Huang Fengying, Cao Jing, Liu Qiuhong, Zou Ying, Li Hongyun, Yin Tuanfang
Department of Obstetrics and Gynecology, The Second Xiangya Hospital, Central South University Changsha, Hunan, China, 410011.
Int J Clin Exp Pathol. 2013 Sep 15;6(10):2129-36. eCollection 2013.
Now there are more and more evidences that Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis of endometriosis (EMs). Vascular endothelial growth factor (VEGF) has a potent angiogenic activity. However, it is worth studying about the regulating mechanism of COX-2/COX-1 and VEGF in the development of human endometriosis in vitro. The current study was designed to investigate the effect of 4 cytokines on COX-2/COX-1 expression and the effect of IL-1β on VEGF release in human endometriosis stromal cells (ESC), and to explore the related signaling pathways involved in vitro.
Isolation, culture and identification of ESC. Cells were treated with 4 cytokines, and the inhibitor mitogen-activated protein-Erk (MEK) and the inhibitor p38 mitogen-activated protein kinase (MAPK) prior to adding cytokine IL-1β. COX-2 protein expression was measured by western blot and VEGF secretion was determined by ELISA.
Among four kinds of cytokines, IL-1β treatment increased COX-2 protein expression and VEGF release in three ESC, and TNF-α had the same effect on COX-2 protein level as IL-1β only in ectopic and eutopic ESC, and MCSF had only slight effect on ectopic ESC. In contrast, cytokines had no effect on COX-1 expression. We also demonstrated that MAPK reduced the synthesis of COX-2 by IL-1β induced. COX-2 inhibitor reduced VEGF release by IL-1β induced.
i) In human ESC in vitro, IL-1β up-regulated the COX-2 expression through the activation of p38 MAPK pathway, and not to COX-1. ii) Up-regulation of VEGF level by IL-1β treatment was found in human endometriosis stromal cell and COX-2 inhibitor was involved in this process.
目前越来越多的证据表明，环氧化酶-2（COX-2）在子宫内膜异位症（EMs）血管生成中起重要作用。血管内皮生长因子（VEGF）具有强大的血管生成活性。然而，COX-2/COX-1与VEGF在人子宫内膜异位症体外发生发展中的调控机制值得研究。本研究旨在探讨4种细胞因子对人子宫内膜异位症基质细胞（ESC）中COX-2/COX-1表达的影响以及白细胞介素-1β（IL-1β）对VEGF释放的影响，并在体外探索相关信号通路。
ESC的分离、培养与鉴定。细胞用4种细胞因子处理，并在加入细胞因子IL-1β之前用丝裂原活化蛋白激酶-Erk（MEK）抑制剂和p38丝裂原活化蛋白激酶（MAPK）抑制剂处理。通过蛋白质免疫印迹法检测COX-2蛋白表达，通过酶联免疫吸附测定法测定VEGF分泌。
在4种细胞因子中，IL-1β处理可增加3种ESC中COX-2蛋白表达和VEGF释放，肿瘤坏死因子-α（TNF-α）仅在异位和在位ESC中对COX-2蛋白水平有与IL-1β相同的作用，巨噬细胞集落刺激因子（MCSF）仅对异位ESC有轻微作用。相反，细胞因子对COX-1表达无影响。我们还证明，MAPK可降低IL-1β诱导的COX-2合成。COX-2抑制剂可降低IL-1β诱导的VEGF释放。
i）在体外培养的人ESC中，IL-1β通过激活p38 MAPK通路上调COX-2表达，而非COX-1。ii）在人子宫内膜异位症基质细胞中发现IL-1β处理可上调VEGF水平，且COX-2抑制剂参与此过程。
