血管紧张素转换酶和血管紧张素Ⅱ1型受体双基因沉默对自发性高血压大鼠血压及主动脉重构的影响
|
摘要
目的用RNA干扰(RNAi)技术同时下调血管紧张素转换酶(ACE)和血管紧张素Ⅱ1型受体(AT_1R),观察其对自发性高血压大鼠(SHR)血压及主动脉重构的影响。方法 30只SHR随机分为5组,每组6只:空白对照组(注射生理盐水);病毒对照组(注射对照腺病毒);重组腺病毒载体(Ad5)-ACE-短发夹环RNA(shRNA)治疗组;Ad5-AT_1R-shRNA治疗组;Ad5-ACE-AT_1R-shRNA治疗组(分别注射表达ACE基因、AT_1R基因、ACE和AT_1R基因特异shRNA的重组腺病毒载体);同时设WKY正常血压对照组(注射生理盐水)。以上各组大鼠均于实验的第1、17天各注射1次,干预前后检测血压、心率的变化。于首次注射后第3天,用实时荧光定量聚合酶链式反应(PCR)检测主动脉ACE和AT_1R mRNA的表达;用Western blot检测主动脉ACE和AT_1R蛋白的表达。实验结束时做主动脉组织光镜病理检查观察主动脉结构。结果首次注射后第3天,Ad5-ACE-shRNA、Ad5-AT_1R-shRNA、Ad5-ACE-AT_1R-shRNA治疗组尾动脉压分别下降(25.2±5.3)、(23.5±4.8)、(27.1±6.4)mm Hg,3个治疗组降压效应可持续15 d,SHR空白对照组和病毒对照组尾动脉压持续升高,正常血压对照组尾动脉压无明显变化,治疗组较空白对照组和病毒对照组下降(均P<0.05)。Ad5-ACE-shRNA、Ad5-AT_1R-shRNA、Ad5-ACE-AT_1R-shRNA治疗组主动脉组织中的ACE、AT_1R mRNA和蛋白表达明显低于空白对照组、病毒对照组(均P<0.05)。光镜观察显示3个治疗组主动脉结构明显改善,Ad5-ACE-AT_1R-shRNA治疗组最显著。结论 ACE和AT_1R双基因沉默,产生较单基因位点的阻断更明显的降压及逆转主动脉重构效应。
Objective To observe the effects of simultaneously down-regulate angiotensin converting enzyme(ACE) and angiotensin Ⅱ type 1 receptor(AT_1R) by RNA interference(RNAi) technique on blood pressure and aortic remodeling in spontaneously hypertensive rats(SHR). Methods Thirty SHR were randomly divided into control group(i.v. injection of saline, n=6), virus control group(injection of adenovirus, n=6), Ad5-ACE-shRNA [i.v. injection of recombinant adenovirus vector(Ad5)-ACE-short hairpin RNA(shRNA), n=6], AT_1R-shRNA treatment group(n=6), and Ad5-ACE-AT_1R-shRNA treatment group(n=6); WHY rats were served as normal blood pressure control group(injection of saline, n=6). The changes of blood pressure(BP) and heart rate(HR) were measured before and after miRNA or saline injected at the 1 st and 17 th day of the experiment. The expression of mRNA and protein of ACE and AT_1R in the aorta were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR) and Western blot on the third day after the first injection. The aortic structure of aortic tissue was examined by light microscopy at the end of the experiment. Results The BP of Ad5-ACE-shRNA group, Ad5-AT_1R-shRNA group and Ad5-ACE-AT_1R-shRNA group decreased respectively by(25.2±5.3),(23.5±4.8) and(27.1±6.4) mm Hg compared with control group or virus control group(P<0.05). The antihypertensive effects in Ad5-ACE-shRNA group, Ad5-AT_1R-shRNA group and Ad5-ACE-AT_1R-shRNA group continued for 15 days. Conversely, the BP in SHR blank control group or SHR virus control group increased significantly, though the BP in WKY control group didn't alter significantly. Compared with blank control group and virus control group, the mRNA, protein expression of ACE and AT_1R in Ad5-ACE-shRNA, Ad5-AT_1R-shRNA and Ad5-ACE-AT_1R-shRNA group decreased significantly(P<0.05). Furthermore, it showed that the structure of the aorta was significantly improved in the three treatment groups, especially in Ad5-ACE-AT_1R-shRNA treatment group. Conclusion ACE and AT_1R double gene silencing produced more obvious antihypertensive effects and reversed more aortic remodeling than single gene silencing.
