Network Pharmacology-Based Analysis of the Effects of Corydalis decumbens (Thunb.) Pers. in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is one of the most malignant tumors worldwide. The main treatment for NSCLC is based on Western medicine; however, the overall effect is unsatisfactory. This study aimed to investigate the potential therapeutic targets and pharmacological mechanisms of action of the traditional Chinese medicine Corydalis decumbens (Thunb.) Pers. in NSCLC based on network pharmacology and bioinformatics. The overlapping genes between Corydalis decumbens (Thunb.) Pers. and NSCLCs were screened using Venn analysis. Cytoscape 3.7.1 software was used to analyze the overlapping target protein-protein interaction (PPI) network. Gene ontology and pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomics database were performed to exploring biological functions of the overlapping genes. The gene expression profiling interactive analysis dataset was used to analyze the correlation between hub gene expression and disease. This study revealed 38 nodes with 191 edges, which may be therapeutic targets for NSCLC. PPI network analysis showed that the most likely association was between the genes AR and NCOA2, NCOA2, and RXRA and ESR1 and NCOA2. These overlapping genes were mainly enriched in the estrogen signaling pathway, calcium signaling pathway, cholinergic synapse, and PI3K-Akt signaling pathway. ESR2 mRNA levels were signiﬁcantly downregulated in patients with lung adenocarcinoma (LUAD) getting worse, and KDR levels were lower in lung squamous cell carcinoma (LUSC) than those in normal tissue. PTGS2 expression was correlated with the median survival time of LUAD, and ESR1 expression was correlated with the median survival time of LUSC. The application of network pharmacology revealed the potential mechanism underlying the effects of Corydalis decumbens (Thunb.) Pers. in NSCLC treatment and provided a theoretical basis for further in-depth research in this field.


Introduction
Lung cancer is one of the most malignant tumors with high morbidity and mortality rates worldwide. It is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) based on its different morphological characteristics. NSCLC accounts for 80% of all lung cancer cases and mainly includes lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large cell lung cancer [1]. Recent research showed that the main treatment for NSCLC is based on Western medicine, including surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy [2,3]. However, the treatment for this disease is often accompanied by unsatisfactory outcomes, such as adverse reactions, easy recurrence and metastasis, poor prognosis, and high costs [4]. us, actively exploring safe and effective treatment methods is necessary to prevent and treat lung cancer [5].
Traditional Chinese medicine (TCM) has a history of more than 2000 years in China and mainly includes botanical, animal, and mineral medicines. It has been widely used to prevent and treat diseases, particularly in Asia [6,7]. Studies have shown that Chinese herbal medicines can directly inhibit malignant tumor growth and proliferation [6]. For example, botanical ingredients such as alkaloids, dietary polyphenols, and saponins are the main inhibitors of lung cancer [8][9][10]. Corydalis decumbens ( unb.) Pers. is botanical medicine that is rich in alkaloids and has mainly focused on treating cardiovascular and cerebrovascular diseases, arthritis, and myopia [11][12][13]; however, its use has been scarce in tumors. e current study, using network pharmacology and bioinformatics, aimed to address the following questions: does Corydalis decumbens ( unb.) Pers. play role in treating NSCLC? Which ingredients of Corydalis decumbens ( unb.) Pers. have an anti-NSCLC effect? What is the molecular mechanism underlying the effect of Corydalis decumbens ( unb.) Pers. in NSCLC treatment?
e Chinese pinyin name "xiatianwu" was entered in the "herb name" column. e filter conditions were oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18.

Drug Ingredient Target Network Construction.
e corresponding targets of the active ingredients were obtained from TCMSP, SymMap (https://www.symmap.org/), and the Encyclopedia of Traditional Chinese Medicine (ETCM, http://www.tcmip.cn/ETCM/index.php/Home/ Index/index.html). e reviewed human database from UniPort (http://www.Uniport.org/) was used to translate target names to gene names. Cytoscape 3.7.1 software was used to construct the drug ingredients target network.

