Investigating the mechanism of luteolin in the treatment of inflammatory bowel disease based on network pharmacology, normal mode analysis and experimental validation

Abstract

This study aimed to explore the therapeutic mechanism of luteolin in the context of inflammatory bowel disease (IBD) by integrating in vitro experiments, network pharmacology, molecular docking, and normal mode analysis. The impacts of luteolin on cell toxicity and survival were assessed by a CCK-8 assay using a LPS-induced HT-29 cell model of inflammation. Flow cytometry was utilized to analyze cell apoptosis. Levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were quantified using commercially available test kits, and intracellular reactive oxygen species (ROS) were detected via flow cytometric analysis. Putative targets of luteolin were sourced from the ECTM and SuperPred databases, while genes related to IBD were acquired from the GeneCards database. Common genes between the candidate targets of luteolin and those associated with IBD were discerned by employing a Venn diagram. Subsequent functional enrichment analyses, covering both Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were executed using the DAVID platform. A network of protein-protein interactions (PPI) was constructed with the STRING database and graphically represented in Cytoscape software (v3.10.2) to identify central targets. Molecular docking investigations were conducted using AutoDockTools 1.5.7, and normal mode analysis was performed on the iMODS server. The results indicated that 2.0 μg/mL of luteolin inhibited cell viability (83.66 % vs 100 %, P < 0.05); therefore, subsequent experiments utilized concentrations of 0.1, 0.5, and 1.0 μg/mL. In comparison with the LPS model group, 0.5 and 1.0 μg/mL of luteolin enhanced cell viability (92.67 % vs 78.00 %, 97.00 % vs 78.00 %, P < 0.05) and SOD levels (18.00 U/mg vs 10.00 U/mg, 21.00 U/mg vs 10.00 U/mg, P < 0.05), while reducing apoptosis (15.00 % vs 29.00 %, 8.00 % vs 29.00 %, P < 0.05), MDA content (13.33 mmol/mg vs 20.00 mmol/mg, 10.50 mmol/mg vs 20.00 mmol/mg, P < 0.05), and ROS levels (27.50 % vs 41.50 %, 14.16 % vs 41.50 %, P < 0.05). No significant differences in these indices were observed with the treatment with 0.1 μg/mL of luteolin ( P > 0.05). Network pharmacology analysis identified 25 common targets for luteolin and IBD targets. These key targets were primarily associated with signaling pathways, such as the PI3K-AKT pathway, cancer, and cellular aging. Further, the results revealed a high binding affinity between luteolin and the AKT1 protein. The findings suggest that luteolin may exert its therapeutic effects on IBD by targeting AKT1 and modulating the PI3K-AKT signaling pathway, thereby inhibiting oxidative stress injury.