Bioactive components and the molecular mechanism of Shengxian Decoction against lung adenocarcinoma based on network pharmacology and molecular docking

Abstract

Objective: The aim of this study was to identify the active components of Shengxian Decoction (SXT) and to elucidate the multi-component, multi-target, and multi-pathway regulatory mechanisms underlying the efficacy of SXT in treating lung adenocarcinoma (LUAD).

Methods: The effects of SXT extract on proliferation, migration, and invasion capabilities of human LUAD cells were determined through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound healing, and Transwell assays. High-Performance Liquid Chromatography (HPLC) was employed to pinpoint the primary active constituents of SXT. The SXT-active component-target-pathway network and protein-protein interaction (PPI) network were constructed based on network pharmacology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID. The clinical significance of key targets was assessed using several external databases, and molecular docking confirmed the binding affinities between key targets and SXT active components.

Results: SXT significantly inhibited the proliferation, migration and invasion of human LUAD cells. HPLC identified and quantified seven active SXT components. Network pharmacology yielded 197 targets, 128 signaling pathways, and 448 GO terms. The PPI network and external validation underscored 13 key targets significantly associated with the influence of SXT on LUAD progression. Molecular docking demonstrated strong interactions between SXT active components and key targets.

Conclusion: SXT treats LUAD through a multifaceted approach involving various components, targets, and pathways. This research offers novel insights into the constituents and molecular mechanisms of SXT in LUAD therapy.

Keywords: Shengxian Decoction (SXT); active components; lung adenocarcinoma (LUAD); molecular docking; network pharmacology.

Figure 1

The schematic diagram of the network pharmacology of Shengxian Decoction (SXT) for lung adenocarcinoma (LUAD).

Figure 2

Cytotoxicity effects of Shengxian Decoction (SXT) extract on human lung adenocarcinoma cells and non-carcinoma cells. A. Cell viability assays of control and 24 h/48 h SXT extract-treated cells showing a dose-dependent cytotoxicity in A549 and NCI-H1299, while the killing effect on WI-38 cells was relatively light (mean ± SD; n = 3). ***P ≤ 0.001; B. Morphological changes in each group of cells after 48 h SXT extract treatment was observed under ×100 microscope (scale bar, 200 μm).

Figure 3

Effect of SXT extract on the migration capacity of A549 and NCI-H1299 cells. Wound healing assay and its quantitation showing strong inhibition with 5 mg/mL of SXT extract treatment (magnification, ×40; scale bar, 200 μm). Quantitative data are represented as mean ± SD (n = 3). ***P ≤ 0.001. SXT, Shengxian Decoction.

Figure 4

Effect of SXT extract on the invasion capacity of A549 and H1299 cells. Invasion assays and its quantitation showing strong inhibition with 24 h treatment of SXT extract (5 mg/mL) (magnification, ×200; scale bar, 200 μm). Quantitative data are represented as mean ± SD (n = 3). ***P ≤ 0.001. SXT, Shengxian Decoction.

Figure 5

Identification of anti-cancer phytochemicals in SXT extract by HPLC analysis. A. Potential chemical compounds in SXT extract responsible for anti-cancer effect; B. HPLC chromatogram of SXT extract; C. HPLC chromatogram of mixed-standard substance; Peak assignment as follows: 1. Neomangiferin, 2. Mangiferin, 3. Calycosin 7-O-glucoside, 4. Isoferulic acid, 5. Luteolin, 6. Formononetin, 7. Saikosaponin A. SXT, Shengxian Decoction; HPLC, High-Performance Liquid Chromatography.

Figure 6

Target selection. A. Volcano maps of LUAD differential expressed genes. Red, upward; Blue, downward; B. Venn diagram of active component targets in SXT and LUAD targets. SXT, Shengxian Decoction; LUAD, lung adenocarcinoma.