Potential Target Genes of Corydalis decumbens ( unb.)
Pers. against Lung Cancer. We used online tools such as GeneCards (https://www.Genecards.org) to identify the symbols of genes reported in NSCLC using keyword "NSCLC." UniProt online databases were used to translate symbol names to gene names. e overlapping genes of Corydalis decumbens ( unb.) Pers. and NSCLC were identified by drawing a Venn diagram. e overlapping genes were considered as potential Corydalis decumbens ( unb.) Pers. genes playing a role in treating NSCLC. Cytoscape 3.7.1 software was used to construct the ingredients-potential target network.

Construction of the Overlapping Target Protein-Protein Interaction (PPI) Network.
e STRING database (http:// www.string-db.org/) and Cytoscape 3.7.1 software were used to elucidate the molecular mechanism underlying the anti-NSCLC effect of Corydalis decumbens ( unb.) Pers. According to the STRING database with the species limited to "Homo sapiens" and a confidence score >0.4, the interaction proteins were screened. e PPI network was constructed using Cytoscape 3.7.1 for further visualization and analysis (PPI node degree, node interaction scores, and hub genes).

Functional Enrichment Analysis.
e Database for Annotation, Visualization and Integrated Discovery (DA-VID, https://david.ncifcrf.gov) was used for functional enrichment analysis of potential genes. "Homo sapiens" was selected as the species and "OFFICIAL GENE SYMBOL" was used for identification. Gene ontology (GO) functions and the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway enrichment analysis in the DAVID online tool were performed for exploring the biological function of the genes used in the treatment of NSCLC; P < 0.05 was considered as the cutoff criterion. In particular, GO functions include biological process (BP), molecular function (MF), and cellular component (CC). P < 0.01 was used as the cutoff criterion.

Correlation Analysis of Hub Gene Expression and Disease.
e top 10 hub genes in the PPI network were verified using the Cytoscape 3.7.1 plugin cytoHubba. e gene expression profiling interactive analysis dataset (GEPIA, http://gepia. cancer-pku.cn/) was used to analyze the expression of hub genes, such as the differential expression analysis between NSCLC tissues and normal tissues, differential expression analysis according to pathological stages of cancer, and the expression analysis associated with overall survival (OS) of the NSCLC patients (P < 0.05). "MCC" was used as the calculate mode, and the "display the shortest path" as the displayed options, LUAD and LUSC, were selected as the cancer type dataset. Log2 FC cutoff � 2, q value cutoff � 0.05, and the other options were set as the default values.

Potential Target Genes of Corydalis decumbens ( unb.)
Pers. A total of 6 ingredients and 67 targets of Corydalis decumbens ( unb.) Pers. were screened (Table 1, Figure 1, Table S1). A total of 3475 NSCLC target genes were obtained from the GeneCards database. After the comparative analysis, the overlapping genes, which might be potential therapeutic targets for NSCLC, were also obtained. e Venn diagram showed that 32 overlapping genes of Corydalis decumbens ( unb.) Pers. participated in the regulation of NSCLC progression ( Figure 2, Table S2). e generated visual PPI network of potential therapeutic targets for Corydalis decumbens ( unb.) Pers. contained 38 nodes and 191 edges ( Figure 3).

