: PPI graph node degree value

3.5 GO function enrichment and KEGG pathway enrichment analysis

On-line GO function enrichment and KEGG pathway enrichment analysis were performed through the DAVID database. The GO enrichment analysis obtained a total of 678 entries. Among them, there are 60 related signaling pathways with corrected P value less than 0.05, including 56 BPs, 1 CC, 3 MFs. Involved in inhibiting abnormal proliferation and differentiation of hepatocellular carcinoma cells, regulating the cell cycle and promoting apoptosis, and also related to the cell metabolism and stimulated reaction of hepatocellular carcinoma cells. Enrichment analysis of KEGG pathway, screening for entries with corrected P value of less than 0.05, of which there are 8 significantly different signal pathways, which are p53 signaling pathway, Calcium signaling pathway, ErbB signaling pathway, VEGF signaling pathway, MAPK signaling pathway, Insulin signaling pathway, Toll-like receptor signaling pathway and NOD-like receptor signaling pathway. These signaling pathways are closely related to hepatocellular carcinoma and will be the focus of our discussion below.

4. Discussion

In this study, a network pharmacology method was used to study the mechanism of Aidi injection in the treatment of hepatocellular carcinoma. Through a topological analysis of the drug active ingredient and target interaction network, it was found that the active ingredients that may play an important role in Aidi injection are: quercetin, kaempferol, beta-sitosterol, isorhamnetin and the like. Quercetin is a bioactive flavonoid that has been shown to protect hepatocellular carcinoma with antioxidant activity. In addition to its antioxidant capacity, quercetin can also inhibit the activity of CK2α which is ubiquitous and carcinogenic in hepatocellular carcinoma, and can inhibit the proliferation and induce apoptosis of hepatocellular carcinoma cells by blocking Notch pathway and Hh pathway [11]; Kaempferol is a flavonoid with antioxidant and antitumor properties. Studies have shown that it can inhibit the proliferation, migration and invasion of HepG2 cells by downregulating miR-21, up-regulating PTEN and inactivating the PI3K / AKT / mTOR signaling pathway [12]. Other studies have shown that it can inhibit human HCC cells by activating the AMPK signaling pathway [13]. Beta-Sitosterol is one of the phytosterols, which has a strong inhibitory effect on the growth of human hepatocellular carcinoma line HepG2 cell, and induces these cells apoptosis through the mitochondrial pathway and membrane death receptor pathway [14]. VEGFA is a member of the VEGF growth factor family. This gene is up-regulated in many known tumors, and its expression is closely related to tumor stage and progression [15]. JUN (c-Jun) is a cell transcription factor-related protein that can promote cell proliferation. Studies have shown that the expression of c-Jun in patients with hepatocellular carcinoma is promoted, leading to the proliferation and immune escape of hepatocellular carcinoma cells and promoting the development of hepatocellular carcinoma [16]. The EGFR (epidermal growth factor receptor) family belongs to the tyrosine kinase family and is the expression product of the proto-oncogene c-erbB1. It is involved in activating a series of complex cellular signal transduction pathways, and regulates important physiological processes such as cell growth, division, and differentiation in normal tissues. The abnormal expression and activation of the EGFR family is closely related to tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis [17]. PTGS1 and PTGS2 are nicknames for the cyclooxygenases COX1 and COX2, and are the main rate-limiting enzymes for prostaglandin synthesis. They play an important role in cancer, inflammation, cardiovascular and cerebrovascular diseases, and autoimmune diseases [18]. In addition, IL6, CASP3, MAPK8, MYC, FOS and other important targets have also played an important role in the treatment of hepatocellular carcinoma by Aidi injection. The drug active ingredient and target interaction network in this study reflects the characteristics of Aidi injection's multi-component, multi-target, multi-pathway treatment of hepatocellular carcinoma. This coincides with the overall concept of Chinese Medicine, dialectical treatment, and the compatibility of prescriptions. At the same time, it shows the good binding ability between the active ingredients of Aidi injection and key targets, suggesting that the results of this study are reliable and have high reference value.

