1. Beijing University of Chinese Medicine,Beijing 100029,China

2. Department of Oncology,Dong Fang Hospital,Beijing University of Chinese Medicine,Beijing 100078,China

Keywords:Network pharmacology Lung cancer Target prediction Xingxiao Pill Molecular docking

ABSTRACT Objective: To analyze the potential targets and mechanism of Xingxiao Pill for the treatment of lung cancer based on network pharmacology and molecular docking, providing reference for the research and clinical development of Xingxiao Pill. Method: The main chemical constituents and their targets of Xingxiao Pill was obtained through BATMAN-TCM database.Effective compounds were screened based on SwissADME and their targets were predicted by SwissTargetPrediction. Main lung cancer targets were obtained and integrated through GeneCards, OMIM(OMIM-Gene-Map-Retrieval), TTD and DRUGBANK databases. The intersection targets of Xingxiao Pill for lung cancer were obtained by Venn. The String platform was used to obtain the intersection targets. The core targets of Xingxiao Pill for lung cancer were screened by BisoGenet3.0. The Metasacpe database was used to analyze the biological processes and pathways involved in intersection targets. Cytoscape3.7.2 software was used to construct the "active ingredients of Xingxiao Pill-targets-pathways" network. Finally, Autodock was used for molecular docking verification. Results: The core active components of Xingxiao Pill for the treatment of lung cancer were morin, testosterone, 17-Beta-Estradiol, alpha-Estradiol, muscone, commiferin, commisterone, octyl acetate, etc. The primary pathways mainly included PI3K/Akt, MTOR, MAPK signaling pathway and MicroRNAs in cancer,etc. It has been proved that the combination of testosterone and estradiol with AKT1, SIRT7 and MDM2 was stable and could be used as reference. Conclusion: This study preliminarily revealed the mechanism of Xingxiao Pill on treating lung cancer with multiple components,targets and pathways, providing some ideas and references for clinical application and basic research in the future.

1. Introduction

Lung cancer is the most common cancer and the most lethal malignancy in the world. In 2018, GLOBOCAN estimated that there were 2.09 million new cases of lung cancer, accounting for 11.6% of all cancer cases, and 1.76 million new deaths, accounting for 18.4%. The incidence and death rates of lung cancer in China account for 36.98% and 39.21% of the global cases respectively, and the incidence and mortality rates are increasing year by year. The overall 5-year survival rate is about 19.7%, and the median survival time is no more than 2 years, which seriously endangers human health[1, 2] .Traditional treatment modes of lung cancer mainly include surgery, radiotherapy or chemotherapy, etc. However, 70%of patients are in advanced stage when diagnosed[3], coupled with adverse reactions of radiotherapy and chemotherapy and other deficiencies, so many patients lose the opportunity of treatment.Therefore, it is urgent to provide new treatment strategies for the elderly and frail, middle-advanced patients and patients who have failed conventional therapy.

Professor Kevin Hu, PhD supervisor, founder of Green Therapeutics of Oncology. Currently, he is the vice president of Dongfang Hospital of Beijing University of Chinese Medicine and the director of the Department of Traditional Chinese Medicine Oncology of Beijing University of Chinese Medicine. In 2003, he first proposed and founded the green therapy of cancer. The "green treatment" of tumor takes "minimally invasive surgery + traditional Chinese medicine" as the main treatment means. Through the combination of traditional Chinese medicine and western medicine,local and systemic combination, and internal and external treatment,the patients' life quality can be guaranteed and improved while their life span can be prolonged. The minimally invasive local treatment and low harm systemic treatment reflect the advantages of "green treatment", which is "low injury, easy tolerance and good efficacy",has brought good news to a large number of cancer patients.Malignant tumor is a systemic disease characterized by local lesions.According to traditional Chinese medicine, tumor is characterized by "Yin in body and Yang in use" and has vital properties. It is easy to consume blood and seize qi, resulting in the imbalance between the whole body and the local part of the body, resulting in the overall deficiency and cold, and local solid and hot manifestations[4].Xingxiao Pill comes from the《Wai Ke Zheng Zhi Quan Sheng Ji》by Wang Weide in the Qing Dynasty. The whole prescription consists of realgar, musk, mastic and myrrh, which is good at reducing swelling, dispersing constipation, promoting blood circulation and relieving pain. It has the effect of promoting metabolism of the body and reconciling Yin and Yang ,Qi and blood, which is in accordance with the concept of "emperor's way" in the third stage of green treatment model[5].Clinical studies have shown that realgar is antitoxic and anti-cancer, combined with dry varnishing is effective in treating lung cancer[6]. Liushen pill (containing musk and realgar)can effectively improve clinical symptoms of patients with malignant tumors[7]. Xingxiao Pill (containing musk, mastic and myrrh) can significantly improve clinical benefit rate, and stabilize the condition and improve the quality of life[8].