Objective To observe the effects of simultaneously down-regulate angiotensin converting enzyme(ACE) and angiotensin Ⅱ type 1 receptor(AT_1R) by RNA interference(RNAi) technique on blood pressure and aortic remodeling in spontaneously hypertensive rats(SHR). Methods Thirty SHR were randomly divided into control group(i.v. injection of saline, n=6), virus control group(injection of adenovirus, n=6), Ad5-ACE-shRNA [i.v. injection of recombinant adenovirus vector(Ad5)-ACE-short hairpin RNA(shRNA), n=6], AT_1R-shRNA treatment group(n=6), and Ad5-ACE-AT_1R-shRNA treatment group(n=6); WHY rats were served as normal blood pressure control group(injection of saline, n=6). The changes of blood pressure(BP) and heart rate(HR) were measured before and after miRNA or saline injected at the 1 st and 17 th day of the experiment. The expression of mRNA and protein of ACE and AT_1R in the aorta were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR) and Western blot on the third day after the first injection. The aortic structure of aortic tissue was examined by light microscopy at the end of the experiment. Results The BP of Ad5-ACE-shRNA group, Ad5-AT_1R-shRNA group and Ad5-ACE-AT_1R-shRNA group decreased respectively by(25.2±5.3),(23.5±4.8) and(27.1±6.4) mm Hg compared with control group or virus control group(P<0.05). The antihypertensive effects in Ad5-ACE-shRNA group, Ad5-AT_1R-shRNA group and Ad5-ACE-AT_1R-shRNA group continued for 15 days. Conversely, the BP in SHR blank control group or SHR virus control group increased significantly, though the BP in WKY control group didn't alter significantly. Compared with blank control group and virus control group, the mRNA, protein expression of ACE and AT_1R in Ad5-ACE-shRNA, Ad5-AT_1R-shRNA and Ad5-ACE-AT_1R-shRNA group decreased significantly(P<0.05). Furthermore, it showed that the structure of the aorta was significantly improved in the three treatment groups, especially in Ad5-ACE-AT_1R-shRNA treatment group. Conclusion ACE and AT_1R double gene silencing produced more obvious antihypertensive effects and reversed more aortic remodeling than single gene silencing.
引文
[1] 王文. 中国高血压事业发展60年[J].中华心血管病杂志,2015,43(2):101-103.
[2] Mancia G, Fagard R, Narkiewicz K, et al. 2013 ESH/ESC guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC)[J].J Eur Heart,2013,34(28):2159-2219.
[3] Xie X, Liu Y, Perkovic V, et al. Renin-angiotensin system inhibitors and kidney and cardiovascular outcomes in patients with CKD: a Bayesian network meta-analysis of randomized clinical trials[J].Am J Kidney Dis,2016,67(5):728-741.
[4] Ferrario CM, Ahmad S, Nagata S, et al. An evolving story of angiotensin Ⅱ forming pathways in rodents and humans[J].Clin Sci(London, England:1979),2014,126(7):461-469.
[5] 宋铁山,李旭光,唐茂林.导入人内皮型一氧化氮合酶基因对高盐饮食大鼠的血压及肾脏功能的影响[J].中华高血压杂志,2007,15(4):303-306.
