Studies On The Interactions Between TCDCA And Glucocorticoid Receptor And Its Inntlence On Activator Protein-1

As a kind of nature bioactive substances of animal bile acids, taurocholiccheno-deoxycholic acid (TCDCA) showed noticeably anti-inflammatory and immuno-modulatory effects. TCDCA with cyclopentane multiple hydrogen phenanthrene on the basic structure, was similar with steroid hormones on their chemical structure. Fibroblast-like synoviocytes (FLS) of adjuvant arthritis (AA) was used as the research subjects to investigate the anti-inflammatory and immunomodulatory mecheanism of TCDCA in depth, and interaction between TCDCA and glucocorticoid receptor (GR) and effects of TCDCA on transcription factor activator protein-1(AP-1) and apoptosis of AA FLS were discussed in this study. The details are as follows:(1) The interaction between TCDCA and GRAutodock software was used to dock TCDCA and GR. Activation of GR was determined by luciferase reporter gene assay. The process of GR translocation was observed by fluorescence microscopy. The results showed that TCDCA could interact with GR at Leu732, and the binding Gibbs free energy change was-4.51kcal/mol, Kd=498.39μM. Besides, TCDCA at the concentration of1×10-4M, compared with the control group, could significantly activate GR and translocation was observed.(2) The influences of TCDCA on the expression and the activities of AP-1and intercellular adhesion molecule-1(ICAM-1)Western blot was used to measure the expressions of c-Fos, c-Jun and p-c-Jun (Ser63) in AA FLS. ELISA was used to measure the AP-1DNA binding activation in AA FLS. The results showed that TCDCA at the concentration1×10-6～1×10-4M could significantly inhibit the protein expressions of c-Fos, c-Jun and p-c-Jun (Ser63) and the DNA binding activation of AP-1.Quantitative PCR (qPCR) and ELISA was used to measure the mRNA and protein expression of ICAM-1respectively in AA FLS. The results showed that TCDCA at the concentration1×106～1×10-4M could significantly inhibit the mRNA and protein expressions (P<0.05), and the inbition of TCDCA could be blocked by mifepristone (RU486).(3) The effects of TCDCA on the apoptosis of AA FLSApoptosis rate of AA FLS induced by TCDCA was determined by flow cytometric analysis. Gene expression levels and the activities of Caspase-3and Caspase-8were evaluated using qPCR and ELISA. The results showed that TCDCA at the concentration of400μg/mL could markedly increase the apoptosis rate of AA FLS (P<0.01). Both caspase-3and caspase-8mRNA levels of AA FLS, comparaed with control group, were markedly increased by treatment with400μg/mL TCDCA. The activities of caspase-8and caspase-3, comparaed with control group, were significantly increased by treatment with50～200μg/mL TCDCA and100～400μg/mL TCDCA, respectively.Conclusions of this study are as follows:(1) TCDCA could bind with GR and activate GR.(2) TCDCA could significantly inhibit the protein expression of c-Jun、p-c-Jun、 c-Fos and the DNA binding activation of AP-1in AA FLS.(3) TCDCA could significantly inhibit both the gene and protein expressions of ICAM-1in AA FLS.(4) TCDCA could significantly increase the gene expression and activation of Caspase-3and Caspase-8in AA FLS, and promote apoptosis of AA FLS.(5) The anti-inflammatory and immunomodulatory effects of TCDCA were related with its influence on GR mediated genomic signaling pathway.