The clinical application of Xingxiao Pill can adjust the physique of patients with lung cancer, improve the symptoms of cold and heat of lung cancer, and improve the quality of life.However, medical evidence is still lacking at present, so this paper intends to predict the targets and pathways of Xingxiao Pill in the treatment of lung cancer through network pharmacology and molecular docking, so as to fill in the molecular mechanism and provide reference for subsequent basic experimental studies.

2. Materials and methods

2.1 Collection of active ingredients and screening of potential targets of Xingxiao Pill

Realgar belongs to mineral medicine. There was no active ingredient and target of realgar when consulting TCM database platform, and its active ingredient in literature were As2S2 and As4S4[9, 10]. According to SwissADME and SwissTargetPrediction,no effective target could be obtained, and the target prediction tool or platform was yet to be developed. Therefore, this paper intended to carry out related studies on musk, frankincense and myrrh, and preliminarily predicted and analyzed the targets of Xingxiao Pill in the treatment of lung cancer. BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm)[11] was an online analysis platform for the molecular mechanism of traditional Chinese medicine, through which this article was going to find the compounds of Xingxiao Pill. PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was a subsidiary of NCBI organic small molecule biological active database, including structure, activity, sources and experiment data[12]. this paper was aimed to obtain chemical structural formula of each component of Xingxiao Pill respectively. SwissADME (www.SwissADME.ch)[13] was used to screen and obtain the effective components,SwissTargetPrediction(www.SwissTargetPrediction.ch)[14] was used to predict the targets of effective ingredients, and integrated into EXCEL file. Finally, potential targets were selected from the Uniprot Protein Database (https://www.uniprot.org)[15, 16] on the conditions that were "reviewed" "Homo sapiens", and the information on the protein targets was standardized and named as gene symbol.

2.2 Screening related targets of lung cancer

Taking "lung cancer" was the key word, potential targets of lung cancer were searched in OMIM-Gene-Map-Retrieval(https://www.omim.org)[17], TTD Database(http://bidd.nus.edu.sg/group/cjttd)[18], DRUGBANK Database(https://www. drugbank.ca)[19] and GeneCards Database(https://www.genecards.org). Afterwards, all targets were combined and duplicated targets were deleted and targets of lung cancer were acquired.

2.3 Intersection targets PPI network construction and core targets screening of Xingxiao Pill and lung cancer

Venn tool was used to intersect musk, frankincense and myrrh with lung cancer targets and drew Venn plot. Then the intersection targets were submitted to STING platform to construct PPI network .With the BisoGenet3.0 plug-in, the PPI network of Xingxiao Pill and lung cancer targets were constructed respectively, by which the Merged network was constructed. According to the Degree, Betweenness Centrality , Closeness Centrality and Local Average Connectivity[20],medians were screened and the core targets was obtained.

2.4 Enrichment analysis of targets’ function and pathways between Xingxiao Pill and lung cancer

Metascape Database(http://metascape.org/)[21] is a bioinformatics database that provides a comprehensive annotated list of genes and analysis resources, mainly for functional and pathway enrichment analysis of differential genes. Intersection targets were imported into Metascape online, and their main Biological Processes and metabolic pathways were enriched and analyzed. Go Biological Processes ,GO Cellular Components, GO Molecular Functions and KEGG Pathway of the targets were plotted by HiPlot online[22].

2.5 Network construction belong to active ingredients of Xingxiao Pill, targets and pathways

Cytoscape3.7.2 is a software for graphical display, analysis and editing of the network.The active ingredients of musk, frankincense and myrrh as well as their targets and signaling pathways for lung cancer were integrated and imported into the software, resulting in the network diagram for visual analysis.

