This study reveals the mechanism of Trichosanthes–Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes–Fritillaria thunbergii in treating lung adenocarcinoma.
We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.
Lung cancer refers to malignant tumors originating from the lung bronchial mucosa, including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and small cell carcinoma1. Lung adenocarcinoma (LUAD) is the most common type of lung cancer, accounting for about 40% of the total lung cancer cases2. Most patients are diagnosed at an advanced stage or have remote metastasis and, thus, lose the opportunity of surgery3. In current clinical treatment, concurrent chemoradiotherapy is the most common strategy for treating LUAD, but its application is limited due to serious adverse reactions4.
Traditional Chinese medicine (TCM) can effectively relieve the clinical symptoms of LUAD patients and reduce the adverse reactions caused by radiotherapy and chemotherapy and has, thus, become a research hotspot5,6,7. In traditional Chinese medicine, lung cancer belongs to the category of "lung accumulation" and "pulmonary petrous". The deficiency of Qi and the interaction of phlegm, stasis, and poison are important in the pathogenesis of lung cancer. Therefore, tonifying Qi and eliminating phlegm and blood stasis are the main clinical treatment8 methods for lung cancer according to TCM theory9. Trichosanthes kirilowii Maxim (Gualou) and Fritillaria thunbergii Miq (Zhebeimu) represent a common drug pair in treating lung cancer, and this combination has the effects of clearing heat and reducing phlegm10,11,12. However, its mechanism of action is still unclear, and further research needs to be conducted.
Network pharmacology is a comprehensive method based on the theory of systems biology and multidirectional pharmacology that aims to reveal complex network relationships between multiple drugs and diseases13. Traditional Chinese prescriptions have the characteristics of being multi-component and multi-target, meaning they are very suitable for the study of network pharmacology14,15. Recently, network pharmacology has emerged as a powerful approach in the study of TCM formulas and has become a research hotspot16,17.
However, to the best of our knowledge, all of the research on network pharmacology are presented as text. Presenting this technology through video will greatly reduce the learning threshold and facilitate the promotion of this technology, which is one of the advantages of this article. In this study, we took Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma as an example to carry out network pharmacology prediction and experimental validation.
All the network pharmacology procedures were carried out in accordance with the Guidelines for Network Pharmacology Evaluation Methods18. All the experimental procedures were performed in accordance with the laboratory management regulations of the Beijing University of Chinese Medicine.
1. Network pharmacological prediction
2. Experimental verification
3. Molecular docking
4. Statistical analysis
A total of 31 Trichosanthes-Fritillaria thunbergii-related active components were identified, including 21 Trichosanthes and 10 Fritillaria thunbergia components, as well as 144 corresponding targets. Overall, 9,049 and 67 LUAD-related genes were extracted from the GeneCards database and the OMIM database, respectively. After deleting duplicated genes, 9,057 genes related to LUAD were identified. The intersection of the LUAD-related genes and Trichosanthes-Fritillaria thunbergii active component-related targets was conducted to obtain potential therapeutic targets. The drug-component-disease-target interaction network of Trichosanthes-Fritillaria thunbergii against LUAD is shown in Figure 1A. In the interaction network, the top ten active components were kaempferol, hydroxygenkwanin, genistein, diosmetin and β-sitosterol, palmitoleicacid, mandenol, hexadecanoic acid, carproic acid, and capric acid, which were identified as the key active components of the action of Trichosanthes-Fritillaria thunbergii in treating LUAD (Figure 1B). The PPI network included 122 functional proteins and 210 interaction relationships, and the visualization results are shown in Figure 2A. The top ten core proteins by degree (parameter used for visualization analysis in Cytoscape software) in descending order included ESR1, VEGFA, PPARA, CYP3A4, AR, APP, FGF2, CREB1, and CYP1A1, which are mainly involved in neovascularization, cell proliferation, apoptosis, and cell membrane transport30,31,32,33,34,35,36,37,38,39(Figure 2B). Of the top 20 pathways ranked by KEGG, the PI3K/AKT signaling pathway40, Rap1 signaling pathway41, phospholipase D signaling pathway42, and MAPK1 signaling pathway43 are closely associated with lung cancer, among which the PI3K/AKT pathway ranked the first and, thus, was used for subsequent verification (Figure 3).