PPI Network Analysis.
Based on the potential pharmacodynamics of Corydalis decumbens ( unb.) Pers. against NSCLC, the interacting proteins were screened using the STRING online tool. We found 31 nodes (PRSS1 did not interact with any of the genes) with 90 PPI relationships in the network (Figure 4(a)). e PPI network was constructed using Cytoscape 3.7.1 software for further visualization and analysis (Figure 4(b)). e results showed 15 genes whose degree was greater than or equal to 5.8 (average score) (Table S3) and 40 edges whose combined score was greater than or equal to 0.69 (average score) (Table S4). We found possible PPIs between Corydalis decumbens ( unb.) Pers. and NSCLC-associated targets. e most likely association was between AR and NCOA2 (score � 0.999). e remaining interactions were as follows: NCOA2 (interacts with) RXRA, ESR1 (interacts with) NCOA2, CALM1 (interacts with) NOS2, CALM1 (interacts with) NOS3, ESR1 (interacts with) NOS3.  were used for further GO function and pathway enrichment analyses using the DAVID online tool. e results showed that the genes could be assigned to different GO terms, which mainly involved biological processes (BP) (90 items), cell components (CC) (27 items), and molecular functions (MF) (41 items). e cutoff for statistical significance was set P < 0.01, and the top 20 items with significant enrichment in biological processes, cell components, and molecular functions were screened, as shown in Figure 5. For biological processes, potential therapeutic targets of Corydalis decumbens ( unb.) Pers. were significantly enriched in "adenylate cyclase-inhibiting G-protein coupled acetylcholine receptor signaling pathway" and "phospholipase C-activating G-protein coupled acetylcholine receptor signaling pathway." For cell components, "axon terminus" and "asymmetric synapse" were mainly enriched. In addition, "RNA polymerase II transcription factor activity," ligand-activated sequence-specific DNA binding," "steroid hormone receptor activity", and "Gprotein coupled acetylcholine receptor activity" belonged to   Evidence-Based Complementary and Alternative Medicine molecular functions. Additionally, the KEGG pathway enrichment analysis (P < 0.01) showed that these overlapping genes were mainly enriched in pathways such as the estrogen signaling pathway, calcium signaling pathway, cholinergic synapse, cAMP signaling pathway, PI3K-Akt signaling pathway, and VEGF signaling pathway ( Figure 6).

Hub Genes Analysis.
e hub genes of Corydalis decumbens ( unb.) Pers. for treating NSCLC were selected based on the Cytoscape 3.7.1 plugin cytoHubba ( Figure 7). Potential therapeutic targets of Corydalis decumbens ( unb.) Pers. included CALM1, AR, ESR1, ESR2, FOS, GJA1, KDR, NOS3, OPRM1, and PRKACA. Using the GEPIA dataset, we compared the mRNA expression of hub genes among NSCLC (LUAD and LUSC) at different stages. e results showed that not all of the ESR2 mRNA expression levels were the same in different stage LUAD of patients (P < 0.05) (Figure 8). Additionally, we compared the transcriptional levels of hub genes between NSCLC and normal samples using the GEPIA dataset. e results indicated that the expression levels of KDR were lower in LUSC than in normal tissue (Figure 9). We further explored the critical efficiency of hub genes in the survival of patients with NSCLC. GEPIA "Survival analysis" tools were used to analyze the correlation of hub genes' mRNA levels in the survival of patients with NSCLC (LUAD and LUSC). e Kaplan-Meier curve and log-rank test analyses revealed that the expression of PTGS2 was correlated with the median survival time of LUAD and the expression of ESR1 was correlated with the median survival time of LUSC. e median survival time of the group with a high expression of PTGS2 mRNA in LUAD was lower than that of the group with low expression of PTGS2 (Figure 10(a)). e group with high expression of ESR1 in LUSC had a lower median survival time than the group with low expression of ESR1 (Figure 10(b)).

Discussion
Lung cancer is one of the most malignant cancers worldwide, and the effect of clinical treatment for this disease is not satisfactory [4]; therefore, this study explored the potential therapeutic effects of Corydalis decumbens ( unb.)

