脑内ACE2基因过表达对高血压前期大鼠血压进展和氧化应激的影响

Effects of ACE2 gene over-expression in brain on prehypertension pro⁃gression and oxidative stress in prehypertensive rats

目的:观察下丘脑室旁核内血管紧张素转换酶2(angiotension-converting enzyme 2,ACE2)基因过表达对高血压前期大鼠血压进展和中枢氧化应激的影响,并探讨ACE2基因中枢降压的分子机制。方法:ACE2基因以慢病毒为载体,载体上携带增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP),由上海吉凯基因化学技术有限公司构建。对6周龄雄性自发性高血压大鼠(spontaneously hypertensive rats,SHR)进行2周跑台运动,随后随机分成SHR组、ACE2基因过表达(SHR-ACE2)组和病毒载体(SHR-eGFP)组,每组10只,同时选取10只血压正常的Wistar-Kyoto(WKY)大鼠不进行任何干预,作为对照组。将ACE2基因和病毒载体分别注射到8周龄SHR的下丘脑室旁核。每4周测1次血压,8周后测定动脉压力反射敏感性(baroreflex sensitivity,BRS),以及血浆去甲肾上腺素(norepinephrine,NE)和室旁核血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)水平;二氢乙啶(dihydroethidium,DHE)染色法检测室旁核活性氧(reactive oxygen species,ROS)水平;Western blot检测室旁核ACE2和NADPH氧化酶(NADPH oxidase,NOX)的蛋白表达。结果:SHR组和SHR-eGFP组室旁核ACE2蛋白表达显著低于WKY组(P<0.01),而SHR-ACE2组ACE2蛋白表达显著高于SHR组(P<0.05),且与WKY组相比无显著差异。与WKY组相比,SHR组和SHR-eGFP组收缩压显著升高,伴有BRS显著降低(P<0.01),而SHR-ACE2组收缩压较SHR组降低(P<0.01),高血压前期进展延缓,且BRS提高(P<0.01)。SHR组和SHR-eGFP组血浆NE浓度、室旁核Ang Ⅱ和ROS水平及NOX2和NOX4蛋白表达均高于WKY组(P<0.01),而ACE2基因过表达显著逆转上述变化(P<0.05)。结论:SHR室旁核过表达ACE2基因能延缓高血压前期进展,改善动脉血压调节,并抑制交感神经活动,其机制可能与降低脑内Ang Ⅱ水平,减轻中枢氧化应激有关。

AIM:To investigate the effects of angiotensin-converting enzyme 2(ACE2)gene over-expression in hypothalamic paraventricular nucleus(PVN)on the progression of hypertension and central oxidative stress in prehypertensive rats and its molecular mechanisms.METHODS:ACE2 gene was constructed with lentiviral vector carrying enhanced green fluorescent protein(eGFP)by Shanghai Genechem Co.,Ltd.Six-week-old male spontaneously hypertensive rats(SHR)underwent the treadmill exercise for 2 weeks.At 8 weeks of age,the SHR were randomly divided into 3 groups:SHR group,vector(SHR-eGFP)group and ACE2 gene over-expression(SHR-ACE2)group(n=10).Normotensive Wistar-Kyoto(WKY)rats(n=10)were used as control group(without intervention).The lentiviral vectors with or without ACE2 gene were microinjected into the PVN of SHR.Systolic blood pressure(SBP)was measured every 4 weeks.Eight weeks after ACE2 gene transfection,the baroreflex sensitivity(BRS),and the levels of plasma norepinephrine(NE)and angiotensinⅡ(AngⅡ)in the PVN were assessed.The level of reactive oxygen species(ROS)was detected by dihydroethidium(DHE)staining.The protein levels of ACE2 and NADPH oxidase(NOX)subunits in the PVN were determined by Western blot.RESULTS:Compared with WKY group,SBP was markedly increased(P<0.01),and the BRS was significantly decreased in SHR group and SHR-eGFP group(P<0.01).SBP was decreased,and the BRS was markedly improved in SHR-ACE2 group compared with SHR group(P<0.01),which postponed the progression of prehypertension.The levels of plasma NE,and AngⅡand ROS in the PVN were significantly increased in SHR group and SHR-eGFP group(P<0.01).However,ACE2 over-expression greatly inhibited above changes in SHR group(P<0.01).ACE2 protein expression was down-regulated,and the protein levels of NOX subunits(NOX2 and NOX4)were up-regulated in the PVN of SHR group and SHR-eGFP group compared with WKY group(P<0.01),while ACE2 over-expression reversed above changes(P<0.01).CONCLUSION:ACE2 gene over-expression in the PVN of SHR postpones the progression of prehypertension,improves the arterial blood pressure regulation,and depresses the sympathetic nerve activity,which may be associated with the reduction of AngⅡlevel and oxidative stress in the brain.