ACE2激活对大鼠重度肺动脉高压的影响及机制研究

发布时间：2018-05-04 11:47

本文选题：肺动脉高压 + 肺血管重构　；参考：《北京协和医学院》2012年博士论文

【摘要】：背景和目的致病因素的作用下,肺循环调节功能的失衡启动一系列病理生理改变,包括内皮功能障碍、炎症激活等,引起肺血管异常收缩,进而平滑肌细胞增殖、血管重构,最终导致肺动脉高压。肾素血管紧张素系统是心血管系统的重要调节系统,它在肺动脉高压发病过程中发挥重要作用,但是干预经典的肾素血管紧张素系统(ACE-Ang Ⅱ-AT1R轴)治疗肺动脉高压的效果欠佳。血管紧张素转化酶2(ACE2)及其参与的ACE2-Ang-(1-7)-Mas轴是肾素血管紧张素系统的新成员,起着拮抗经典肾素血管紧张素轴的作用,而且在呼吸系统疾病中发挥着广泛的作用。本研究应用ACE2激动剂Resorcinolnaphthalein(Rec)干预野百合碱联合肺叶切除构建重度肺动脉高压模型,探讨ACE2激活在预防和逆转重度肺动脉高压中的作用及其机制。方法 1.实验动物和分组：SD大鼠,随机分为6组：空白对照组,ACE2对照组,肺动脉高压组,ACE2预防组,ACE2治疗组,Mas受体拮抗组(Rec+A-779)。左肺切除后1周,皮下注射野百合碱诱导重度肺动脉高压。ACE2对照和ACE2预防组在野百合碱注射后第1天开始持续注射Rec, ACE2治疗组第14天开始注射Rec, Mas受体拮抗组第1天开始同时注射Rec和Mas受体拮抗剂A-779。 2.干预2周后,除ACE2治疗组外(干预42天),其余各组取8只,心导管测定平均肺动脉压和动脉收缩压,分离右心室测定右心室肥厚指数,弹力纤维染色(Verhoeff-铁苏木素染色)后分析肺动脉(100-200gm)中膜厚度和肺小动脉(50-100gm)内膜梗阻性增生程度。 3.内皮功能检测：肺动脉内注射不同浓度的乙酰胆碱和硝普钠,检测内皮依赖的血管舒张反应的变化。 4.活性荧光共振能量转移法检测肺组织ACE2酶活性。 5.生存分析：采用Kaplan-Meier生存分析图分析。 6. ELISA检测肺组织Ang Ⅱ和Ang-(1-7)浓度,Western blot检测肺组织ACE, ACE2, AT1R, Mas表达水平。 7.免疫组化检测直径50-100μm肺动脉管壁平滑肌抗体(a-SMA)和增殖细胞核抗原表达,分析肺动脉平滑肌细胞增殖情况。 8. RT-PCR检测肺组织炎症因子IL-6, MCP-1, TNF-α, IL-10。结果 1.Rec对大鼠肺组织ACE2酶活性的影响ACE2对照组ACE2酶活性是正常的1.74倍；肺动脉高压组ACE2酶活性下降38.2%,ACE2预防和治疗组ACE2酶活性则分别比肺动脉高压组升高1.87和1.78倍,提示Rec可以激活大鼠肺组织ACE2。 2.ACE2激活对PAH大鼠生存率的影响实验终点时,肺动脉高压大鼠和Mas受体拮抗组大鼠全部死亡,ACE2预防组未出现死亡,ACE2治疗组生存率为80%。统计学分析,ACE2治疗组和预防组与肺动脉高压组差异显著。 3.ACE2激活对血液动力学和右心室的影响大鼠左肺切除联合野百合碱注射2周后,肺动脉压力升高,右心室肥大；ACE2对照组肺动脉压力没有明显变化。ACE2激活可以防止肺动脉高压和右心室肥厚,同样ACE2治疗4周也可以降低肺动脉压力,但是无法逆转肥厚的右心室。各处理组大鼠动脉收缩压没有变化。 4.ACE2激活对肺动脉内皮细胞功能的影响在相同的基础肺动脉压力下,肺动脉高压大鼠乙酰胆碱诱导的内皮依赖的舒张反应减弱,ACE2不但可以抑制它的减弱,而且可以修复受损的舒张反应。硝普钠诱导的肺内皮依赖的舒张反应各组之间没有明显差异。 5.ACE2激活对肺动脉中膜肥厚和内膜增生的影响肺动脉高压大鼠的直径为100-200gm的肺小动脉主要表现为中膜肥厚,ACE2激活可以抑制和逆转中膜肥厚。直径为50-100μm的肺小动脉主要表现为内膜增生,梗阻明显,ACE2预防组大鼠则主要表现为中膜肥厚,ACE2治疗组大鼠小动脉梗阻程度也显著下降。 6.Mas受体对ACE2效果的影响同时应用ACE2激活剂Rec和Mas受体阻断剂A-779,肺动脉高压预防作用消失,包括改善生存率,降低肺动脉压力,抑制右心室肥厚,肺小动脉中膜肥厚和内膜增生。 7.ACE2激活对肾素血管紧张素系统各主要成份的影响肺动脉高压形成以后,ACE和AT1R上升为正常的2.49和2.75倍,Ang IⅡ则由25.06±3.81pg/mg上升到91.00±6.70pg/mg,ACE2激活可抑制ACE(78.5％), AT1R(78.9%)和Ang Ⅱ(46.60±10.00pg/mg)的上升,但是肺动脉形成以后再激活ACE2,ACE和AT1R下降不明显,仅能降低Ang Ⅱ(52.01±7.61pg/mg,). 