低氧诱导因子1参与常氧条件下硫化氢促血管内皮细胞迁移的作用

Involvement of HIF-1 in the migration-promoting effects of hydrogen sulfide in vascular endothelial cells under normoxic conditions

本文旨在探讨常氧条件下硫化氢(H2S)促进血管内皮细胞迁移的分子机制。采用划痕迁移模型研究常氧条件下NaHS(H2S供体)对恒河猴视网膜-脉络膜内皮细胞系RF/6A细胞迁移的作用;用Real-time PCR方法检测RF/6A细胞内低氧诱导因子1α(hypoxia-inducible factor1α,HIF-1α)mRNA水平;用Western blot检测RF/6A细胞内HIF-1α的蛋白水平;用2’,7’-二氯荧光素乙酰乙酸盐(2’,7’-dichlorofluorescein diacetate,DCFH-DA)荧光探针法检测RF/6A细胞内活性氧(reactive oxygen species,ROS)水平。结果显示,不同浓度(10～100μmol/L)NaHS对RF/6A细胞有显著的促迁移作用,该作用可以被HIF-1抑制剂氯化镉(CdCl2,50μmol/L)所阻断。NaHS能够在常氧培养条件下呈剂量和时间依赖性地显著提高HIF-1α的蛋白水平,并且对HIF-1αmRNA水平有快速、持久的提高作用。另外,NaHS能够显著降低常氧条件下RF/6A细胞中的ROS水平。以上结果提示,HIF-1参与常氧条件下H2S促内皮细胞迁移的作用,而ROS作为HIF-1α的上游调节因子,可能也参与H2S促进内皮细胞迁移的过程。

The purpose of this study was to investigate the molecular mechanisms whereby hydrogen sulfide （H2S） exerts the promoting effect on vascular endothelial cells migration. We used wound healing assay to study the effect of NariS （H：S donor） on the migration ability of rhesus retinal pigment epithelial cell line, RF/6A cells, under normoxic conditions. Real-time PCR was used to measure hypoxia-inducible factor 1α （HIF-1α） mRNA level. Western blot was used to measure the expression of HIF-1α protein. The probe 2＇,7＇-dichlorofluorescein diacetate （DCFH-DA） was used to measure intracellular reactive oxygen species （ROS） level. The results showed that NariS （10-100 μmol/L） could significantly promote RF/6A cells migration under normoxic conditions, and this effect could be inhibited by 50 μmol/L HIF-1 inhibitor, CdCl：. NariS increased the protein level of HIF-1α in a dose- and time-dependent manner, and up-regulated the mRNA level of HIF-1α quickly and continuously. Moreover, NariS could significantly decrease ROS levels in RF/6A cells under normoxic conditions. These results suggest HIF-1 may mediate the promoting effect of H2S on vascular endothelial cells migration under normoxic conditions. ROS, as an upstream regulator of HIF-1α, may be involved in the migration- promoting effect of H2S.