[Enhancement by FK506 of triptolide-induced inhibition of expression of COX-2 and iNOS in human rheumatoid arthritis synovial fibroblasts].
To explore the effects of FK506 on the inhibition by triptolide (TP) of cell proliferation and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their inducing products PGE2, NO in human rheumatoid arthritis synovial fibroblasts (RASF), and to study the mechanisms of combination of FK506 and TP in RA therapy, RASF used in the experiments were obtained from synovial tissue of patients with RA and were cultured. RASF were pretreated with FK506(10-1000 nmol/L)for 2 h, then the cells were stimulated with TNF alpha(20 microg/L) in the presence or absence of TP (10 microg/L). The RASF proliferation was determined by [(3)H]-TdR incorporation, and the productions of PGE2 and NO in culture supernatants of RASF were detected with competitive ELISA and enzyme reduction of nitrate. Expression of COX-2 and iNOS mRNA in RASF were analyzed by semi quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western blot method and cellular enzyme immunoassay in synovial fibroblasts. NF-kappa B activity in whole-cell extract of RASF was also measured by an ELISA-based method. Results showed that neither FK506 nor TP at lower concentration (10 microg/L) alone affected TNF alpha-induced COX-2, iNOS expression and production of PGE2, NO in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 microg/L) down-regulated COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was positively correlated with FK506 concentrations (10-1000 nmol/L). NF-kappa B activity in TNF alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 microg/L) treatment than those with TP (10 microg/L) alone. No change was observed in inhibition of proliferation of synovial cells after combined treatment of FK506 and TP. In conclusion, FK506 enhanced TP-mediated down-regulation of COX-2, iNOS and their inducing products PGE2, NO in human RASF by suppressing the activity of NF-kappa B.
Yao
Hang-Ping
HP
Institute of Immunology, College of Medical Sciences, Zhejiang University, Hangzhou 310031, China. yaohp2001@yahoo.com.cn
Shao
Xue-Ting
XT
Sun
Wen-Ji
WJ
Shao
Chuan-Sen
CS
Zhang
Li-Huang
LH
D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D015237:Dinoprost / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004852:Epoxy Compounds; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010616:Phenanthrenes; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D016559:Tacrolimus / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004224:Diterpenes; D004852:Epoxy Compounds; D007166:Immunosuppressive Agents; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D010616:Phenanthrenes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C001899:triptolide; D009569:Nitric Oxide; D015237:Dinoprost; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D016559:Tacrolimus