基于生物信息学及实验验证探讨宣肺化浊方缓解急性肺损伤的潜在成分及作用机制OA

Objective To explore the potential components and mechanisms of Xuanfei Huazhuo Prescription(XFHZP)in alleviating acute lung injury(ALI)using bioinformatics combined with cellular experiments.Methods(1)The blood-absorbed components of XFHZP were characterized using ultra performance liquid chromatography Triple time of flight mass spectrometry(UPLC-Triple TOF-MS/MS).The targets of these components were predicted using the Swiss Target Prediction and SEA databases.ALI-related targets were retrieved from the Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),DrugBank,and GeneCards databases.Overlapping targets between blood-absorbed components and ALI were subjected to protein-protein interaction(PPI)analysis via the STRING database to identify key targets.A"herb-component-target"network was constructed using Cytoscape,and topological analysis was performed with the NetworkAnalyzer plugin to identify hub targets.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted using the DAVID database.ALI microarray data were obtained from the GEO database.Hub targets that were present in both the network and GEO datasets were further analyzed using random forest model,least absolute shrinkage and selection operator(LASSO)regression,and support vector machine recursive feature elimination(SVM-RFE)to screen core targets.Molecular docking was performed using Schrödinger software to identify potential components.(2)An apoptosis model was established in alveolar epithelial cells(MLE-12)induced by lipopolysaccharide(LPS),and cells were treated with different concentrations of the potential components.Cell proliferation was assessed using the CCK-8 assay;apoptosis was detected by flow cytometry;protein expression levels of PI3K,p-PI3K,Akt,and p-Akt were measured by Western Blot.Results(1)A total of 17 prototype components of XFHZP were absorbed into the blood,and 118 potential targets were identified for ALI treatment.PPI and network analysis indicated that PIK3CA,AKT1,etc.,were hub targets,mainly involved in biological processes such as phosphorylation and negative regulation of apoptosis,and mediated signaling pathways including PI3K/Akt,MAPK,and apoptosis.Machine learning identified AKT1 as the core target of XFHZP in treating ALI.Molecular docking showed that piceatannol and chrysin exhibited more stable binding affinity with AKT1.(2)Piceatannol and chrysin effectively promoted the proliferation of LPS-induced MLE-12 cells.Concentrations of 0.25 and 1 μmol·L-1 for piceatannol,and 24 μmol·L-1 for chrysin were selected for subsequent experiments.Compared with the blank group,the model group showed a significant increase in apoptosis rate(P＜0.01)and upregulation of phosphorylation levels of Akt and PI3K(P＜0.01).Compared with the model group,both piceatannol and chrysin at different concentrations inhibited apoptosis(P＜0.01).Furthermore,piceatannol downregulated the ratios of p-Akt/Akt and p-PI3K/PI3K(P＜0.01),and 4 μmol·L-1 chrysin inhibited the phosphorylation activation of the PI3K/Akt pathway(P＜0.01).Conclusion The blood-absorbed components piceatannol and chrysin from XFHZP effectively reduce apoptosis of alveolar epithelial cells and exhibit a prominent effect in alleviating epithelial injury,possibly through inhibiting the phosphorylation activation of the PI3K/Akt pathway.