2.6 Molecular docking verification

The PDB Protein Database (https://www.rcsb.org/)[23] was a protein crystal structure database that provides structural information of crystal complexes of protein targets. PyMol software was a molecular visualization software for viewing protein three-dimensional structure, isolating protein and small molecules. Open Babel GUI software was used to convert formats. AutoDock4.2.6 was used to predict the interaction between ligands and biomacromolecule targets, including AutoGrid and AutoDock. AutoDock Tools1.5.6 is an auxiliary software for the graphical and visual docking of molecules in structures. The core components of musk, frankincense and myrrh were docked and verified with Autodock Tools.

3. Results

3.1 Active ingredients and potential targets of musk,frankincense and myrrh were obtained

Enter "SHE XIANG", "RU XIANG" and "MO YAO" in the "Cluster Name" of BATMAN-TCM platform, and set the "Score cutoff"value to 20, and adjust the P value to 0.05 to obtain the compound components of musk, frankincense and myrrh. Among them, there are 30, 19 and 15 ingredients of musk, frankincense and myrrh respectively, which are abbreviated as "SX1～SX30, RX1～RX19,MY1～MY15", as shown in Table 1. Some of the compounds have not been labeled because there is no unified standard name and attribute value. The chemical structure formulas of musk, mastic and myrrh compounds were obtained through PubChem, which were imported into the SwissADME platform for screening, and those whose "gastrointestinal absorbability" was "high" and "drug-like" had two or more of the five filter property values of "yes" were passed the screening. Lastly, 20 kinds of active chemical constituents from musk, 8 kinds of active chemical constituents from frankincense and 13 kinds of active chemical constituents from myrrh were obtained,as shown in Table 1. The targets of each effective component were predicted through SwissTargetPrediction, and the "Probability ＞ 0"was set as the effective targets, which is integrated into the Excel file. Among them, there were 769 active targets of musk, 273 of frankincense and 469 of myrrh, and 121 targets were obtained by Uniprot screening after the combination of component targets and deletion of duplicative values.

3.2 Related targets of lung cancer were screened

Disease-related targets were screened from GeneCards, and targets whose Score was greater than the median were set as potential disease targets. According to lung cancer related targets, the median was 16.855, and 1449 targets were obtained. Combined with OMIMGene-Map-Retrieval, DRUGBANK and TTD Databases, all lung cancer targets were merged and duplicated values were deleted to 1914 relevant targets.

3.3 Intersection targets PPI network and core targets of Xingxiao Pill and lung cancer were constructed and screened respectively

Venn was used as the intersection of lung cancer-related targets and active ingredient targets of Xingxiao Pill, and 50 intersection targets were obtained, as shown in Figure 1.The PPI network of intersection targets is constructed on the String platform, as shown in Figure 2.The size and color of nodes as well as the thickness and color of the wires in the figure reflected the interconnection between targets.The larger the nodes were, the brighter the nodes were, the thicker and darker the wires were, representing the more critical locations of these targets. The intersection targets and their components are shown in Table 2. The PPI network of Xingxiao Pill component targets and lung cancer targets was obtained by Bisogne3.0, which were merged into the merged network. After screening the two times median of Degree, the Hithubs core network was extracted, and the core network was obtained by screening according to Degree,BC, CC, Lac and other parameters. As shown in Figure 3, 18 core targets were obtained. Among them, 8 targets were related to the mechanism of lung cancer and supported by more literatures, which were selected for subsequent prediction and research, as shown in Table 3.

Figure. 1 Venn diagram of intersection targets of Xingxiao Pill in the treatment of lung cancer

Figure2 PPI network diagram of intersection targets of Xingxiao Pill in the treatment of lung cancer

Figure3 Screening flow chart of core targets of Xingxiao Pill in the treatment of lung cancer

