Department of Respiratory Diseases, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China.
Chin Med J (Engl). 2010 Sep;123(17):2432-9.
Thrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury. In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species (ROS) can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.
ROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin (20 U/ml) exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 (ERK1/2) signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.
Thrombin, at 20 U/ml, stimulated human lung fibroblasts (HLF) to generate ROS in a time dependent manner. The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.
The activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.
凝血酶是一种多功能丝氨酸蛋白酶，在组织损伤后止血过程中起着至关重要的作用。除了促凝作用外，凝血酶还是一种有效的间充质细胞有丝分裂原，因此在组织修复过程中，它在间充质细胞的局部增殖中起着重要作用。活性氧（ROS）可以诱导一些人类细胞以较低的速度增殖，而在较高浓度下，它们会促进细胞凋亡或坏死。越来越多的证据表明，凝血酶可以诱导一些细胞产生 ROS。基于这些观察结果，我们提出了一个假设，即凝血酶可以刺激人肺成纤维细胞产生 ROS，ROS 在人肺成纤维细胞增殖中发挥重要作用。
用流式细胞术检测凝血酶（20U/ml）作用 30 分钟和 60 分钟后成纤维细胞中 ROS 的产生。用商业试剂盒检测不同浓度凝血酶处理后肺成纤维细胞中还原型谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）的比值。用 Western blot 检测凝血酶刺激肺成纤维细胞后 NADPH 氧化酶和细胞外调节激酶 1/2（ERK1/2）信号通路。
凝血酶（20U/ml）以时间依赖的方式刺激人肺成纤维细胞（HLF）产生 ROS。用凝血酶处理的成纤维细胞中 GSH/GSSG 的比值明显下降。NADPH 氧化酶被激活，ERK1/2 信号通路参与了凝血酶处理的成纤维细胞的增殖过程。
凝血酶激活 NADPH 氧化酶导致 ROS 的产生，通过激活 ERK1/2 信号通路促进成纤维细胞增殖。
