RNA干扰血管紧张素Ⅱ1a型受体对肾血管性高血压大鼠血压及心肌肥厚的影响

目的用RNA干扰技术下调血管紧张素Ⅱ1a型受体(AT1a)表达,观察其对肾血管性高血压及其心肌肥厚重构的影响。方法构建两肾一夹(two-kidney,one-clip:2K1C)高血压大鼠模型,用携带U6启动子和AT1a特异短发夹RNA(shRNA)编码序列的质粒pAT1a-shRNA1,pAT1a-shRNA2单次尾静脉注射给药,以含非特异性shRNA编码序列的无关质粒pGenesil-Con(pCon)、选择性AT1受体拮抗剂缬沙坦每日灌胃给药干预3周,无干预为对照,检测尾动脉压变化和颈动脉压水平、左心室重量与体重之比(LV/BW),并用Western-blot分析组织AT1受体表达情况。结果尾动脉压(与干预前比较):Blank组及pCon组继续升高25mmHg左右,pAT1a-shRNA1、pAT1a-shRNA2组下降15~16mmHg,缬沙坦组下降约30mmHg;颈动脉压和左室/体重之比:pAT1a-shRNA1(194mmHg±5mmHg;2·27±0·37)、pAT1a-shRNA2(200mmHg±5mmHg;2·31±0·26)、缬沙坦组(164mmHg±5mmHg;2·26±0·39)显著低于对照组(234mmHg±10mmHg;3·24±0·38)及pCon组(232mmHg±7mmHg;2·94±0·06);与对照组相比,pAT1a-shRNA1、pAT1a-shRNA2组左心室(分别下降53·3%和47·8%)和主动脉(分别下降58·7%和49·3%)组织内AT1受体表达显著减少。结论RNA干扰AT1a受体有效地抑制了肾血管性高血压进展及其心肌肥厚重构。RNA干扰技术可能成为高血压病基因治疗的一种新策略。

Effects of RNA interference targeting angiotensin 1a receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C hypertension

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.