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研究了FK5 0 6对雷公藤内酯醇抑制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 (iNOS)及合成前列腺素E2 (PGE2 )、一氧化氮 (NO)的影响 ,探讨了FK5 0 6联合雷公藤抗类风湿作用的机制。分离培养人类风湿关节炎滑膜成纤维细胞 ,用不同浓度的FK5 0 6处理后 ,加或不加雷公藤内酯醇 (TP)和TNFα ,分别用 [3H] TdR法、竞争ELISA和硝酸酶还原法、半定量逆转录 聚合酶链反应 (RT PCR)法、细胞酶免疫法及Western印迹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iNOSmRNA表达水平及蛋白质表达水平。同时提取各组细胞蛋白质 ,测定其核转录因子NF κB活性。结果表明 ,FK5 0 6( 10～ 10 0 0nmol/L)及低浓度的TP( 10 μg/L)单独对TNFα活化的RASF表达COX 2、iNOS及合成PGE2、NO均无明显的抑制作用 ;FK5 0 6与低浓度的TP联合作用后 ,对活化的RASF具有不同程度的抑制作用 ,并与联合的FK5 0 6浓度 ( 10～ 10 0 0nmol/L)呈明显正相关性 ,且两者联合对TNFα激活的RASF核转录因子NF κB活性有明显的抑制作用。实验未观察到FK5 0 6对TP抑制滑膜细胞增殖的影响。以上结果说明 ,FK5 0 6可显著增强TP对滑膜细胞COX 2和iNOS的表达及其产物PGE2和NO合成
To study the effects of FK506 on triptolide inhibition of rheumatoid arthritis synovial fibroblast (RASF) proliferation and expression of cyclooxygenase 2 (COX 2 ), inducible nitric oxide synthase (iNOS) and synthetic prostaglandins Effects of E2 (PGE2) and nitric oxide (NO) on the mechanism of anti-rheumatoid effect of FK506 combined with Tripterygium wilfordii. Human rheumatoid arthritis synovial fibroblasts were isolated and cultured, treated with different concentrations of FK506, with or without triptolide (TP) and TNFα, respectively, using [3H] TdR method, competitive ELISA, and nitrate reduction The proliferative activity of synovial cells, the levels of PGE2 and NO in cell culture supernatants, and the synoviocytes COX 2 and cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT PCR), cellular enzyme immunoassay and Western blotting. iNOS mRNA expression levels and protein expression levels. At the same time, each group of cellular proteins was extracted to determine its nuclear transcription factor NFκB activity. The results showed that FK506 (10～100 nmol/L) and low concentration of TP (10 μg/L) alone had no significant inhibitory effect on the expression of COX2, iNOS, PGE2, and NO by activated TNFα; FK5 Combined with low concentration of TP, the combination of 0.6 and activated RAS has different degrees of inhibitory effect on the activated RASF, and has a positive correlation with the combined FK506 concentration (10～100 nmol/L). TNFα activated RASF nuclear transcription factor NF κB activity has a significant inhibitory effect. No effect of FK506 on TP inhibition of synoviocyte proliferation was observed in the experiment. The above results show that FK506 can significantly enhance the expression of COX 2 and iNOS in synovial cells and the synthesis of PGE2 and NO in synovial cells.