金雀异黄素对脂多糖诱导的巨噬细胞MAPK和TLR信号转导通路的影响

Effect of genistein on the TLR and MAPK transduction cascades in lipopolysaccharide-stimulated macrophages

目的研究金雀异黄素对脂多糖(LPS)诱导的巨噬细胞丝裂原活化蛋白激酶(MAPK)信号转导通路和Toll样受体(TLR)通路的影响。方法用100 ng/mL脂多糖和金雀异黄素分别处理RAW264.7巨噬细胞不同时间后,采用Western blot法检测对MAPK信号通路蛋白磷酸化的影响;采用RT2 ProfilerTMPCR芯片检测金雀异黄素对LPS诱导的TLR信号转导通路基因表达的影响。结果 LPS能够显著诱导蛋白p38和p42/44磷酸化,激活MAPK信号通路,金雀异黄素能够加强其作用,同时,LPS能够显著诱导TLR信号转导通路的细胞因子基因表达,包括IFN-β、IL-10、IL-1α、IL-1β、IL-6、TNF-α、集落刺激因子2(CSF-2)、CSF-3、趋化因子CCL2和CXCL10、环氧合酶2(COX-2)、NF-κB1和IκB-α等,金雀异黄素能够显著降低这些上调基因的表达。结论金雀异黄素能够显著增强LPS激活的MAPK信号通路并抑制TLR信号通路的激活。

Objective To delineate and confirm the signaling processes involving mitogen-activated protein kinase （MAPK） and Toll-like receptor （TLR） in the regulation of lipopolysaccharide （LPS）-induced activity of macrophages by genistein at the protein and transcriptional levels. Methods RAW264.7 macrophages were treated with genistein and then stimulated with LPS （100 ng/mL） for different time duration. We evaluated the induction, of MAPK phosphorylation by Western blot analysis; RT2 ProfilerTM PCR array was used to investigate the expressions of TLR pathway-related genes after different treatments. Results LPS led to the phosphorylation of Raf, MEK1/2 and ERK1/2 at 15 minutes post-stimulation and it lasted till 30 minutes. Phosphorylation of p38 was also observed, but it was not as obvious as p-ERK1/2. Addition of genistein further increased the phosphorylation of ERK1/2, its downstream protein p90rsk and p38. Cells treated with LPS and LPS together with genistein demonstrated significant number of genes to be differentially regulated as compared with control cells. Genistein alone could up-regulate the gene CD80, MEKK1, c-los, Rela, and Ticam2. LPS could up-regulate 20 genes including cytokines IFN-β, IL-10, IL-1α, IL-1β, IL-6, TNF-α, colony stimulating factor 2 （CSF-2） and CSF-3, chemokines CCL2 and CXCL10, transcription factor NF-KB1, IKB-a and cyclooxygenase 2 （COX-2）. The presence of genistein led to a strong inhibition of the expressions of these genes and up-regulated the transcription factors IκB-β and c-Rel, a subunit of NF-KB. Conclusion Genistein strongly enhances the LPS-induced activities of MAPK transduction cascades and inhibits TLR pathway.