肺动脉高压大鼠肺组织ACE2降低为正常的41.67%,而预防和治疗组ACE2则达到肺动脉高压大鼠的2.52和2.35倍。肺动脉高压对Mas和Ang-(1-7)的影响不大,ACE2激活后,Mas则分别升高了的2.43和2.51倍,Ang-(1-7)也相应升高。肺动脉高压大鼠肺组织ACE/ACE2和AT1R/Mas比值明显升高,而预防组和治疗组这两个比值则降低。 8.ACE2激活对肺动脉平滑肌细胞增殖的影响各组直径为50-100gm肺小动脉壁均广泛表达平滑肌细胞抗体,提示平滑肌细胞在其病变中占重要地位。肺动脉高压大鼠肺动脉增殖细胞核抗原阳性率显著升高,ACE2激活后可以降低平滑肌细胞的增殖细胞核抗原阳性率,抑制增殖。 9.ACE2激活对肺组织炎症因子的影响大鼠肺动脉高压形成后,IL-6上升47倍,MCP-1上升31倍,TNF-a上升16倍,IL-10下降79%；而预防组IL-6降低85%,MCP-1降低86%, TNF-α降低82%,IL-10上升11倍,治疗组则IL-6降低82%,MCP-1降低80%, TNF-α降低78%,IL-10升高9倍。结论 1.Rec持续注射可以在大鼠体内有效的激活肺组织ACE2 2.ACE2激活可预防左肺切除联合野百合碱注射诱导的大鼠重度肺动脉高压形成 3.ACE2激活一定程度上逆转大鼠重度肺动脉高压的血液动力学和病理学改变 4.ACE2激活通过调控肾素血管紧张素系统内缩血管／促增殖轴(ACE-AngⅡ-AT1R轴)和血管保护轴(ACE2-Ang-(1-7)-Mas轴)之间的平衡起作用。 5.在大鼠肺动脉高压的形成和进展过程中激活ACE2,可修复内皮依赖的血管舒张反应,改善内皮功能；可减少新生内膜中平滑肌细胞PCNA的表达,抑制平滑肌细胞增生；可降低肺组织促炎因子IL-6, MCP-1, TNF-α,升高抗炎因子IL-10,从而减轻炎症反应。
[Abstract]:Background and purpose
Under the action of pathogenic factors, the imbalance of pulmonary circulation regulating function starts a series of pathophysiological changes, including endothelial dysfunction, inflammation activation and so on, causing abnormal contraction of pulmonary vessels, proliferation of smooth muscle cells, vascular remodeling, and pulmonary arterial hypertension. Renin angiotensin system is an important regulatory system of the cardiovascular system. It plays an important role in the pathogenesis of pulmonary hypertension, but the intervention of the classical renin angiotensin system (ACE-Ang II -AT1R axis) is not good for the treatment of pulmonary hypertension. Angiotensin converting enzyme 2 (ACE2) and the ACE2-Ang- (1-7) -Mas axis involved in the renin angiotensin system are new members of the renin angiotensin system and play an antagonism to the classical renin blood vessels. In this study, the ACE2 agonist Resorcinolnaphthalein (Rec) was used in this study to construct a model of severe pulmonary hypertension with monocrotaline combined with lobectomy, and to explore the role and mechanism of ACE2 activation in the prevention and reversal of severe pulmonary vein pressure.
