Vergne P, Liagre B, Bertin P, Cook-Moreau J, Treves R, Beneytout J L, Rigaud M
Department of Rheumatology, CHRU Dupuytren, and the University of Medicine of Limoges, France.
J Rheumatol. 1998 Mar;25(3):433-40.
Our objective was to characterize the effect of methotrexate (MTX) on prostaglandin E2 (PGE2) synthesis in cultured human rheumatoid synovial cells. Prostaglandins (PG) are important mediators of inflammation and joint destruction in rheumatoid arthritis (RA). Two isoforms of cyclooxygenase (COX), the key enzyme in PG synthesis, have been characterized: a constitutively expressed form, COX-1, and an inducible form, COX-2. The mechanisms of action of low dose MTX in RA treatment are still poorly understood. As the clinical effects are often first noticed within a month of starting MTX therapy, an antiinflammatory action has been proposed.
Adherent synovial cells were obtained by collagenase digestion of rheumatoid synovium, isolated from patients with RA undergoing synovectomy. Between passages 3 and 6, cultured synovial cells were incubated with or without MTX for 54 h, at various concentrations. Interleukin (IL)-1beta (1 ng/ml) was added or not for the last 6 h of incubation. Supernatants were harvested and assayed for PGE2 by enzyme immunoassay (EIA). Exogenous [1-14C]arachidonic acid metabolism of synoviocytes was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). COX-1 and COX-2 mRNA expression was determined by total RNA extraction and reverse transcription polymerase chain reaction.
Cellular viability was not affected by MTX. EIA showed that MTX decreased IL-1beta induced PGE2 production by synoviocytes in a dose dependent manner. RP-HPLC analysis confirmed the inhibition of PGE2 and (12S)-12-hydroxy-5,8,10-heptadecatrienoic acid production. COX-1 and IL-1beta induced COX-2 mRNA expression were not inhibited by MTX.
MTX has an inhibitory effect on IL-1beta stimulated production of PGE2 by cultured human rheumatoid synoviocytes, without affecting either COX mRNA expression. Among various biochemical and immunologic events, MTX could have an antiinflammatory action by decreasing PGE2 release.
我们的目的是研究甲氨蝶呤（MTX）对培养的人类风湿性滑膜细胞中前列腺素E2（PGE2）合成的影响。前列腺素（PG）是类风湿性关节炎（RA）中炎症和关节破坏的重要介质。已鉴定出PG合成中的关键酶环氧化酶（COX）的两种同工型：一种组成型表达形式COX-1和一种诱导型COX-2。低剂量MTX在RA治疗中的作用机制仍知之甚少。由于临床效果通常在开始MTX治疗后的一个月内首次显现，因此有人提出了其抗炎作用。
通过胶原酶消化类风湿性滑膜组织获得贴壁滑膜细胞，该滑膜组织取自接受滑膜切除术的RA患者。在第3至6代之间，将培养的滑膜细胞在不同浓度下与MTX一起或不与MTX一起孵育54小时。在孵育的最后6小时添加或不添加白细胞介素（IL）-1β（1 ng/ml）。收集上清液并通过酶免疫测定（EIA）测定PGE2。通过反相高效液相色谱（RP-HPLC）分析滑膜细胞的外源性[1-14C]花生四烯酸代谢。通过总RNA提取和逆转录聚合酶链反应测定COX-1和COX-2 mRNA表达。
细胞活力不受MTX影响。EIA显示MTX以剂量依赖性方式降低IL-1β诱导的滑膜细胞PGE2产生。RP-HPLC分析证实了对PGE2和（12S）-12-羟基-5,8,10-十七碳三烯酸产生的抑制作用。MTX未抑制COX-1和IL-1β诱导的COX-2 mRNA表达。
MTX对培养的人类风湿性滑膜细胞中IL-1β刺激的PGE2产生具有抑制作用，而不影响COX mRNA表达。在各种生化和免疫事件中，MTX可能通过减少PGE2释放而具有抗炎作用。
