Zhang Jing-qun, Sun Hong-lei, Ma Ye-xin, Wang Dao-wen
Department of Cardiology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Yi Xue Za Zhi. 2006 Apr 25;86(16):1138-43.
To investigate the effects of RNA interference (RNAi) targeting angiotensin 1a (AT1a) receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C (2-kidney, 1-clip) hypertension.
Two kinds of RNAi plasmids, pAT1a-shRNA1 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 928 - 946 and pAT1a-shRNA2 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 978 - 996, and a blank plasmid pCon carrying a nonspecific shRNA-coding sequence were constructed. Thirty Sprague-Dawley rats underwent clipping of the left renal artery so as to establish two-kidney, one-clip (2K1C) hypertension models and then were randomly divided into 5 equal groups: pAT1a-shRNA1 group (injected with pAT1a-shRNA1 4 mg/kg only one time), pAT1a-shRNA2 group (injected with pAT1a-shRNA2 4 mg/kg only one time), pCon group (injected with pCon 4 mg/kg only one time), valsartan group (perfused into the stomach with valsartan, a AT1 receptor inhibitor 30 mg.kg(-1).d(-1), for 3 weeks), and control blank group (without any treatment). Three weeks later, the systolic pressure of the caudal artery was measured, catheterization through carotid artery was conducted to measure the systolic blood pressure (SBP) and diastolic blood pressure (DBP), and the left ventricular pressure curve was drawn. Then the rats were killed; the weight of the heart was measured, the ratio of left ventricle weight to body weight (LV/BW) was calculated, and pathological examination of the heart and thoracic aorta was performed. Western blotting was used to detect the protein expression of AT21 in the ventricle and aorta. Six age-matched healthy rats were used as normal controls.
There was no significant difference in the caudal artery pressure among the 5 groups (all P > 0.05) before intervention. Three weeks later the caudal artery pressures of the blank control group and pCon group continued to significantly increase by about 25 mm Hg compared to the values before the intervention (both P < 0.001) and without significant difference between these 2 groups; however, the caudal artery pressures of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 15.1 mm Hg +/- 5.4 mm Hg, 16.4 mm Hg +/- 8.4 mm Hg, and 30.6 mm Hg +/- 18.2 mm Hg lower than those before the intervention respectively (all P < 0.01); and were also significantly lower than those of the blank groups (P < 0.01 or P < 0.05). There was no significant differences in the +/- dp/dt value and indicators of renal function among these groups. The carotid artery pressure of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 194 mm Hg +/- 5 mm Hg, 200 mm Hg +/- 5 mm Hg, and 164 mm Hg +/- 5 mm Hg, all significantly lower than those of the blank and pCon groups (234 mm Hg +/- 10 mm Hg and 232 mm Hg +/- 7 mm Hg respectively, all P < 0.01). The LV/BW of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 2.27 +/- 0.37, 2.31 +/- 0.26, and 2.26 +/- 0.39, all significantly lower than that of the blank and pCon groups (3.24 +/- 0.38 and 2.94 +/- 0.06, respectively, all P < 0.01), similar to that of the normal control group (P > 0.05). The myocardiocytes were significantly hypertrophic and the arterial tunica media was significantly thickened in the blank group and such changes were all improved to different degrees in the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups. The protein expression levels of AT1 receptor in the myocardium of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 53.3% and 47.8% respectively than that of the blank group, and the protein expression levels of AT1 receptor in the thoracic aorta of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 58.7% and 49.3% respectively than that of the blank group (all P < 0.01); however, there were no significant difference in the protein expression levels of AT1 receptor in the myocardium and thoracic aorta between the valsartan and blank groups (both P > 0.05).
RNA interference targeting AT1a receptor inhibits the development of renovascular hypertension and the accompanying cardiac hypertrophy. The RNAi technology may become a new strategy of gene therapy for hypertension.
研究靶向血管紧张素1a（AT1a）受体的RNA干扰（RNAi）对二肾一夹（2K1C）高血压大鼠血压及心脏肥大的影响。
构建两种RNAi质粒，携带U6启动子及对应928 - 946位点的AT1a特异性shRNA编码模板序列的pAT1a - shRNA1，以及携带U6启动子及对应978 - 996位点的AT1a特异性shRNA编码模板序列的pAT1a - shRNA2，还有携带非特异性shRNA编码序列的空白质粒pCon。30只Sprague - Dawley大鼠行左肾动脉夹闭术以建立二肾一夹（2K1C）高血压模型，然后随机分为5组：pAT1a - shRNA1组（仅一次注射4 mg/kg pAT1a - shRNA1）、pAT1a - shRNA2组（仅一次注射4 mg/kg pAT1a - shRNA2）、pCon组（仅一次注射4 mg/kg pCon）、缬沙坦组（用AT1受体抑制剂缬沙坦30 mg·kg⁻¹·d⁻¹灌胃3周）和对照空白组（未作任何处理）。3周后，测量尾动脉收缩压，经颈动脉插管测量收缩压（SBP）和舒张压（DBP），绘制左心室压力曲线。然后处死大鼠；测量心脏重量，计算左心室重量与体重之比（LV/BW），并对心脏和胸主动脉进行病理检查。采用蛋白质印迹法检测心室和主动脉中AT1的蛋白表达。选取6只年龄匹配的健康大鼠作为正常对照。
干预前5组尾动脉压力无显著差异（均P > 0.05）。3周后，空白对照组和pCon组尾动脉压力较干预前继续显著升高约25 mmHg（均P < 0.001），且两组间无显著差异；然而，pAT1a - shRNA1组、pAT1a - shRNA2组和缬沙坦组尾动脉压力分别比干预前降低15.1 mmHg ± 5.4 mmHg、16.4 mmHg ± 8.4 mmHg和30.6 mmHg ± 18.2 mmHg（均P < 0.01）；且也显著低于空白组（P < 0.01或P < 0.05）。这些组间±dp/dt值及肾功能指标无显著差异。pAT1a - shRNA1组、pAT-1a - shRNA2组和缬沙坦组颈动脉压力分别为194 mmHg ± 5 mmHg、200 mmHg ± 5 mmHg和164 mmHg ± 5 mmHg，均显著低于空白组和pCon组（分别为234 mmHg ± 10 mmHg和232 mmHg ± 7 mmHg，均P < 0.01）。pAT1a - shRNA1组、pAT1a - shRNA2组和缬沙坦组的LV/BW分别为2.27 ± 0.37、2.31 ± 0.26和2.26 ± 0.39，均显著低于空白组和pCon组（分别为3.24 ± 0.38和2.94 ± 0.06，均P < 0.01），与正常对照组相似（P > 0.05）。空白组心肌细胞显著肥大，动脉中膜显著增厚，而pAT1a - shRNA1组、pAT1a - shRNA2组和缬沙坦组这些改变均有不同程度改善。pAT1a - shRNA1组和pAT1a - shRNA2组心肌中AT1受体蛋白表达水平分别比空白组降低53.3%和47.8%，pAT1a - shRNA1组和pAT1a - shRNA2组胸主动脉中AT1受体蛋白表达水平分别比空白组降低58.7%和49.3%（均P < 0.01）；然而，缬沙坦组与空白组心肌和胸主动脉中AT1受体蛋白表达水平无显著差异（均P > 0.05）。
靶向AT1a受体的RNA干扰可抑制肾血管性高血压及伴随的心脏肥大的发展。RNAi技术可能成为高血压基因治疗的新策略。
