Elucidation of signaling pathways involved in effects of plant flavonoids in human primary macrophages

Background: Apigenin and luteolin are plant flavonoids demonstrating antitumorigenic , antioxidant, and anti-inflammatory properties. Previously, we showed an influence of apigenin and luteolin on FOXO1 nuclear translocation and target gene expression in liver cells. However, there is little information about the effects of apigenin und luteolin in human primary cells. Methods: Human monocyte-derived macrophages were treated with flavonoids for 1h followed by stimulation with 100 ng/ml LPS for 4h or 24h. Gene expression of FOXO1 target genes and cytokines was determined by qRT-PCR. Cytokine expression in culture medium was measured using Luminex technology. For the screening of signaling pathways involved in flavonoid effects, apigenin/luteolin pretreated cells were stimulated with 100nM insulin or 100 ng/ml LPS for 30 min, and protein lysates were analyzed using PathScan Intracellular Signaling Array. Results: Apigenin (10, 20µM) and luteolin (20, 30µM) completely blocked the LPS-induced increase of mRNA expression and secretion of cytokines TNFalpha, MCP1, IL6 and IL10 after 4h and 24h. For most cytokines studied, suppression was found already by treatment with 0.05µM of flavonoids. Moreover, apigenin or luteolin treatment also reduced the mRNA expression of the LPS receptor TLR4 , antioxidative gene SOD , and cell cycle modulating genes GADD45 and CCNG2 . Furthermore, PathScan array revealed decreased phosphorylation of key proteins of the insulin signaling pathway (Akt, S6 Ribosomal protein, PRAS40), as well as proteins involved in inflammatory signaling (ERK1/2) and cell survival (Bad, GSK3beta). Conclusion: Our data suggest anti-inflammatory effects of apigenin and luteolin in primary human macrophages and elucidate a range of signalling pathways involved.