Method
1. experimental animals and groups: SD rats were randomly divided into 6 groups: blank control group, ACE2 control group, pulmonary hypertension group, ACE2 prevention group, ACE2 treatment group, Mas receptor antagonist group (Rec+A-779). 1 weeks after left pneumonectomy, the subcutaneous injection of monocrotaline induced severe pulmonary arterial hypertension,.ACE2 control and ACE2 prevention group began to hold on first days after the injection of lilocinine. Continuous injection of Rec, ACE2 treatment group began Rec injection on the fourteenth day, while Mas receptor antagonist group started injecting Rec and Mas receptor antagonist A-779. on the first day.
2. after 2 weeks of intervention, 8 were taken out of the other groups except the ACE2 treatment group (42 days of intervention). The mean pulmonary artery pressure and arterial systolic pressure were measured by cardiac catheterization. Right ventricular hypertrophy index was measured by the separation of right ventricle. The middle membrane thickness of pulmonary artery (100-200gm) and the intimal obstruction hyperplasia of pulmonary artery (50-100gm) were analyzed after elastin staining (Verhoeff- ferulin staining). Degree.
3. endothelial function test: different concentrations of acetylcholine and sodium nitroprusside were injected into the pulmonary artery to detect the changes of endothelium-dependent vasodilatation.
4. activated fluorescence resonance energy transfer assay was used to detect the activity of ACE2 in lung tissue.
5. survival analysis: Kaplan-Meier survival analysis.
6. ELISA was used to detect the concentration of Ang II and Ang- (1-7) in lung tissue. Western blot was used to detect the expression levels of ACE, ACE2, AT1R and Mas in lung tissue.
7. immunohistochemistry was used to detect the expression of smooth muscle antibody (a-SMA) and proliferating cell nuclear antigen (PCNA) of pulmonary artery smooth muscle cells in diameter of 50-100 m, and to analyze the proliferation of pulmonary artery smooth muscle cells.
8. RT-PCR was used to detect inflammatory cytokines IL-6, MCP-1, TNF- alpha, IL-10. in lung tissue.
Result
The effect of 1.Rec on the activity of ACE2 enzyme in the lung tissue of rats was 1.74 times as high as that of the normal ACE2 control group; the activity of ACE2 enzyme in the pulmonary hypertension group decreased by 38.2%, and the activity of ACE2 enzyme in the ACE2 prevention and treatment group was 1.87 and 1.78 times higher than that in the pulmonary hypertension group, suggesting that Rec could activate the lung tissue ACE2. in rats.
The effect of 2.ACE2 activation on the survival rate of PAH rats
At the end of the experiment, all the rats of the pulmonary hypertension rats and the Mas receptor antagonist group died, the ACE2 prevention group did not die, the survival rate of the ACE2 treatment group was 80%. statistical analysis, and the difference between the ACE2 treatment group and the prevention group was significantly different from the pulmonary hypertension group.
Effects of 3.ACE2 activation on hemodynamics and right ventricle
After 2 weeks of left lung resection combined with monocrotaline injection, pulmonary arterial pressure increased and right ventricular hypertrophy was increased. Pulmonary arterial pressure in ACE2 control group was not significantly changed by.ACE2 activation to prevent pulmonary hypertension and right ventricular hypertrophy. Similarly, the pulmonary artery pressure could be reduced by the treatment of ACE2 for 4 weeks, but the right ventricle could not be reversed by the hypertrophy of the right ventricle. Rats in each group were treated. The systolic pressure of the arteries did not change.
Effect of 4.ACE2 activation on the function of pulmonary artery endothelial cells
Under the same basic pulmonary arterial pressure, the endothelium dependent relaxation response induced by acetylcholine in the pulmonary hypertension rats weakened, and ACE2 not only inhibited its weakening, but also repaired the damaged diastolic response. There was no significant difference in the endothelium dependent diastolic response induced by sodium nitroprusside.