3.4 Biological functions and pathways of targets were finished by enrichment analysis

The intersection targets of musk, frankincense, myrrh and lung cancer were submitted to Metascape platform for enrichment andcluster analysis, and the results were visualized. Results of targets signaling pathway showed that the core targets of Xingxiao Pill in the treatment of lung cancer mainly involved EGFR tyrosine kinase inhibitor resistance, PI3K-Akt, proteoglycan, MAPK, cell cycle, Th17 cell differentiation, transcription disorders, microRNA,mTOR, non-small cell lung cancer, central carbon metabolism and other signaling pathways in cells, reflected in Table 4 and Figure 4. GO functional annotations showed the main involved biological processes included the transcription of RNA polymerase II promoter, epithelial cell growth, cell's response to nitrogen compounds, negative regulation of cell differentiation, neural process positive adjustment, mature cells, cell adhesion adjustment,glial cell differentiation, autophagy adjustment, positive regulation of cellular catabolism, hormone level adjustment, etc. The main cell components, means the cell locations of the targets, were microtubule tissue center, centrosome, chromosome region, dendrite,cell membrane region, cytoplasmic cystic cavity, spindle axis, etc.The primary molecule functions of the targets were adjusting protein kinase transferase, phosphoric acid, serine/threonine kinase and tyrosine kinase, transmembrane receptor protein kinases, the cell cycle dependent protein, group of protein kinase, REDOX enzyme activity, and transcription factors, RNA polymerase II transcription factors, growth factors, hormones, etc. As shown in figure 5.

Table 2 The intersection targets corresponding to the composition of Xingxiao Pill

Table 3 Core targets of Xingxiao Pill in the treatment of lung cancer

Table 4 Signaling pathways of targets of Xingxiao pill in treatment of lung cancer

Figure4 enrichment analysis of the signal pathways of the targets of Xingxiao Pill in the treatment of lung cancer

Figure5 enrichment analysis of biological process, cell component and molecular function of the targets of Xingxiao Pill in treatment of lung cancer

3.5 Network of active ingredients of Xingxiao Pill-targetspathways was established

Cytoscape3.7.2 was used to construct the “active ingredients of Xingxiao Pill, targets and pathways” network of Xingxiao Pill and lung cancer. Network Analysis was used to screen the core components of Xingxiao Pill in the treatment of lung cancer according to Degree, BC and CC. The predicted core components were morin, myrrh terpenol, ecdysis hormone, testosterone, octyl acetate, diphenylcyclopropenone, methyl eugenol, α-estradiol,estradiol, eugenol, muskone, etc., as shown in Table 5. In the“active ingredients of Xingxiao Pill, targets and pathways” network,blue squares represent the targets, purple diamonds represent the pathways, pink, yellow and green hexagons represent the core components of musk, frankincense and myrrh respectively, and yellow squares represent the common components of frankincense and myrrh. The mutual network among active ingredients , targets and pathways is shown in Figure 6.

Figure6 “active ingredients, targets and pathways” network diagram of Xingxiao Pill in the treatment of lung cancer

Table 5 Core components of Xingxiao Pill in the treatment of lung cancer

3.6Molecular docking results of core components of Xingxiao Pill and targets

Open Babel GUI software was used to convert the compound structure of the core components of musk, frankincense and myrrh into PDB files. PyMol software was used to modify the structure of the core protein, and the intersection targets and core targets that could be found in the structure were selected (see Table 6).AutoDock was used for docking analysis. The results of molecular butt binding energy are shown in Table 6. Be≤-5.5 (kJ mol-1) is the stable binding conformation, the bold part in Table 6 is the stable binding conformation, and Figure 7 is the partial conformational binding diagram. The R language is used to draw a heatmap to directly reflect the docking situation between drugs and targets, as shown in Figure 8. The brightness and shade of the heatmap reflect the size of binding energy. The darker the color, the smaller the binding energy and the more stable the binding conformation. Thus,the conformation of testosterone binding to SIRT7, Akt1, VEGFA and MDM2 and estradiol binding to AR, Akt1, SIRT7, MDM2 and XPO1 were stable.

