Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

Summary

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.

Abstract

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.

Introduction

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 reactions

Protocol

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

1. Selection of active components
   1. Open the HERB database (http://herb.ac.cn)¹⁹, and u.......

Discussion

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 se.......

Materials

Name	Company	Catalog Number	Comments
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