Effect of 5.ACE2 activation on pulmonary artery intima thickening and intimal hyperplasia
The pulmonary arterioles with the diameter of 100-200gm in the pulmonary hypertension rats were mainly the middle membrane hypertrophy, and the ACE2 activation could inhibit and reverse the middle membrane hypertrophy. The pulmonary arterioles with the diameter of 50-100 u m were mainly intima hyperplasia, and the obstruction was obvious. The rats in the ACE2 prevention group were mainly the middle membrane fat, and the degree of the small artery obstruction in the ACE2 treatment group was also obvious. It's falling.
The effect of 6.Mas receptor on the effect of ACE2
At the same time, ACE2 activator Rec and Mas receptor blocker A-779 were used, and the prevention of pulmonary hypertension disappeared, including improving survival rate, reducing pulmonary artery pressure, inhibiting right ventricular hypertrophy, pulmonary arterioles hypertrophy and intima hyperplasia.
Effects of 7.ACE2 activation on the main components of renin angiotensin system
After the formation of pulmonary hypertension, ACE and AT1R increased to 2.49 and 2.75 times as normal, Ang I II increased from 25.06 + 3.81pg/mg to 91 + 6.70pg/mg, ACE2 activation inhibited ACE (78.5%), AT1R (78.9%) and Ang II (46.60 + 10.00pg/mg), but pulmonary artery formation reactivated ACE2. .01 + 7.61pg/mg,).
ACE2 in pulmonary tissue of pulmonary hypertension rats decreased to 41.67% of normal, while ACE2 in prevention and treatment group reached 2.52 and 2.35 times that of pulmonary hypertension rats. Pulmonary hypertension had little effect on Mas and Ang- (1-7). After ACE2 activation, Mas increased by 2.43 and 2.51 times respectively, and Ang- (1-7) increased correspondingly.
The ratio of ACE/ACE2 to AT1R/Mas in lung tissue of rats with pulmonary hypertension increased significantly, while the two ratios in the prevention group and the treatment group decreased.
Effect of 8.ACE2 activation on proliferation of pulmonary artery smooth muscle cells
The expression of smooth muscle cell antibody was widely expressed in the wall of 50-100gm pulmonary arterioles in each group, suggesting that smooth muscle cells play an important role in its pathological changes. The positive rate of proliferating cell nuclear antigen (PCNA) in pulmonary artery of pulmonary hypertension rats was significantly increased. ACE2 activation could reduce the positive rate of proliferating cell nuclear antigen (PCNA) of smooth muscle cells and inhibit the proliferation.
Effect of 9.ACE2 activation on inflammatory factors in lung tissue
After the formation of pulmonary hypertension, IL-6 increased 47 times, MCP-1 increased 31 times, TNF-a increased 16 times, IL-10 decreased by 79%, IL-6 decreased by 85%, MCP-1 decreased by 86%, TNF- alpha was 82%, IL-10 increased 11 times, IL-6 decreased by 82%, MCP-1 decreased 80%, TNF- alpha decreased 78%, IL-10 increased 9 times.
conclusion
Continuous injection of 1.Rec can effectively activate lung tissue ACE2 in rats.
2.ACE2 activation prevents the formation of severe pulmonary hypertension induced by left lung resection combined with monocrotaline injection in rats
3.ACE2 activation can reverse the hemodynamic and pathological changes of severe pulmonary hypertension in rats to some extent.
4.ACE2 activates the balance between the regulation of the renin angiotensin system (ACE-Ang II -AT1R axis) and the vascular protective axis (ACE2-Ang- (1-7) -Mas axis) by regulating the renin angiotensin system.
5. the activation of ACE2 in the formation and progression of pulmonary hypertension in rats can repair endothelium dependent vasodilatation, improve endothelial function, reduce the expression of PCNA in the smooth muscle cells in neointima, inhibit the proliferation of smooth muscle cells, and reduce the IL-6, MCP-1, TNF- alpha, and anti-inflammatory factors IL-10 of lung tissue, thus reducing the anti-inflammatory factor IL-10. Inflammatory reaction.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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