Table 6 Docking and binding energy table between main components of Xingxiao Pill and the targets

Figure7 Docking structure diagram between main components of Xingxiao Pill and targets

Figure8 Heatmap of core components of Xingxiao Pill docking with targets

4. Discussions

Xingxiao Pill was effective surgical treatment for ulcer swelling,now it was found that had certain curative effect in lung cancer therapy in clinical, which could hit the local pathogens, maintain the body upright, hence to extend its application range to lung cancer,in order to apply old drug into new use. A large number of database resources were integrated through network pharmacology[24], in which the therapeutic targets and pathways were studied, reflecting inheritance innovation. In lung cancer clinical application, realgar,the gentleman medicine of Xingxiao Pill, can clear phlegm and be good at detoxification. Musk is official medicine, can invigorate blood and unblock the collaterals. Frankincense and myrrh are adjuvant, which can activate blood circulation to remove blood stasis and pain. The whole recipe group plays a role in promoting blood circulation to remove blood stasis and nourishing body by dredging collaterals. Therefore, the body's metabolism can be promoted, Yin and Yang can be regulated, chills and fever can be balanced, in line with the "emperor" stage of "green therapy" concept, which mainly adjusts physique, harmonizes qi and blood, Yin and Yang, reflecting the theoretical innovation and is worth for further study.

In this study, several active components of Xingxiao Pill were screened for the treatment of lung cancer. Among them, morin[25]and octyl acetate[26] play a role in regulating cancer cell cycle,estradiol[27], muskone[28, 29] and eugenol[30] are related to inducing tumor cell apoptosis and inhibiting angiogenesis, and elevated testosterone levels are positively correlated with increased risk of lung cancer[31, 32].Studies have shown that realgar has the effect of inducing apoptosis of lung cancer cells and inhibiting tumor growth[33], musk plays an anti-cancer effect by regulating IL-24,TNF family genes and MAPK pathway[34], frankincense is related to apoptosis, autophagy and cell cycle regulation of lung cancer cells[35]. And myrrh or the combination of myrrh and frankincense have a variety of anti-tumor activities ,including inhibition of tumor cell proliferation, induction of cell differentiation and apoptosis,generation of cytotoxicity, inhibition of tumor angiogenesis[35, 36],further supporting the anti-tumor mechanism and efficacy of the core components mentioned in this paper.

This study found that 10 components of musk were mapped to AR, and one of them was related to IL-6 through the construction of PPI network of intersection targets. The active components of frankincense are targeted to HSP90AA1, STAT3 and AR, and myrrh is associated with VEGFA, IL-6, mTOR and AR. The literature suggests that AR is an androgen receptor that is significantly associated with cancer risk. HSP90AA1 is associated with the proliferation ,apoptosis or signal transduction of lung cancer cells[37]. STAT3[38, 39] and VEGFA play an important role in tumor angiogenesis and metastasis[40]. IL-6 regulates tumor immunity and inflammatory microenvironment[41]. MTOR can regulate the growth, metabolism, senescence and autophagy of tumor cells, and has been used as a therapeutic target in immunotherapy[42]. The core targets were screened by Bisogenet3.0 plug-in and mapped to the components of Xingxiao Pill, and it was found that AkT1 is a core element that regulates cancer cell survival, cell cycle, ribosomal biogenesis, protein synthesis and other functions, and has become a cancer therapeutic target at present[43].

KEGG results showes that PI3K/Akt/mTOR[44, 45], proteoglycan[46],

MAPK[47], microRNA and central carbon metabolism[48] play an important role in the growth and metabolic reprogramming of cancer cells. Proteoglycan and MAPK signaling pathways[49] have the function of regulating tumor angiogenesis. EGFR[50] and PI3K/Akt[51] play an important role in overcoming multidrug resistance.MicroRNA[52], cell cycle, and Th17 cell pathway play important roles in tumor cell cycle, apoptosis, inflammation, and senescence.

In conclusion, this paper has preliminarily explored the anti-cancer targets and pathways of Xingxiao Pill in the treatment of lung cancer based on network pharmacology. It is preliminarily predicted that the anti-cancer mechanism of Xingxiao Pill involves the regulation of apoptosis, cycle, angiogenesis, metabolic reprogramming and other pathways of lung cancer cells with the combination of molecular docking and literature research results. MTOR, AKT1, VEGFA,SIRT7, PI3K/Akt/mTOR and MAPK were possible targets and pathways. In future, further experimental studies will be conducted to explore the mechanism of Xingxiao Pill in regulating lung cancer.

Author’s Contribution ZHOU Xiang-nan was in charge of statistical data, making charts and writing, HU Kai-wen was responsible for theoretical guidance of Chinese medicine, ZHOU Tian was in charge of full text review and guidance, LI Zhi-ming was in charge of foreword and discussion,GAO Lei was in charge of method guidance.