Proteomics-based characterization of apigenin-mediated anti-inflammatory effects in TNF-α-stimulated ARPE-19 cells

Tumor necrosis factor-α (TNF-α)-induced inflammation in retinal pigment epithelial (RPE) cells contributes to retinal degenerative disorders. Here, we investigated the anti-inflammatory effects of apigenin, a flavonoid compound, in human ARPE-19 cells and explored the underlying mechanisms. Apigenin cytotoxicity was evaluated using the MTT assay, and ARPE-19 cells were then pretreated with apigenin prior to TNF-α stimulation. Apigenin significantly and dose-dependently suppressed TNF-α-induced secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and soluble intercellular adhesion molecule 1 (sICAM1). At the signaling level, apigenin reduced phosphorylation of inhibitor of κBα (IκBα) and p65 and attenuated activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38), while exerting minimal effects on inhibitor of κB kinase (IKK) and extracellular signal-regulated kinase (ERK). Label-free quantitative proteomics was performed to compare TNF-α-stimulated cells with control cells and apigenin-pretreated cells with TNF-α-stimulated cells. Differentially expressed proteins from both comparisons were subjected to integrative bioinformatic analysis, including functional enrichment and protein-protein interaction assessment, which identified an overlapping subset of TNF-α-responsive proteins modulated by apigenin and primarily associated with inflammatory and interferon-related processes. Based on these analyses, six representative proteins-ICAM1, STAT1, OAS3, IFIT3, ISG15, and SQSTM1/p62-were selected and further validated by Western blotting. Collectively, these findings demonstrate that apigenin attenuates TNF-α-driven inflammatory responses in RPE cells through partial suppression of NF-κB and MAPK signaling and regulation of interferon-related protein expression.