Enhancement by FK506 of Triptolide-induced Inhibition of Expression of
COX-2 and iNOS in Human Rheumatoid Arthritis Synovial Fibroblasts

( Institute of
Immunology, College of Medical Sciences, Zhejiang University, Hangzhou 310031，
China；
¹ The First Affiliated Hospital, College of Medical Sciences, Zhejiang University,
Hangzhou 310003, China )

Abstract
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 fribroblasts (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 TNFα(20 μg/L) in the presence or absence of TP (10 μg/L).
The RASF proliferation was determined by [³H]-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-κB activity in whole-cell extract
of RASF was also mea-sured by an ELISA-based method. Results showed that neither
FK506 nor TP at lower concentration (10 μg/L) alone affected TNFα-induced
COX-2, iNOS expression and production of PGE2, NO in synovial cells. Combined
treatment of FK506 and a lower concentration of TP (10 μg/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-κB activity in TNFα-stimulated synovial
cells was suppressed more profoundly by FK506 plus TP (10 μg/L) treatment
than those with TP (10 μg/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-κB.