木犀草素通过靶向Akt信号通路诱导骨髓增生异常综合征细胞线粒体依赖性凋亡与衰老OA

Objective Myelodysplastic syndromes(MDS)are associated with a high risk of progression to leukemia,highlighting the critical need to explore novel therapeutic agents.Based on network pharmacology prediction and molecular docking,we hypothesized that luteolin(LUT)might induce cellular senescence and apoptosis in MDS cells by targeting the Akt signaling pathway.This study aims to verify its targets and molecular mechanisms through experimental studies.Methods By integrating multiple databases,such as PharmMapper and GeneCards,common targets of LUT and MDS were screened.A protein-protein interaction(PPI)network was constructed using the STRING database and visualized with Cytoscape software to identify key targets.The DAVID database was employed for the functional enrichment analysis of Gene Ontology(GO)and the pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)for the common targets.Molecular docking between LUT and the key targets was performed using AutoDock software,and the docking efficacy was evaluated by analyzing the binding energy.Subsequently,molecular dynamics simulations were carried out using Gromacs to observe the ligand-receptor interactions and conformational changes.MUTZ-1 and MOLM-13 cells were used as experimental models.After treating the cells with different concentrations of LUT for 24 h(MUTZ-1:0、15、30、60 μmol/L,MOLM-13:0、4、8、16 μmol/L),cell viability was measured by CCK-8 assay,DNA synthesis capacity was assessed by EdU assay,colony formation was examined by soft agar assay,morphological changes were observed by transmission electron microscopy(TEM),intracellular reactive oxygen species(ROS),mitochondrial ROS,mitochondrial membrane potential,and apoptosis rates were detected by flow cytometry,senescence was assessed by senescence-associated β-galactosidase(SA-β-Gal)staining,mRNA expression levels of senescence-associated secretory phenotype(SASP)factors were determined by RT-qPCR,and protein expression levels were examined by Western blotting.Results A total of 568 LUT targets and 2 348 MDS-related targets were screened,yielding 228 intersecting targets.The intersecting targets were imported into the DAVID database.GO functional enrichment analysis demonstrated that biological processes were enriched in the responses to oxidative stress and cellular senescence;cellular components were enriched in protein kinase complex and secretory granule lumen;and molecular functions were enriched in protein kinase activity and cytokine receptor binding.KEGG pathway enrichment analysis revealed predominant enrichment in key pathways including the Akt signaling pathway,cellular senescence,and apoptosis.The PPI network constructed using the STRING database and Cytoscape software identified the top 10 key targets,as IL6,NFKB1,STAT3,TNF,JUN,BCL2,ALB,CASP3,Akt1 and IL1B.Molecular docking results indicated that the binding energy between LUT and Akt1 was-6.25 kcal/mol,suggesting a strong interaction.The molecular dynamics simulation results demonstrated stable binding between LUT and the Akt1 target.In vitro experimental results showed that LUT inhibited the proliferation of both MUTZ-1 and MOLM-13 cells in a dose-dependent manner.The half-maximal inhibitory concentration(IC ₅ ₀)of LUT acting on MUTZ-1 cells for 24 h was 29.960 μmol/L,and that on MOLM-13 cells for 24 h was 8.654 μmol/L.Treatment of MUTZ-1 and MOLM-13 cells with various concentrations of LUT for 24 h significantly inhibited DNA synthesis and colony formation(P<0.01).The treatment of LUT at different concentrations for 24 h resulted in significant increases in intracellular ROS and mitochondrial ROS levels in a dose-dependent manner(P<0.05),decrease in mitochondrial membrane potential in a dose-dependent manner(P<0.05),and elevation of apoptosis rates in a dose-dependent manner(P<0.05).TEM revealed obvious apoptotic morphological features in MUTZ-1 and MOLM-13 cells after 24 h of LUT treatment.The protein levels of Cleaved PARP and Cleaved caspase-3,and the pro-apoptotic protein BAX were significantly upregulated,and that of the anti-apoptotic protein BCL-2 was significantly downregulated.SA-β-Gal staining showed that the proportion of senescent cells was significantly increased in MUTZ-1 and MOLM-13 cells after LUT treatment for 24 h,along with enhanced expression of related SASP factors(P<0.001),and significant upregulation of senescence-associated proteins p53,p21 and p16.After LUT treatment for 24 h,the protein expression of p-Akt(Ser473)and p-MDM2(Ser166)was significantly downregulated,while the total Akt protein expression showed no significant change.Conclusion LUT may induce mitochondria-dependent apoptosis and senescence in MDS cells by targeting the Akt signaling pathway,thereby inhibiting the proliferation of MDS cells.