Figure 7

GO functional enrichment analysis and KEGG analysis of 197 common targets. A. The Bar chart of the top 10 terms of biological processes, cellular components and molecular functions extracted according to the P value based on GO enrichment analysis. In order to be able to show it in the figure, RNA polymerase II transcription factor activity and ligand-activated sequence-specific DNA binding to RNA polymerase II transcription factor activity were deleted in the GO analysis; B. The Bar chart of the top 30 terms extracted according to the P value based on KEGG enrichment analysis. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 8

The interaction network of GO terms generated by the Cytoscape plug-in ClueGO. A. The functional networks with the most terms enriched by biological processes are shown; B. The functional networks with the most terms enriched by molecular functions are shown. The GO terms are rendered as nodes, with the size of the node representing importance. GO, Gene Ontology.

Figure 9

Construction and pathway analysis of the SXT-active component-target-pathway. A. SXT-active component-target-pathway. The nodes with different colors and shapes represent the herbs, components, targets, and pathways, and an edge is an association between the nodes; B. PI3K-Akt signaling pathway. The red nodes represent potential targets of SXT active component for the treatment of LUAD, arrows represent the activation effect, T arrows represent the inhibition effect and segments show the activation effect or inhibition effect. SXT, Shengxian Decoction; PI3K, phosphoinositide 3-kinase; Akt, protein kinase; LUAD, lung adenocarcinoma.

Figure 10

PPI network to demonstrate SXT active component targets for LUAD treatment. A. PPI network diagram of common targets; B. The PPI network diagram of important targets above the median of degree centrality, closeness centrality and betweenness centrality, with the center and first circle representing the key targets. PPI, protein-protein interaction; SXT, Shengxian Decoction; LUAD, lung adenocarcinoma.

Figure 11

Validation of the mRNA expression of key targets in UALCAN. A. The mRNA levels of 13 key targets in LUAD tissues and normal lung tissues. B. The mRNA levels of 13 key targets in different tumor stages of LUAD (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). LUAD, lung adenocarcinoma; ALB, albumin; AKT1, serine/threonine protein kinase 1; EGFR, epidermal growth factor receptor; SRC, steroid receptor coactivator; HSP90AA1, heat shock protein 90 alpha family class A member 1; CASP3, caspase-3; MMP9, matrix metalloproteinase-9; HRAS, harvey rat sarcoma viral oncogene homolog; ESR1, estrogen receptor 1; IGF1, insulin like growth factor 1; RHOA, ras homolog gene family member A; MAPK1, mitogen-activated protein kinase 1; ANXA5, annexin A5.

Figure 12

Immunohistochemical images (magnification, ×20) of key target protein expression levels in the Human Protein Atlas database. Scale bar, 300 μm. The abbreviations in the figure are the same as in Figure 11.

Figure 13

Overall survival analysis of 13 key targets in LUAD in Kaplan-Meier mapper database. *P ≤ 0.05, **P ≤ 0.01. HR, hazard ratio. Other abbreviations in the figure are the same as in Figure 11.

Figure 14

Genetic alterations in 13 key targets in LUAD patients in cBioPortal. A. OncoPrint visual summary of genetic alterations detected in 13 key targets. B. Summary of alterations in 13 key targets in LUAD. The abbreviations in the figure are the same as in Figure 11.

Figure 15

Molecular docking heat map. The abbreviations in the figure are shown in Figure 11.

Figure 16

The picture of molecule docking model of (A) Neomangiferin with AKT1, (B) Calycosin 7-O-glucoside with AKT1, (C) Neomangiferin with EGFR, and (D) Luteolin with EGFR. The green model represents the SXT active components, and the grey model indicates the key target proteins. The hydrogen bonds were represented by yellow dotted lines, and the length was marked around the lines. AKT1, serine/threonine protein kinase 1; EGFR, epidermal growth factor receptor; SXT, Shengxian Decoction; GLU, glutamic acid; ARG, arginine; THR, threonine; ASP, aspartic acid; ILE, isoleucine; SER, serine; LYS, lysine; CYS, cysteine; ASN, asparagine; PRO, proline; MET, methionine.

Figure 17

Schematic representation of the multi-component and multi-target of SXT intervening in the development of lung adenocarcinoma. SXT, Shengxian Decoction; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide 3-kinase; AKT, serine/threonine protein kinase; mTOR, mammalian target of rapamycin.