We obtained the effects of Aidi injection involving multiple biological processes through GO function enrichment analysis, mainly focusing on inhibiting abnormal proliferation and differentiation of hepatocellular carcinoma cells, regulating the cell cycle, and promoting apoptosis, and also related to the cell metabolism and stimulated reaction of hepatocellular carcinoma cells. KEGG enrichment analysis was performed on related targets in order to further explore the possible biological pathways in the action mechanism of Aidi injection. The results show that the treatment of Aidi injection for hepatocellular carcinoma mainly involves 8 related signaling pathways. By searching relevant literature and analyzing,we found that the P53 signaling pathway, Toll-like receptor signaling pathway, VEGF signaling pathway, MAPK signaling pathway, and ErbB signaling pathway are closely related to hepatocellular carcinoma and may be the most important pathways for Aidi injection. It is well known that the tumor suppressor gene P53 is a major functional protein that mediates apoptosis, can sense DNA damage and transmit intracellular apoptosis signals [19, 20]. It is functionally the "main guardian of the genome", including controlling the cell cycle, apoptosis and maintaining genome stability. It is widely accepted that p53 dysfunction is closely related to the occurrence and development of cancer. Mutations or deletions of p53 have been observed in nearly half of human cancers including hepatocellular carcinoma, suggesting that it may play an important role in the pathogenesis of hepatocellular carcinoma [21]. Wang Yuxuan and others found that Huangqi protein causes programmed necrosis of hepatocellular carcinoma cell line HepG2 and is closely related to the p53 signaling pathway [22]. Liu and others [23] demonstrated that the mechanism of inhibition and apoptosis of hepatocellular carcinoma HepG2 cells induced by 12C6 + heavy ion beam irradiation is through the biological function of the p53 signaling pathway (including extrinsic and intrinsic apoptosis pathways) to mediate. Toll-like receptors (TLRs) are a class of noncatalytic receptors that function as innate immune pathogen pattern recognition receptors. Studies have found that the TLR signaling pathway is closely related to tumorigenesis and development [24]. Not only related to tumor growth and immune suppression, but also to apoptosis and activation of the immune system. TLR can induce apoptosis and activate innate and adaptive immune responses, all of which may make it a target for anti-tumor therapy [25]. A large number of studies have shown that TLR is expressed on the surface of hepatocellular carcinoma cells and is involved in regulating the proliferation, metastasis and immune escape of cancer cells, and affects the progression of hepatocellular carcinoma [26]. The latest research results show that the increased expression of TLR2 and TLR4 on peripheral blood mononuclear cells may reflect the occurrence and development of hepatocellular carcinoma and may indicate a poor prognosis, which can provide new therapeutic targets for hepatocellular carcinoma [27]. Hepatocellular carcinoma is a highly vascularized solid tumor and angiogenesis plays a vital role in the development of hepatocellular carcinoma [28]. VEGF (vascular endothelial growth factor) is a key mediator of angiogenesis, which can regulate the proliferation, migration, and survival of vascular endothelial cells and cause changes in vascular permeability and control tumor growth by controlling angiogenesis. Signal pathways such as P13K-Akt, MAPK, HIF-1, and VEGF downstream of VEGF are also involved in tumor angiogenesis. The combination of VEGF and VEGFR2 causes phosphorylation of P13K and Akt / PKB and then induces the growth, migration and tubular formation of endothelial cells. Kaempferol in Aidi injection inhibits the proliferation, migration and invasion of HepG2 cells by inhibiting the P13K-Akt signaling pathway downstream of VEGF. The importance of VEGF in tumor metastasis makes it a routine target for anticancer therapies. For example, bevacizumab is the most commonly used angiogenesis inhibitor that directly targets VEGF. Sorafeni (VEGFR tyrosine kinase inhibitor) is an angiogenesis inhibitor that acts on other signaling molecules in the VEGF pathway. Both of them have good therapeutic effect on hepatocellular carcinoma. MAPK (mitogen-activated protein kinase) is a class of protein kinases, mainly including three subfamilies of ERK, JNK and p38, which regulates multiple life processes such as cell growth, differentiation, and apoptosis [29]. In the ERK signaling pathway, after the upstream RAS and RAF are activated, they regulate downstream proteins (such as c-Jun, c-Myc, SRF, c-Fos, c-met, etc.), thereby promoting the invasion and metastasis of hepatocellular carcinoma cells. JNK and p38-MAPK signaling pathways mainly affect autophagy and apoptosis of hepatocellular carcinoma cells [30], and Ras / MAPK signaling pathways are abnormally activated in 50% to 100% of hepatocellular carcinomas and are associated with poor prognosis of hepatocellular carcinoma [31]. To sum up, the abnormal activation of the MAPK signaling pathway is closely related to the occurrence, development, invasion, metastasis and tumor angiogenesis of hepatocellular carcinoma [32,35]. Studies have shown that chemotherapy drugs such as mitomycin and cisplatin can activate the p38 / MAPK signaling pathway and promote apoptosis of hepatocellular carcinoma cells [36]. A newly synthesized derivative of glycyrrhizin, ONTD, can activate the JNK and p38-MAPK signaling pathways and induce apoptosis of hepatocellular carcinoma cells, and play a role in inhibiting hepatocellular carcinoma [37]. The ErbB family includes four tyrosine kinase receptors, which are HER1 (ErbB1, EGFR), HER2 (ErbB2, NEU), HER3 (ErbB3) and HER4 (ErbB4). When bound to its ligands (such as AR, EGF, TGF, etc.), activate downstream related targets (such as Akt, MAPK, etc.), thereby regulating cell proliferation, differentiation, migration and apoptosis activities [38]. ErbB is involved in some physiological activities in normal tissues. Its mutations are related to the occurrence of multiple cancers, and its high expression is also related to various solid tumor such as hepatocellular carcinoma [39], nonsmall cell lung cancer [40], gastric cancer [41], breast cancer [42] etc. Studies have shown that inhibition of the ERBB2 gene can inhibit the growth of hepatocellular carcinoma cells and block the cell cycle progression [39].

In this study, a network pharmacology research method was used to establish the "component-target-disease" co-expression network of Aidi injection, and its complex network relationship of multi-component, multi-target and multi-pathway was studied. Aidi injection can treat hepatocellular carcinoma through the active ingredients such as quercetin, kaempferol, beta-sitosterol, isorhamnetin to act on VEGFA, EGFR, JUN, PTGS1, PTGS2 and other key targets, and regulate the P53 signaling pathway, VEGF signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, ErbB signaling pathway and other signaling pathways to play a direct or indirect therapeutic role. It mainly focuses on inhibiting the abnormal proliferation and differentiation of hepatocellular carcinoma cells, inhibiting its angiogenesis, regulating the cell cycle, and promoting apoptosis. And also related to the cell metabolism, stimulated reaction and biological regulation of hepatocellular carcinoma cells. This study lays the foundation for further experimental research. However, as the data in the database are still being updated, while the contents of traditional Chinese medicine components, interactions and other pharmacokinetic effects in the body are ignored, this study still has certain limitations and the specific mechanism needs further research.