For stopping of the reaction, we added the stop solution to each well

an IL-1b or TNF-a, or vehicle control for 4 hours. The luciferase activity was detected on a Glomax Multi-functional Plate Reader using the Promega Luciferase Assay System kit according to manufacturer’s instructions. The relative luciferase activity of each group was compared to control vehicle. Statistical analysis Assessment of IL-1b and IL-6 mRNA stability Cells were pretreated with apigenin for 2 h and then treated with LPS or vehicle control for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19672638 2 h before addition of actinomycin D. The cells were harvested for isolation of total cellular RNA at 0.5, 1, 2, 4 and 6 h after addition of actinomycin D. The mRNA stability of IL-1b and IL-6 were quantified with real-time PCR as described previously. All of the experiments were repeated at least three times and the data were expressed as mean 6 SD. One-way ANOVA was employed to analyze the differences between sets of data. To confirm the differences occurred between groups, post hoc tests were used for follow-up test. Statistics were performed using GraphPad Prism 5.0. A value of p, 0.05 was considered statistically significant. Western blot analysis Total cell lysates were prepared as previously described. The protein concentration was determined using Bio-Rad protein assay reagent. A total of 70 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19673983 mg of proteins was resolved on 10% BisTris gels and transferred to nitrocellulose membranes. The protein levels of target genes were detected using specific primary antibodies and IRDye secondary antibodies on Odyssey Fluorescence Imaging System. The density of the immunoblots was analyzed using Odyssey V3.0 software and normalized with b-Actin. Results Identification of the target genes regulated by apigenin in LPS-mediated immune response in macrophages Although apigenin has been BAY 41-2272 supplier reported to have anti-inflammatory activities and is able to inhibit the expression of several proinflammatory cytokines such as TNF-a and IL-6, there is no information available regarding the effect of apigenin on LPSinduced immune response. In order to determine the effect of apigenin on LPS-induced inflammatory response and identify the specific target genes, we did the quantitative real-time PrimePCR array using the Bio-Rad predesigned assay specifically for inflammatory immune response. The differentiated THP-1 macrophages were pretreated with apigenin for 2 h and then treated with LPS or vehicle control for 24 h. Total cellular RNA was isolated and reverse transcribed into firststrand cDNA. A total of 20 ng of cDNA was used to run a realtime PrimePCR array according to the protocol recommended by the manufacturer. The results indicated that in LPS-stimulated macrophages, more than two dozen genes were significantly upregulated including IL-6, IL-8, IL1b, IL12b, NFKBIA, and NFKB1. But IL-10 and TLR4 were significantly down-regulated. Cytokines are important immunomodulation agents in regulating host responses to infection, inflammation, sepsis, and cancer. As shown in Fig.3, apigenin not only significantly inhibited LPS-induced up-regulation of pro-inflammatory cytokines, such as IL-1b, IL-6, and IL-12b, but also reduced LPS-induced increase of inflammatory chemokine CCL5 and adhesion molecules. In addition, LPS-induced down-regulation of IL-10 was reversed by apigenin. These results suggest that apigenin may Immunofluorescence staining of ASC Human THP-1-derived macrophages were cultured on 22622mm glass coverslips in 6-well plates. Cells were pretreated with apigenin for 2 h and then treated with LPS or vehicl