The experiments indicated that Trichosanthes-Fritillaria thunbergii extracts at concentrations over 400 µg/mL could inhibit cell proliferation, and the inhibition effect on A549 cells at concentrations up to 800 µg/mL was close to the half inhibitory concentration (IC50) (Figure 4). Thus, 400 µg/mL, 600 µg/mL, and 800 µg/mL were used as the low, medium, and high doses for the subsequent experiments. The intervention of Trichosanthes-Fritillaria thunbergii extracts caused no significant change in AKT protein expression in each group; however, the expression of p-AKT (Ser473) was inhibited and showed a dose-dependent effect (Figure 5). The key components of Trichosanthes-Fritillaria thunbergii in LUAD treatment were molecularly docked with the key proteins of the PI3K/AKT pathway, and the results suggested the binding energies of diosmetin and kaempferol with AKT1 were less than −7, indicating strong binding activity44 (Figure 6).
Figure 1: Diagram of the drug-component-disease-target network. (A) Blue represents the disease; yellow represents the drug; red represents the component; green represents the target. Abbreviations: GL = Trichosanthes; ZBM = Fritillaria thunbergia; LUAD = lung adenocarcinoma. (B) Top ten active ingredients in the network ordered by degree in descending order. Please click here to view a larger version of this figure.
Figure 2: PPI network of Trichosanthes-Fritillaria thunbergii in the treatment of LUAD. (A) Visualization results of the PPI network. The darker the node, the more central the protein is in the network. (B) Top ten targets in the network by degree. Please click here to view a larger version of this figure.
Figure 3: KEGG pathway enrichment of Trichosanthes-Fritillaria thunbergii targets against LUAD. (A) The top 20 KEGG pathways are ranked according to the P-values in ascending order. (B) Map of the PI3K/AKT signaling pathway. Please click here to view a larger version of this figure.
Figure 4: Effects of different concentrations of Trichosanthes-Fritillaria thunbergii extract on A549 cell proliferation (n = 3). Trichosanthes-Fritillaria thunbergii extracts at concentrations over 400 µg/mL could inhibit cell proliferation. The inhibition effect on A549 cells at concentrations up to 800 µg/mL was close to the half inhibitory concentration (IC50). Abbreviation: GL-ZBM = Trichosanthes-Fritillaria thunbergii. Please click here to view a larger version of this figure.
Figure 5: Effects of different concentrations of Trichosanthes-Fritillaria thunbergii extract on AKT protein expression and phosphorylation levels (n = 3). There was no significant change in AKT protein expression in each group, while the protein expression of p-AKT (Ser473) in the medium and high dose groups was significantly down-regulated, and the difference was statistically significant when compared with the control group (*P < 0.05 versus control group). Abbreviation: GL-ZBM = Trichosanthes-Fritillaria thunbergii. Please click here to view a larger version of this figure.
Figure 6: Molecular docking of the related components with core proteins. (A) Heatmap of the binding energy of key components of Trichosanthes-Fritillaria thunbergii for LUAD treatment molecularly docked with key proteins of the PI3K/AKT pathway. (B) Molecular docking diagram of Diosmetin and AKT1 proteins. (C) Molecular docking diagram of kaempferol and AKT1 proteins.The pink lines represent hydrogen bonds, the gray structures represent drug compositions, and the colored structure represents AKT1. Please click here to view a larger version of this figure.
Generally, a complete network pharmacology study includes the identification of active components from databases, the acquisition of targets corresponding to active components and diseases, the construction of a drug-component-disease-target network, and the prediction of core targets and pathways. The association between active components and core proteins (molecular docking) is preliminarily predicted by computer technology, and the final verification is conducted using an experiment.
The selection of relevant databases is the most critical part of network pharmacology, as this determines the quality of the research. The HERB database integrates information from several TCM databases, such as Syµmap, TCMID, TCMSP, and TCM-ID, and is the most comprehensive TCM and ingredient database at present. Therefore, in this study, the HERB database was used for the screening of the active components19.
In this study, 31 active components and 144 intersection targets of Trichosanthes-Fritillaria thunbergii in treating LUAD were identified through related databases. By constructing a drug-component-disease-target network, we explored ten key active components through topological analysis. The core targets of Trichosanthes-Fritillaria thunbergii in the treatment of LUAD were screened out through PPI analysis.