[6] Chen A, Huang BS, Wang HW, et al. Knockdown of mineralocorticoid or angiotensin Ⅱ type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin Ⅱ hypertension in rats[J].J Physiol,2014,592(16):3523-3536.
[7] 周华,边云飞,李茂莲.RNA干扰联合基因沉默ACE和AT1R对自发性高血压大鼠血压及心肌重构的影响[J].中华心血管病杂志,2010,38(1):60-66.
[8] Wang Y, Sun Z. Klotho gene delivery prevents the progression of spontaneous hypertension and renal damage[J].Hypertension,2009,54(4):810-815.
[9] He Q, Fan C, Yu M, et al. Associations of ACE gene insertion/deletion polymorphism, ACE activity, and ACE mRNA expression with hypertension in a Chinese population[J].PLoS One,2013,8(10):e75870.
[10] Chen A, Huang BS, Wang HW, et al. Knockdown of mineralocorticoid or angiotensin Ⅱ type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin Ⅱhypertension in rats[J].J Physiol,2014,592(16):3523-3536.
[11] Gonzalez-Villalobos RA, Satou R, Ohashi N, et al. Intrarenal mouse renin-angiotensin system during Ang Ⅱ-induced hypertension and ACE inhibition[J]. Am J Physiol Ren Physiol,2010,298(1):F150-F157.
[12] 吴宏力,鲁军,佘星星,等. 血管紧张素转换酶2-血管紧张素(1-7)-Mas轴的血压调控作用研究进展[J].中华高血压杂志,2014,22(6):528-532.
[13] Briet M, Barhoumi T, Mian MOR, et al. Aldosterone-induced vascular remodeling and endothelial dysfunction require functional angiotensin type 1a receptors[J].Hypertension,2016,67(5):897-905.
[14] Ferreira AJ, Santos RA, Bradford CN, et al. Therapeutic implications of the vasoprotective axis of the renin-angiotensin system in cardiovascular diseases[J]. Hypertension,2010,55(2):207-213.
[15] Passos-Silva DG, Verano-Braga T, Santos RA. Angiotensin(1-7): beyond the cardio-renal actions[J]. Clin Sci(Lond),2013,124(7):443-456.
[16] Igase M, Strawn WB, Gallagher PE,et al. Angiotensin Ⅱ AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats[J].Am J Physiol Heart Circ Physiol,2005,289(3):H1013-H1019.
[17] Walters PE, Gaspari TA, Widdop RE. Angiotensin (1-7) acts as a vasode-pressor agent via angiotensin Ⅱ type 2 receptors in conscious rats[J].Hypertension,2005,45(5):960-966.
[18] Edis G, Ivana D, Dragic B. Effects of combination of AT1-antagonist candesartan cilexetil and AСЕ-inhibitors in patients with congestive heart failure[J].Srp Arh Celok Lek,2013,141(1/2):29-34.
[19] Kunz R, Friedrich C, Wolbers M, et al. Meta-analysis: effect of monotherapy and combination therapy with inhibitors of the renin angiotensin system on proteinuria in renal disease[J]. Ann Intern Med,2008,148(1):30-48.
[20] Baltatzi M, Savopoulos C, Hatzitolios A. Role of angiotensin converting enzyme inhibitors and angiotensin receptor blockers in hypertension of chronic kidney disease and renoprotection study results[J].Hippokratia,2011,15(1):27-32.
[21] Fitchett D. Clinical trial update: focus on the ONTARGET study[J].Vasc Health Risk Manag,2007,3(6):901-908.
[2] Mancia G, Fagard R, Narkiewicz K, et al. 2013 ESH/ESC guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC)[J].J Eur Heart,2013,34(28):2159-2219.
[3] Xie X, Liu Y, Perkovic V, et al. Renin-angiotensin system inhibitors and kidney and cardiovascular outcomes in patients with CKD: a Bayesian network meta-analysis of randomized clinical trials[J].Am J Kidney Dis,2016,67(5):728-741.
