NEDD4调控AMPK活性参与心肌细胞慢性缺氧适应的研究

NEDD4 participates in myocardial adaptation to chronic hypoxia by regulating AMPK

目的探讨NEDD4在心肌细胞慢性缺氧适应过程中的作用及其可能的机制。方法1收集手术矫正的先心病患儿32例,其中紫绀型先心病18例,非紫绀型先心病14例。取术中切除的右室流出道心肌组织作为标本,用免疫组化染色检测NEDD4在心肌细胞中表达的分布,Western blot检测NEDD4在心肌组织中的表达情况;2将H9c2心肌细胞分为常氧组、慢性缺氧组和阳性对照组,其中慢性缺氧组又分为对照组、空白转染组和干扰转染组,设计合成以心肌细胞NEDD4基因为靶标的siRNA,通过阳离子脂质体将合成的siRNA转染至H9c2心肌细胞中,常氧培养(74%N2,5%CO2,21%O2)48 h后开始对慢性缺氧组心肌细胞进行缺氧刺激(94%N2,5%CO2,1%O2),缺氧刺激72 h后Western blot检测NEDD4、p-AMPK表达水平变化,流式细胞术检测缺氧刺激所致心肌细胞损伤情况。结果 1紫绀组患儿术前血氧饱和度明显低于非紫绀组(P<0.05);免疫组化染色结果显示NEDD4主要分布于心肌细胞质中;与非紫绀组相比,紫绀组患儿心肌组织中NEDD4表达水平显著下降[NEDD4/β-actin条带灰度比值:非紫绀组(0.72±0.07);紫绀组(0.42±0.06),P<0.05];2与常氧组相比,对照组心肌细胞死亡比例显著增加(P<0.05);但干扰NEDD4表达后,缺氧所致心肌细胞损伤比例降低(P<0.05)。Western blot检测结果显示,与空白转染组相比,干扰转染组AMPK磷酸化水平显著增加[p-AMPK/AMPK条带灰度比值:空白转染组(0.21±0.01),干扰转染组(0.88±0.04),P<0.05]。结论 NEDD4可能通过调控AMPK活性参与慢性缺氧情况下心肌细胞代谢的适应调节。

Objective To determine the role of neuronal precursor cell expressed developmentally down-regulated 4 （NEDD4） in myocardial adaptation to chronic hypoxia and investigate the underlying mechanisms. Methods ①A total of 32 children with congenital heart diseases [ cyanotic （ n = 18 ） or acyanotic cardiac defects （n = 14） ] admitted in our institute were recruited in this study. Their tissue samples of the right ventrieular myocardium taken immediately after aortic clamping were employed to detect the distribution and protein expression of NEDD4 by immunohistochemical assay and Western blotting respectively. ② Cardiomyocytes were cultured under normoxic or hypoxic condition. The hypoxic ceils were transfected with NEDD4 siRNA by Lipofectamine RNAiMAX Reagent to knockdown the expression of NEDD4. The cells without transfection or transfected with non-targeting control siRNA served as control cells. Then the cells were cultured under normoxia （74% N2, 5% CO2 and 21% O2） for 48 h followed by hypoxia （94% N2, 5% CO2 and 1% O2 ） for 72 h, Western blotting and flow cytometry were used to detect the protein expression of NEDIM and phospho-AMPK and the proportion of damaged myocardial cells respectively. Results ① Cyanotic children had significantly lower oxygen saturation than non-cyanotic group before surgery （P 〈 0. 05 ）. NEDIM protein was mainly located in the cytoplasm. And its protein level was obviously lower in the cyanotic children than the acyanotic ones （0.42 ±0.06 vs 0. 72 ± 0.07, P 〈 0.05 ）. ② The proportion of dead cardiomyocytes was larger in the cells under hypoxia than those under normoxic culture （P 〈 0.05 ）. But NEDD4 siRNA transfection inhibited the cell death induced by hypoxia （P 〈 0.05 ）. Western blotting showed that the level of p-AMPK was significantly higher in the transfected cells than in the blank cells （0.88 ± 0.04 vs 0. 21 ± 0.01, P 〈 0.05 ）. Conclusion NEDIM may participate in myocardial adaptation to chronic hypoxia by regulating the activation of AMPK.