Among the top 20 pathways from the KEGG results, the PI3K/AKT signaling pathway, MAPK1 signaling pathway, Rap1 signaling pathway, and PPAR signaling pathway are closely related to tumors according to the literature40,41,42. MAPK1, a classic cancer-related signaling pathway, is an important regulator of cell growth and differentiation. When activated, this signaling pathway leads to uncontrolled cell proliferation, cell cycle extension, and tumor occurrence and development45. The Rap1 signaling pathway is an important regulator of the NF-κB signaling pathway and MAPK1 signaling pathway and is closely related to cell adhesion in lung cancer46. Additionally, it has been confirmed that inhibiting the Rap1 signaling pathway can improve tumor metastasis in lung carcinoma47. The PPAR signaling pathway is associated with cell proliferation, energy homeostasis, tumorigenesis, and metabolic disorders48. Studies have confirmed its function in promoting tumor angiogenesis and tumor growth49.
The PI3K/AKT signaling pathway mainly affects the metabolism, proliferation, apoptosis, and vascularization of tumors through the phosphorylation and activation of AKT. Previous studies have shown that the PI3K/AKT signaling pathway plays a regulatory role in the MAPK1 signaling pathway, Rap1 signaling pathway, and PPAR signaling pathway50,51,52. Moreover, the KEGG prediction results showed that the PI3K/AKT signaling pathway was most closely related to the action of Trichosanthes-Fritillaria thunbergii against LUAD, so it was selected for subsequent experimental verification.
The PPI-predicted core targets, VEGFA and CREB1 in the PI3K/AKT signaling pathway, as well as the key proteins PI3K and AKT1 in the PI3K/AKT signaling pathway, were included for the molecular docking. In a previous study, a docking binding energy less than −7 was considered significant44. The results of this study suggested that the binding energy of kaempferol and diosmetin with AKT1 exceeded this threshold, suggesting that the effects of these two components on AKT may be the key to the action of Trichosanthes-Fritillaria thunbergii against LUAD.
In further experimental verification, we investigated the effect of Trichosanthes-Fritillaria thunbergii extract on the phosphorylation level of AKT. The results showed that different concentrations of Trichosanthes-Fritillaria thunbergii extract had no significant effect on the expression of the AKT protein, but Trichosanthes-Fritillaria thunbergii extract could significantly inhibit the phosphorylation of AKT protein in a dose-dependent manner. These results suggest that the inhibition of the PI3K/AKT pathway is the key mechanism of action of Trichosanthes-Fritillaria thunbergii against LUAD.
In conclusion, through network pharmacology and experimental validation, this study has verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in the treatment of LUAD.
There are still some shortcomings in this study. The study only included high-bioavailability active components without discussing the possible biotransformation of active molecules in the colon, intestinal cells, and liver, which is not comprehensive enough. In addition, the effect of Trichosanthes-Fritillaria thunbergii on the proliferation and PI3K/AKT pathway of lung adenocarcinoma cells was only experimentally verified in vitro. This study provides a theoretical basis for developing new drugs and expanding clinical applications. In the future, further experimental validations of these prediction results will be performed to support assessments of potential clinical applications.
The authors have nothing to disclose.
This study was supported by the Innovation Training Program of Beijing University of Chinese Medicine (No: 202110026036).
0.25% trypsin-EDTA | Gibco | R001100 | |
A549 cell line | Procell | CL-0016 | |
AKT antibody | CST | 4691S | |
BCA Protein Assay Kit | Solarbio | PC0020 | |
Chemiluminescence detection system | Shanghai Qinxiang Scientific Instrument Factory | ChemiScope 6100 | |
Dulbecco's modified eagle medium (DMEM) | Solarbio | 11995 | |
Enhanced chemiluminescence (ECL) kit | ABclonal | RM00021 | |
Fetal bovine serum | ScienCell | 0025 | |
HRP Goat Anti-Rabbit IgG (H+L) | ABclonal | AS014 | |
MTS assay kit | Promega | G3580 | |
p-AKT antibody | CST | 6040S | |
Penicillin streptomycin | Gibco | C14-15070-063 | |
Phenylmethanesulfonyl fluoride (PMSF) | Solarbio | P0100 | |
Phosphatase inhibitor | Beyotime | P1081 | |
Phosphate buffered saline (PBS) | Solarbio | P1020 | |
Polyvinylidene difluoride (PVDF) membranes | Millipore | ISEQ00010 | |
RIPA lysis solution | Solarbio | R0010 | |
Rotary evaporator | Shanghai Yarong Biochemical Instrument Factory | RE52CS-1 | |
Vacuum freeze-drying mechanism | Ningbo Scientz Biotechnology | SCIENTZ-10 | |
β-Actin antibody | ABclonal | AC026 |