[4] Ferrario CM, Ahmad S, Nagata S, et al. An evolving story of angiotensin Ⅱ forming pathways in rodents and humans[J].Clin Sci(London, England:1979),2014,126(7):461-469.
[5] 宋铁山,李旭光,唐茂林.导入人内皮型一氧化氮合酶基因对高盐饮食大鼠的血压及肾脏功能的影响[J].中华高血压杂志,2007,15(4):303-306.
[6] Chen A, Huang BS, Wang HW, et al. Knockdown of mineralocorticoid or angiotensin Ⅱ type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin Ⅱ hypertension in rats[J].J Physiol,2014,592(16):3523-3536.
[7] 周华,边云飞,李茂莲.RNA干扰联合基因沉默ACE和AT1R对自发性高血压大鼠血压及心肌重构的影响[J].中华心血管病杂志,2010,38(1):60-66.
[8] Wang Y, Sun Z. Klotho gene delivery prevents the progression of spontaneous hypertension and renal damage[J].Hypertension,2009,54(4):810-815.
[9] He Q, Fan C, Yu M, et al. Associations of ACE gene insertion/deletion polymorphism, ACE activity, and ACE mRNA expression with hypertension in a Chinese population[J].PLoS One,2013,8(10):e75870.
[10] Chen A, Huang BS, Wang HW, et al. Knockdown of mineralocorticoid or angiotensin Ⅱ type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin Ⅱhypertension in rats[J].J Physiol,2014,592(16):3523-3536.
[11] Gonzalez-Villalobos RA, Satou R, Ohashi N, et al. Intrarenal mouse renin-angiotensin system during Ang Ⅱ-induced hypertension and ACE inhibition[J]. Am J Physiol Ren Physiol,2010,298(1):F150-F157.
[12] 吴宏力,鲁军,佘星星,等. 血管紧张素转换酶2-血管紧张素(1-7)-Mas轴的血压调控作用研究进展[J].中华高血压杂志,2014,22(6):528-532.
[13] Briet M, Barhoumi T, Mian MOR, et al. Aldosterone-induced vascular remodeling and endothelial dysfunction require functional angiotensin type 1a receptors[J].Hypertension,2016,67(5):897-905.
[14] Ferreira AJ, Santos RA, Bradford CN, et al. Therapeutic implications of the vasoprotective axis of the renin-angiotensin system in cardiovascular diseases[J]. Hypertension,2010,55(2):207-213.
[15] Passos-Silva DG, Verano-Braga T, Santos RA. Angiotensin(1-7): beyond the cardio-renal actions[J]. Clin Sci(Lond),2013,124(7):443-456.
[16] Igase M, Strawn WB, Gallagher PE,et al. Angiotensin Ⅱ AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats[J].Am J Physiol Heart Circ Physiol,2005,289(3):H1013-H1019.
[17] Walters PE, Gaspari TA, Widdop RE. Angiotensin (1-7) acts as a vasode-pressor agent via angiotensin Ⅱ type 2 receptors in conscious rats[J].Hypertension,2005,45(5):960-966.
[18] Edis G, Ivana D, Dragic B. Effects of combination of AT1-antagonist candesartan cilexetil and AСЕ-inhibitors in patients with congestive heart failure[J].Srp Arh Celok Lek,2013,141(1/2):29-34.
[19] Kunz R, Friedrich C, Wolbers M, et al. Meta-analysis: effect of monotherapy and combination therapy with inhibitors of the renin angiotensin system on proteinuria in renal disease[J]. Ann Intern Med,2008,148(1):30-48.
[20] Baltatzi M, Savopoulos C, Hatzitolios A. Role of angiotensin converting enzyme inhibitors and angiotensin receptor blockers in hypertension of chronic kidney disease and renoprotection study results[J].Hippokratia,2011,15(1):27-32.
[21] Fitchett D. Clinical trial update: focus on the ONTARGET study[J].Vasc Health Risk Manag,2007,3(6):901-908.