雷公藤内酯醇抑制滑膜成纤维细胞COX-2和iNOS表达

Effect of Triptolide on TNFα-induced activation of NF-κB and expression of COX-2 and iNOS in human rheumatoid arthritis synovial fribroblasts

目的 :研究雷公藤内酯醇对类风湿关节炎滑膜成纤维细胞 (RASF)增殖和表达环氧化酶 - 2 (COX- 2 )、诱导型一氧化氮合酶 (i NOS)及合成前列腺素 E2 (PGE2 )、一氧化氮 (NO)的影响 ,并探讨其机制。方法 :分离培养人类风湿关节炎滑膜成纤维细胞 ,用 TNFα(2 0μg/ L)刺激 ,同时加或不加雷公藤内酯醇 (TP,0～ 10 0μg/ L )。分别用 3 H-Td R法、竞争酶联免疫吸附试验 (ELISA)和硝酸酶还原法、半定量逆转录 -聚合酶联反应 (RT- PCR)法、细胞酶免疫法及蛋白免疫印迹 (Western- blot)法 ,检测滑膜细胞的增殖活性、细胞培养上清液中 PGE2和 NO的水平 ,滑膜细胞 COX- 2和 i NOSm RNA表达水平及蛋白表达水平。同时提取各组细胞蛋白 ,测定其核转录因子 NF-κB活性。结果 :TP(>2 0μg/ L)能显著抑制 TNFα诱导的 RASF COX- 2和 i NOS表达 ,并明显减少 PGE2和 NO的生成 ,抑制作用与 TP浓度呈明显相关性。同时观察到 ,TP对 TNFα激活的 RASF核转录因子 NF- κB活性有明显的抑制作用(IC50 ≈ 35 μg/ L) ,TP对 RASF NF- κB活性的抑制程度与其抑制 RASF COX- 2和 i NOS表达的效应相一致。实验未观察到 TP对 RASF增殖的影响。结论 :TP可显著抑制 TNFα刺激的 RASF COX- 2和 i NOS的表达及其诱导产物PGE2和 NO的生成 ,这?

Objective: To explore the effects of Triptolide (TP) on TNFα-induced 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). Methods: Fibroblasts (RASF) were obtained from synovial tissue of patients with RA and were cultured in vitro. RASF were stimulated with TNFα(20 μg/L) in the presence or absence of TP (0～100 μg/L) for 20 h. 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. Expressions 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 measured by an ELISA-based method. Results: TP (>20 μg/L) down-regulated markedly TNFα-induced COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was po-sitively correlated with TP concentrations. NF-κB activity in TNFα-stimulated synovial cells was suppressed profoundly by TP treatment (IC 50≈35 μg/L). The activity of NF-κB was correlated with the levels of COX-2 and iNOS expression in TNFα-stimulated RASF. No change was observed in proliferation of synovial cells after treatment of TP. Conclusion: TP could significantly down-regulate TNFα-induced COX-2, iNOS expression and production of PGE2, NO in human RASF, which is associated with the suppression of NF-κB activity.