siRNA沉默AT2受体通过Ang(1-9)-ACE2-AT2通路抑制心脏微血管内皮细胞NO的生成

The siRNA silencing AT2 receptor inhibits production of NO in cardiac microvascular endothelial cells by Ang(1-9)-ACE2-AT2 pathway

目的研究重组人血管紧张素转化酶2(rh ACE2)作用于心脏微血管内皮细胞后对NO产生的影响,采用siRNA沉默AT2受体研究Ang(1-9)-ACE2-AT2通路在这一过程中的作用。方法体外培养人心脏微血管内皮细胞(CMVEC),分为对照组、AngⅡ组、AngⅡ+rh ACE2组、AT2受体抑制剂组和阴性对照siRNA组。Griess试剂法测定细胞培养上清液中NO的含量,RT-PCR检测HUVEC中eNOS mRNA的表达,检测细胞内NO的生成及内皮型一氧化氮合酶(eNOS)活性。Western blot检测磷酸化eNOS的表达。观察rh ACE2对AngⅡ作用后CMVEC生成NO、eNOS mRNA、eNOS蛋白表达的影响,同时观察加用AT2受体抑制剂PD123319干预后,rh ACE2对磷酸化eNOS蛋白表达的影响。观察siRNA干扰CMVEC中AT2受体蛋白后对eNOS活性、NO的影响。结果 AngⅡ组NO的含量[(3.495±0.362)nmol/L]明显低于对照组[(11.513±0.392)nmol/L,P<0.05];AngⅡ+rhACE2组细胞培养液中NO的含量和磷酸化eNOS表达水平均明显高于AngⅡ组(P<0.05),而eNOS mRNA和非磷酸化eNOS蛋白表达水平与AngⅡ组相比无统计学差异(P>0.05)。经AT2通路抑制剂PD123319干预后的AT2受体抑制剂组磷酸化eNOS表达水平明显低于AngⅡ+rhACE2 30 min组(P<0.05)。Western blot检测结果显示转染AT2siRNA组转染48 h AT2受体蛋白表达降低(P<0.05);与AT2受体抑制剂组和阴性对照siRNA组相比,转染AT2 siRNA组的eNOS活性和NO含量均显著降低。结论 rh ACE2作用于人心脏微血管内皮细胞后,eNOS活性增强,NO生成增加,Ang(1-9)-ACE2-AT2信号通路参与这一过程。

Objective To explore the effect of recombinant human angiotensin converting enzyme 2（ rh ACE2） on NO formation in the cardiac microvascular endothelial cells（ CMVEC）,and the role of Ang（ 1-9）-ACE2-AT2 pathway in the process. Methods Human cardiac microvascular endothelial cells（ CMVEC） were cultured in vitro and randomized into control group,Ang Ⅱ group,Ang Ⅱ ＋rh ACE2 group,AT2 receptor inhibitor group,AT2 siRNA transfection group and negative control siRNA group. Griess reagent measurement was applied to detect NO content in cell culture supernatant. RT-PCR was used to detect the expression of eNOS mRNA in HUVEC. Western blot was used to detect the expression of phospho-eNOS. NO fluorescent probe DAF-FM DA was loaded to detect the intracellular NO formation and the activity of endothelial NO synthase（ eNOS）. Results The content of NO in AngⅡ group was significantly lower than that in control group [（ 3. 495 ± 0. 362） nmol/L vs（ 11. 513 ± 0. 392） nmol/L,P〈0. 05]. NO contents and the phospho-eNOS expression levels of cultured cell liquid in Ang Ⅱ ＋ rh ACE2 group were significantly higher than those in Ang Ⅱgroup（ P〈0. 05）,however,the protein expression levels of eNOS mRNA and non-phospho-eNOS showed no significant difference between two groups（ P〈0. 05）. The expression levels of phospho-eNOS were significantly lower in AT2 receptor inhibitor group than in AngⅡ ＋ rhACE2 group after treatment for 30 min（ P〈0. 05）. Western blot results showed that the protein expression of AT2 receptor decreased in AT2 siRNA transfection group after transfection with siRNA for 48 h（ P〈0. 05）. Compared with AT2 receptor inhibitor group and negative control siRNA group,the eNOS activity and NO levels were significantly reduced in AT2 siRNA transfection group.Conclusion The rhACE2 can increase eNOS activity and NO production in human cardiac microvascular endothelial cells,and Ang（ 1-9）-ACE2-AT2 signaling pathway is involved in the process.