Role of Oe-ME in AP-1 Transmission Transduction To explore which molecule can be targeted by Oe-ME in the AP-1 signaling pathway, RAW264.7 cells were exposed to LPS, and the phosphorylated and total levels of JNK, p38, and ERK were tested. that this anti-inflammatory effect of Oe-ME could be attributed to its control of posttranslational modification and transcription of TAK1. is usually a herb that is commonly used as ethnopharmacological food and medicine in the Mediterranean, North America, and Australia. L. is usually traditionally used to treat hypertension, inflammatory diseases, cardiovascular diseases, malignancy, Magnolol and Magnolol obesity . Due to its numerous biophenols and other bioactive ingredients, it has been identified as a functional food. Special attention is usually paid to the biological and pharmacological properties of those compounds. Oleuropein, secoiridoids, hydroxytyrosol, caffeic acid, L. have been isolated and demonstrated to possess variety of pharmacological activities such as antioxidant , anti-inflammatory, antimicrobial, anti-atherosclerosis , and antiviral properties . Our previous results suggest that the methanol extract of L. (Oe-ME) and its components, kaempferol (KFL), quercetin (QCN), and luteolin, reduce the activation of Src and Syk in the NF-B signaling pathway and protects cells from Rabbit polyclonal to LCA5 excessive inflammation. However, the molecular mechanisms regarding its anti-inflammatory effect in other signaling pathways remains unknown. In this study, we aimed to explore how Oe-ME exerts its anti-inflammatory effect with respect to the AP-1 signaling pathway. 2. Results 2.1. Cytotoxicity and the Effect of Oe-ME on Production of Inflammatory Genes To address our experimental aims, we used two cell lines (RAW264.7 and HEK293T cells) in this study. RAW264.7 cells, mouse mononuclear macrophage-like cells, are immune cells that can show inflammatory responses to pathogens . Since RAW264.7 cells Magnolol show low transfection efficiency, we employed HEK293T cells displaying higher transfection efficiency to validate putative target protein by overexpression of its gene . We found that Oe-ME experienced no cytotoxicity on RAW264.7 and HEK293T cells at the tested concentrations (Determine 1a). LPS enables macrophages to release important inflammatory cytokines and mediators, which is vital to activate subsequent immune responses. Therefore, in our study, LPS was utilized to stimulate RAW267.4 cells. The role of Oe-ME in the production of inflammatory genes was explored by RT-PCR. The mRNA expression levels of IL-2, IL-6, COX-2, MMP-9, ICAM-1, TNF-, and MCP1 were sharply elevated by LPS, while their expression was decreased by Oe-ME treatment in a dose-dependent manner (Physique 1e). The same pattern was obtained of the expression levels of COX-2, TNF-, and IL-6 through quantitative real-time PCR (Physique 1bCd). The mRNA production of COX-2, TNF-, and IL-6 was significantly ( 0.05 and 0.01) decreased by Oe-ME. Pam3CSK4 and poly (I:C) were also applied to induce inflammation. After activation, the enhanced expression of COX-2, MMP-9, and ICAM1 was significantly ( 0.05 and 0.01) diminished by Oe-ME treatment (Physique 1f,g). In the mean time, whether expression levels of COX-2, MCP1, and ICAM1 can be modulated by prednisolone (a steroidal anti-inflammatory drug: positive control drug) or ranitidine (an H2 receptor blocker: unfavorable control drug) was examined. As Physique 1h shows, 100 M of prednisolone but not ranitidine significantly ( 0.01) reduced the expression of both COX-2 and MCP1 but not Magnolol ICAM1. Open in a separate window Open in a separate window Physique 1 Cell viability and the effect of Oe-ME on mRNA expression of inflammation-related genes. (a) RAW264.7 and HEK293T cells were treated with indicated doses of Oe-ME (50, 100, and 200 g/mL) and then incubated for 24 h. Cell viability was measured using an MTT assay. (b,c,d) After pretreatment with Oe-ME for 30 min, RAW264.7 cells were exposed to LPS and real-time PCR were employed to assess Magnolol the production of TNF-, COX-2, and IL-6. (e,f,g,h) RAW264.7 cells were incubated with LPS, poly (I:C), or pam3CSK4 in the absence or presence of various concentrations of Oe-ME. The mRNA expression levels of ICAM-1, IL-2, COX-2, MMP-9, and MCP1 were measured by RT-PCR. Prednisolone and ranitidine were used as positive and negative control drugs, respectively. Band intensity was measured and quantified using ImageJ. ## 0.01.