论文部分内容阅读
目的探讨活性氧(ROS)、转化生长因子β1(TGF-β1)在醛固酮促进系膜细胞生成纤溶酶原激活物抑制剂1(PAI-1)途径中的作用。方法用醛固酮刺激大鼠肾脏系膜细胞。采用激光共聚焦检测系膜细胞内ROS水平。以貂肺上皮细胞(Mvllu)生长抑制MTT法检测上清中TGF-β1活性。然后用抗氧化剂N-乙酰半胱氨酸(NAC)与抗TGF-β1中和抗体阻断醛固酮刺激肾脏系膜细胞所产生ROS和TGF-β1。用RT-PCR观察系膜细胞PAI-1 mRNA的变化。用Western印迹法测定系膜细胞培养上清液中的PAI-1。结果醛固酮刺激大鼠肾脏系膜细胞24 h后,细胞内ROS水平上升到对照组的5倍(P＜0．01)；细胞上清中活性TGF-β1浓度明显升高(P＜0．01)；PAI-1 mRNA与蛋白表达分别上升到对照组的1．8倍(P＜0．01)和1．7倍(P＜0．01)。而采用NAC抑制ROS水平的升高、采用抗TGF-β1中和抗体阻断TGF-β1的作用后,PAI-1 mRNA表达分别下降了32%和30%(P＜0．01)；PAI-1蛋白表达分别下降了21%与11%(P＜0．05),但仍未下降到正常水平。结论ROS、TGF-β1在醛固酮上调系膜细胞的PAI-1表达方面起了重要作用,但醛固酮并非完全依赖ROS、TGF-β1促使PAI-1的表达上调。
Objective To investigate the role of reactive oxygen species (ROS) and transforming growth factor-β1 (TGF-β1) in the pathway of aldosterone-induced plasminogen activator inhibitor-1 (PAI-1) pathway in mesangial cells. Methods Aldosterone was used to stimulate rat mesangial cells. The level of ROS in mesangial cells was detected by laser scanning confocal microscope. The activity of TGF-β1 in the supernatant was detected by Mvllu growth inhibition MTT assay. The aldosterone was then blocked by antioxidants N-acetylcysteine (NAC) and anti-TGF-β1 neutralizing antibody to stimulate ROS and TGF-β1 produced by mesangial cells. The changes of PAI-1 mRNA in mesangial cells were observed by RT-PCR. PAI-1 in mesangial cell culture supernatant was determined by Western blotting. Results After aldosterone stimulated rat mesangial cells for 24 h, the intracellular ROS level increased to 5-fold (P <0.01), and the concentration of active TGF-β1 in the supernatant increased significantly (P <0.01 ); PAI-1 mRNA and protein expression increased 1.8 times (P <0.01) and 1.7 times (P <0.01), respectively, of the control group. However, PAI-1 mRNA expression was decreased by 32% and 30% (P <0.01) by using NAC to inhibit the increase of ROS levels and blocking TGF-β1 with anti-TGF- 1 protein expression decreased by 21% and 11% respectively (P <0.05), but still not decreased to normal level. Conclusions ROS and TGF-β1 play an important role in aldosterone up-regulation of PAI-1 expression in mesangial cells. However, aldosterone is not completely dependent on ROS. TGF-β1 up-regulates PAI-1 expression.