Evidence-Based Complementary and Alternative Medicine
Pers. against NSCLC based on network pharmacology analysis and bioinformatics. Network pharmacology is an interdisciplinary subject that combines systems biology and pharmacology using high-throughput sequencing, genomics, and other technologies to analyze and explore the multichannel regulation of signaling pathways [14]. In this study, we predicted a potential PPI network between NSCLC and Corydalis decumbens ( unb.) Pers. based on network pharmacology analysis. e results showed that the main ingredients of Corydalis decumbens ( unb.) Pers. were PDSP1_000624, palmatine, fumarine, isocorypalmine, bicuculline, and C09367. Studies have shown that alkaloids exhibit good antitumor activity. Bicuculline can effectively inhibit the migration and invasion of human hepatocellular carcinoma MHCC97-H cells which is related to the downregulation of VEGF, MMP-2, and MMP-9 gene expression and inhibition of JAK2/STAT3 signaling pathway activation [15]. Fumarine exerts antiproliferative effects by stimulating the p53 pathway in human colon cancer [16], and the apoptotic rate of HT-29 cells increased significantly after photodynamic treatment with palmatine [17]. A total of 32 overlapping genes were identified between the drug targets and NSCLC molecular targets. GO and KEGG analyses showed that they were mainly assigned to different GO terms and pathways. For example, transcription factor binding, protein binding, drug binding, estrogen signaling pathway, and calcium signaling pathway promote the occurrence and metastasis of NSCLC. Shi et al. [18] found that, in nonsmoking female patients with lung adenocarcinoma, Ca 2+ is an important small-molecule signal that regulates cell function. e high expression of the Ca 2+ pathway transcription factor NFATc2 is considered to be a new regulator for lung cancer to initiate cell phenotype, and high NFATc2 expression predicted poor tumor differentiation, adverse recurrence-free, and cancer-specific overall survivals in human lung cancers [19]. e increase in Ca 2+ concentration in the cytoplasm of cancer cells can also induce apoptosis [20]. Increasing evidence suggests that the estrogen signaling pathway may be a therapeutic target for metastatic NSCLC [21]. Babita et al. [22] indicated that, in a preclinical model of NSCLC, estrogen promoted the growth of NSCLC and increased tumor microvessel density. In the estrogen signaling pathway, estrogen and estrogen receptors are thought to play an important role in NSCLC [23]. Hormone receptors are prognostic factors indicating metastatic tropism to the bones and comparably outcome in NSCLC early stage [24]. e PI3K-AKT signaling pathway plays a major role whole in the occurrence and development of NSCLC [25,26]. erefore, it is speculated that the potentially effective ingredients of Corydalis decumbens ( unb.) Pers. mainly act on the key factors in the above pathways to effectively treat NSCLC.
Studies have found that vascular epidermal growth factor receptor 2 (VEGFR-2) plays a key role in the occurrence and development of tumors including NSCLC [31][32][33]. VEGFR-2 is also known as the KDR in the human body. Several studies have shown that the estrogen receptor beta (ESR2) appeared in many tumors, including lung cancer, wherein ESR2 and interleukin 6 receptor (IL-6R) interacts to mediate lung cancer progression. Cell proliferation, invasion, and cell cycle were significantly increased, and cell apoptosis was markedly inhibited by the concurrent action of ESR2 and IL-6 in A549 cells [34]. Chen et al. [35] showed that phosphorylated ESR1 could directly bind to the promoter of FOSL1 (FOS-like 1) in ESCs/DSCs. Moreover, silencing of

Conclusions
is study is used the network pharmacology analysis to understand the mechanism of action of Corydalis decumbens ( unb.) Pers. and preliminarily verified the effects of Corydalis decumbens ( unb.) Pers. in multicomponent, multitarget, and multichannel treatment of lung cancer. e application of network pharmacology revealed the potential mechanism underlying the treatment of NSCLC by Corydalis decumbens ( unb.) Pers. and provides a theoretical basis for further in-depth research. However, this study did not consider the influence of drug dosage, drug ingredients, and drug delivery methods on the treatment results. erefore, further experimental study is required to verify the results of this study.

Data Availability
e data used to support the findings of this study are included within the Supplementary Materials.

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