基于网络药理学、分子对接及体外实验验证探讨归芪白术方调控PI3K/AKT信号通路诱导MKN-45细胞凋亡的作用机制OA

Objective To investigate the mechanism by which the Guiqi Baizhu Fang(归芪白术方)induces apoptosis in MKN-45 gastric cancer cells via modulation of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway,integrating network pharmacology,molecular docking,and experimental validation.Methods Active components of the Guiqi Baizhu Fang were screened and their potential targets predicted using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Gastric cancer-related genes were retrieved from the GeneCards database,and intersection targets between the drug and disease were identified.Core regulatory pathways were determined via Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Drug-containing serum from rats administered the Guiqi Baizhu Fang was prepared,and its absorbed active components were identified using ultra-performance liquid chromatography coupled with qua-drupole-electrostatic field orbitrap high-resolution mass spectrometry(UPLC-QE-Orbitrap-MS).The binding affinity of absorbed small molecules to PI3K and AKT proteins was verified by molecular docking.For in vitro experiments,MKN-45 cells were treated with 10％drug-containing serum.Cell proliferation viability was detected by the CCK-8 assay;ultrastructural changes in apoptotic cells were observed via transmission electron microscopy;mRNA expres-sion of apoptosis-related genes was measured using quantitative reverse transcription polymerase chain reaction(RT-qPCR);and phosphorylation levels of pathway proteins and expression of apoptosis-related proteins were detected by Western Blotting.Results A total of 141 active components and 872 corresponding targets were identified for the Guiqi Baizhu Fang.1656 gastric cancer-related genes were retrieved,yielding 245 shared drug-disease targets.KEGG enrichment analysis indicated the PI3K/AKT signaling pathway was the most significantly enriched core regulatory pathway.MS analysis identified 1253 compounds in the total Guiqi Baizhu Fang,with 627 absorbable into blood,from which 8 core absorbed active components were screened.Molecular docking results showed that all 8 core absorbed small molecules exhibited good binding activity with PI3K and AKT proteins.In vitro results showed that compared with the blank group,cell proliferation inhibition rates were significantly increased in the high-,medium-,and low-dose Guiqi Baizhu Fang groups and the positive control group(P＜0.05).Under transmission electron microscopy,cells and organelles in the blank group appeared normal.The positive control group showed typical apoptotic features such as blurred nuclear membranes,cytoplasmic vacuolization,and chromatin margination.All Guiqi Baizhu Fang dose groups exhibited apoptotic manifestations,including cell shrinkage,cytoplasmic conden-sation,and nuclear chromatin pyknosis and margination,with the high-dose group showing the most significant effects.RT-qPCR results showed that compared with the blank group,mRNA expression of PI3K,AKT,and B-cell lymphoma-2(Bcl-2)was significantly decreased in the high-and medium-dose formula groups and the positive con-trol group,while mRNA expression of Bcl-2-associated X protein(Bax)was significantly increased in all treatment groups.The low-dose formula group also showed significantly decreased AKT mRNA expression(P＜0.05 or P＜0.01).Western Blotting results indicated that compared with the blank group,the ratios of phosphorylated(p)-PI3K to total PI3K,p-AKT to total AKT,and Bcl-2 to glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were significantly decreased in all treatment groups(P＜0.01),while the Bax/GAPDH ratio was significantly increased(P＜0.05 or P＜0.01).Conclusion The drug-containing serum of Guiqi Baizhu Fang can induce apoptosis in human gastric cancer MKN-45 cells in vitro by inhibiting the phosphorylation and activati(on) of the PI3K/AKT signaling pathway and regulating the balance of the Bcl-2/Bax apoptotic axis.Its multi-component,multi-target characteristics provide a new perspective for gastric cancer treatment.