人脐静脉内皮细胞中还原型辅酶Ⅱ氧化酶对缺氧诱导因子-1α及内皮素-1表达的影响及其机制探讨

Effect of NADPH oxidase on hypoxia-inducible factor-1α and endothelin-1 expression in human umbilical endothelial cells and its possible mechanism

摘要:

目的：研究人脐静脉内皮细胞(HUVECs)中还原型辅酶Ⅱ(NADPH)氧化酶对缺氧诱导因子(HIF)1α和内皮素(ET)1表达的影响及其机制。方法：HUVECs培养液共25瓶,分为A(常氧)组、B(缺氧)组、C(NADPH氧化酶抑制剂夹竹桃麻素+常氧)组、D(可迅速降解HIF1α的外源性H2O2+缺氧)组及E(H2O2+夹竹桃麻素+常氧)组,每组各5瓶。各组处理3h后收集培养上清液及提取细胞总蛋白,分别用WesternBlot法和ELISA法检测细胞中HIF1α蛋白水平和上清液中ET1水平。结果：A组HIF1α灰度扫描比值为0.336±0.012,ET1值为5.87±2.22pg/mL,均呈低水平表达;B及C组HIF1α灰度扫描比值分别为0.773±0.018及0.888±0.022,ET1值分别为95.38±8.06pg/mL及33.67±4.21pg/mL,水平均显著高于A组(均P<0.05);D及E组HIF1α灰度扫描分别为0.330±0.016及0.318±0.034,比值呈低水平表达,但ET1值分别为108.43±8.38pg/mL及109.66±5.80pg/mL,水平均显著高于A组(均P<0.05)。结论：缺氧或夹竹桃麻素可诱导HUVECs的HIF1α和ET1表达升高,H2O2可抑制HIF1α表达,但仍使ET1表达升高。其机制可能是作为氧感受器的NADPH氧化酶通过改变细胞内氧化还原状态而使HIF1α表达发生变化,其下游基因ET1表达除受HIF1α调控外,还存在H2O2的调控机制。

Abstract:

OBJECTIVE: To study the effect of NADPH oxidase on hypoxia-inducible factor (HIF)-1α and endothelin (ET)-1 expression in human umbilical endothelia cells (HUVECs) and its possible mechanism. METHODS: Twenty-five bottles of HUVECs culture fluid were randomly assigned into five groups: group A (normoxic control), group B (hypoxic), group C (NADPH oxidase inhibitor apocynin+normoxic), group D (H_2O_2 which can degrade HIF-1α rapidly+hypoxic) and group E (H_2O_2+apocynin+normoxic), with five bottles in each group. The culture supernates were collected and the total protein was extracted 3 hrs after treatment. Western Blot and ELISA were used to detect the HIF-1α protein expression in HUVECs and the ET-1 level in the culture supernates respectively. RESULTS: There was a lower expression of HIF-1α protein (0.336±0.012) and lower ET-1 levels (5.87±2.22 pg/mL) in group A. The HIF-1α protein expression in groups B and C (0.773± 0.018 and 0.888±0.022) and ET-1 levels (95.38±8.06 and 33.67± 4.21 pg/mL) were noticeably higher than in group A (P< 0.05). The groups D and E had the HIF-1α protein expression levels similar to group A, but the ET-1 levels in group D (108.43±8.38 pg/mL) and group E (109.66±5.80 pg/mL) were significantly higher than in group A (P< 0.05). CONCLUSIONS: Hypoxia or apocynin can increase the HIF-1α and ET-1 expression in HUVECs. H_2O_2 can inhibit the HIF-1α expression but increase the ET-1levels. It is speculated that NADPH oxidase as an oxygen sensor regulates the HIF-1α expression by changing the intracellular redox reaction and that except HIF-1, H_2O_2 might contribute to ET-1 synthesis and release.