【作者】 蒋叶；

【导师】 余书勤；

【作者基本信息】 南京师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 艾拉莫德(T-614)，是一种尚未上市的非甾体抗炎药(NSAIDs)，研究表明，它对类风湿性关节炎和骨关节炎有明显的治疗作用，但迄今尚未有文献报道T-614对肺部炎症的影响。本文从NF-κB途径、p38 MAPK信号通路及炎症因子表达方面研究了T-614对脂多糖(LPS)介导的肺泡巨噬细胞炎症过程的作用，同时在整体水平利用动物急性肺损伤(ALI)模型研究了T-614对肺损伤的干预作用。 1．T-614对LPS激活的大鼠肺泡巨噬细胞系NR8383的作用： 体外培养NR8383经不同浓度的T-614处理，LPS刺激后应用酶联免疫吸附法(ELISA)检测细胞上清中TNF-α的水平，半定量逆转录聚合酶链式反应(RT-PCR)检测TNF-α mRNA水平，ELISA法检测NF-κB的活性，western blotting法检测磷酸化p38 MAPK的表达。结果表明T-614对LPS诱导的NR8383细胞TNF-α mRNA水平和蛋白水平的上调有显著抑制作用，对NF-κB的转录活性也有抑制作用，但对p38 MAPK磷酸化程度无明显作用。 2．T-614对大鼠油酸型ALI模型的干预作用： 本研究首次应用大鼠油酸型ALI动物模型研究T-614的体内效应。T-614于造模前给药，剂量为20、40、80mg／kg，造模后利用动物肺功能测定仪实时测定肺损伤过程中肺容量(V)和动态肺顺应性(Cdyn)的变化，并收集支气管肺泡灌洗液(BALF)，检测灌洗液中蛋白浓度的变化。结果发现T-614可显著改善油酸型肺损伤大鼠的肺功能，明显降低灌洗液中蛋白浓度，表明T-614对油酸所致大鼠ALI具有保护作用。 本研究结果表明T-614可抑制LPS诱导的大鼠肺泡巨噬细胞系NR8383表达TNF-α，此作用可能通过干预NF-κB活性而非p38 MAPK实现；另外，T-614对油酸型ALI大鼠的肺容量、动态肺顺应性及灌洗液中蛋白浓度的影响表明T-614对大鼠油酸型ALI具有一定的保护作用。因此我们推测：T-614可能通过对NF-κB通路的干预抑制炎症反应，因而对急性肺损伤具有治疗潜能。更多还原

【Abstract】 Iguratimod (T-614), is a non-steroidal anti-inflammatory drug that hasn’t come into the market. Studies show that T-614 has obvious curative effect on rheumatoid arthritis and osteoarthritis, but there are no reports about its effect on lung inflammation. In the present study, we investigate the effect of T-614 on lipopolysaccharide (LPS)-mediated inflammatory process on the level of NF-kB pathway, p38 MAPK pathway and inflammatory factors.In addition, we study the intervene effect of T-614 on animal acute lung injury (ALI) model. 1. The effect of T-614 on LPS-induced rat pulmonary alveolar macrophage cell line NR8383:NR8383 cellls were pretreated with T-614, then were stimulated with LPS. The production of TNF-α in the supernatant of NR8383 was determined with enzyme-linked immunosorbent assay (ELISA).The TNF-α mRNA level was determined by a semi-quantitative PCR assay. NF-κB activity in nuclear extract ofNR8383 was detected with ELISA. The phosphorylation of p38 MAPK was measured by western blotting. The findings indicated that T-614 could inhibit LPS-stimulated mRNA expression and production of TNF-α, and also inhibited the activation of NF-κB, but had no obvious inhibitory effect on the phosphorylation of p38 MAPK. 2. The effect of T-614 on oleic acid-induced ALI rat model:We use an oleic acid-induced ALI rat model to investigate the effect of T-614 in vivo for the first time. T-614 was given before injection of oleic acid at doses of 20, 40, and 80 mg/kg. The changes of lung volume and dynamic lung compliance were real time recorded by anrise2005 pulmonary function test apparatus after injection of oleic acid. We also collected bronchoalveolar lavage fluid(BALF), and measured the protein concentration in BALF. The findings showed that pretreatment of T-614 significantly alleviated oleic acid-induced lung injury accompanied by improvement of lung function and decline of protein concentration in BALF, which suggested that T-614 had a beneficial effect on ALI induced by oleic acid in rat.更多还原