Exploring the mechanism of puerarin in regulating autophagy and apoptosis in non small cell lung cancer based on network pharmacology
Objective To explore the regulatory effect of puerarin(PUE)on autophagy and apoptosis in non-small cell lung cancer(NSCLC)cells and its potential mechanisms.Methods PUE targets were screened using databases such as Traditional Chinese Medicine Systems Pharmacology Database(TCMSP).NSCLC-related genes from these databases were integrated to conduct target intersection analysis for identifying common targets.A protein-protein interaction(PPI)network was constructed,followed by Gene Ontology(GO)functional annotation,Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis and molecular docking validation.To validate the aforementioned predictions,NSCLC cell lines A549 and H1299 were selected and divided into control,PUE,rapamycin(Rapa)positive control,gefitinib(EGFRi)and EGFRi+PUE groups.Cell viability was determined using Cell Counting Kit-8(CCK-8)and lactate dehydrogenase(LDH)assays.Real-time quantitative polymerase chain reaction(RT-qPCR)was employed to detect the expression levels of autophagy-and apoptosis-related genes,including unc-51 like autophagy activating kinase 1(ULK1),sequestosome 1(P62),lactate dehydrogenase A(LDHA),B-cell lymphoma-2(BCL2),and AMP-activated protein kinase(AMPK).RT-qPCR was applied to examine the expressions of ULK1,P62,LDHA,BCL2,and AMPK.Western blot was utilized to examine the protein expressions of microtubule-associated protein 1 light chain 3 β(LC3B Ⅱ/Ⅰ),ULK1,P62,LDHA,phosphorylated AMPK(P-AmpK),and ubiquitinated cysteine protease caspase 3.Immunofluorescence was used to observe changes in autophagy-related proteins P62 and LC3B.Flow cytometry was applied to analyze cell apoptosis level,mitochondrial membrane potential and reactive oxygen species(ROS)level.The regulatory effects of PUE on epidermal growth factor receptor(EGFR),intracellular phosphatidylinositol kinase(PI3 K),protein kinase B(Akt),mammalian target protein of rapamycin(mTOR),and proteins in ULK1 signaling pathway were analyzed.Results Nine key target genes of PUE against NSCLC were screened through network pharmacology and molecular docking.It was confirmed that PUE closely bound to multiple key target genes such as EGFR.GO and KEGG analyses revealed that PUE may exert anti-NSCLC effects by regulating signaling transduction,apoptosis,cell proliferation and PI3K/AKT pathways.In vitro cell experiments showed that when compared to control group,median lethal concentration(LC50)of PUE in A549 cells was 0.50 mmol/L(P＜0.05),and LC50 of PUE in H1299 cells was 1.00 mmol/L(P＜0.05).When PUE concentration was 0.25 mmol/L,the numbers of A549 and H1299 cells were decreased by 57.95％and 69.35％,respectively(P＜0.05).PUE treatment upregulated ULK1 and BCL2 expressions in A549 and H1299 cells,downregulated the expressions of LDHA,AMPK,P62,upregulated autophagy-related proteins LC3BⅡ/Ⅰ and ULK1,downregulated P62,LDHA and EFGR proteins(P＜0.05)and upregulated cleaved-caspase 3 protein expression in A549 cells(P＜0.05).Flow cytometry showed that PUE treatment induced early apoptosis of A549 and H1299 cells(P＜0.05),downregulated the levels of ROS and mitochondrial membrane potential(P＜0.05).Immunofluorescence showed that PUE treatment enhanced the LC3B fluorescence signal in A549 and H1299 cells,while weakened P62 fluorescence signal(P＜0.05).PUE,EFGRi and EFGRi+PUE treatments downregulated EGFR,PI3K,AKT,mTOR related pathway genes in A549 and H1299 cells(P＜0.05)and 7 key pathway proteins including P-EGFR,EGFR,P-PI3K,PI3K,P-AKT,AKT,and mTOR(P＜0.05).These effects were enhanced after inhibiting EGFR(P＜0.05),confirming that PUE exerted anti NSCLC effects through EGFR-mediated itPI3K/AKT/mTOR/ULK1 pathway.Conclusion PUE exerts anti-NSCLC effects,and its might mediate PI3K/AKT/mTOR/ULK1 pathway through EGFR,regulate autophagy and apoptosis in NSCLC cell lines A549 and H1299.
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维普
