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染料木黄酮对缺氧性肺动脉高压及右心室肥大的预防作用
摘　要：目的:研究染料木黄酮(genistein,Gen)对慢性缺氧性肺动脉高压(HPH)的预防作用,为防治HPH提供一种新的途径.方法:健康雄性Wistar大鼠,体重(210.3±28.7)g,随机分为5组:①平原对照组(C);②慢性缺氧组(H);③缺氧+Gen低剂量组(H+L):25mg/④缺氧+Gen中剂量组(H+M):50 mg/⑤缺氧+Gen高剂量组(H+H):100mg/C组在平原,②～⑤组每天灌胃大鼠受试物或与受试物等容积的二甲基亚砜(DMSO),置于减压舱,模拟海拔5000m高原,8h/d,持续21d,分别测定肺动脉压、右心室功能和右心室肥大指数.结果:Gen可以显著抑制慢性缺氧引起的肺动脉压升高(P＜0.01).单纯缺氧组右心室肥大指数RV/(LV+Sep)显著增加(P＜0.01);与单纯缺氧组比较,Gen各组RV/(LV+Sep)从低剂量到高剂量呈逐渐降低的趋势(40.53±3.80)%,(39.07±3.69)%,(33.73±3.20)%,但仅高剂量组(100mg/kg bw)有显著性差异(P＜0.01).结论:Gen可以有效防治慢性缺氧性肺动脉高压,对慢性缺氧诱导的右心室肥大的预防作用与剂量有关,其作用机制有待进一步探讨.
优质期刊推荐KeywordsGαq protein,polypeptide,monocrotaline,right ventricle remodeling,resveratrol,apoptosis,pulmonary hypertension,pulmonary artery remodeling
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&&&&&&&&肺动脉高压是进行性和致命性疾病,系多种原因引起的肺循环压力高于正常的病症,可继发右心室重构、右心衰竭,故防治肺动脉高压及右心室重构具有重要的临床意义。现可用的降肺动脉高压药物不仅数量少、疗效有限、且因缺乏针对性,因而存在诸多不良反应。目前对右心室重构的研究相当有限,且临床无针对性抗右心室重构药物,因此,寻找抗肺动脉高压和/或抗右心室重构新药十分必要。肺动脉平滑肌细胞(PASMCs)异常增殖,导致远端肺动脉腔狭窄,甚至阻塞,造成肺血管阻力增加,是肺动脉高压病理生理机制的重要特点。因此,抑制或逆转肺动脉平滑肌增殖,改善远端肺动脉重构,是预防和治疗肺动脉高压的关键措施之一。右心室重构最初是继发于内在刺激和外部刺激的代偿现象,最终会发展成为右心衰竭,是肺动脉高压患者死亡的重要原因。因此,抑制右心室重构发展具有重要意义。心肌细胞凋亡作为细胞死亡的方式之一,与细胞坏死一道,共同调控心肌细胞数量,是各种病因导致心室重构的重要环节。汇利心康(GCIP-27)是我室针对心肌肥大发生、发展过程中具有重要作用的Gαq羧基末端,克隆制备的一种G蛋白竞争性抑制性肽。既往研究证实:GCIP-27能有效抑制AngII,NA诱导的离体心肌细胞肥大反应,抑制不同动物模型的左心室重构,并且GCIP-27抑制自发性高血压大鼠(SHR)左心室重构几乎不依赖于其降低体循环动脉压作用。另外,GCIP-27还可抑制SHR主动脉平滑肌重构效应以及抑制AngII所诱导体外血管平滑肌细胞增殖效应。为此,本课题拟对GCIP-27防治MCT所诱导的肺动脉高压及右心室重构作用及机制进行了探索性研究。白藜芦醇(resveratrol,Res)是一种天然多酚类化合物,对心血管有多种益处。Res能抑制Ang-II,PE所诱导的离体心肌细胞肥大反应及抑制心肌成纤维细胞增殖和分化,抑制不同模型的左心室重构;另外,还具有保护肺血管内皮及抑制Ang-II等引起的体外的大鼠主动脉平滑肌细胞增殖作用;Res苷具有抑制急、慢性低氧型肺动脉高压作用。鉴于目前尚缺乏针对性防治肺动脉高压和右心室重构药物,为了拓宽筛选领域,本课题一并研究Res对MCT所诱导的大鼠肺动脉高压及右心室重构的防治影响。方法:♂SD大鼠随机分为正常对照组(control组,n=8)、MCT模型组(MCT组,n=12)、GCIP-27低剂量预防处理组(GCIP-27-P-L组,n=8)、GCIP-27高剂量预防处理组(GCIP-27-P-H,n=8)、GCIP-27治疗处理组(GCIP-27-T-H,n=8)、Res低剂量预防处理组(Res-P-L组,n=8),Res高剂量预防处理组(Res-P-H组,n=8),Res治疗处理组(Res-T-H组,n=8)。大鼠颈背部一次性皮下注射MCT(50 mg/kg)造模,于造模后1-21d或14-21d分别给GCIP-27(30, 90μg/kg,ip,bid)或Res(10, 30 mg/kg, ig, bid)处理。在实验过程中,观察动物一般状况及动物死亡情况;在实验第21d,用彩色多普勒超声成像技术(CDFA)和肺脉冲多普勒技术(PW)观察所有存活动物的肺动脉加速时间(PAAT)、右心室游离壁厚度(RVFWT)、和三尖瓣返流等指标的变化;在实验第22d用右心导管术检测肺动脉收缩压(PASP)、肺动脉舒张压(PADP)、平均肺动脉压(MPAP)、右心室收缩压( RVSP )等指标;用称重法测量和计算右心室肥大指数[RVHI=RV/(LV+SEP)]、右心室质量指数(RVMI=RV/BW)、肺重指数(LI=LUNG/BW);用石蜡包埋处理肺或右心室标本;用偏振光显微镜观察右心室及肺HE染色形态学改变及免疫组化技术处理的相关抗原表达等变化;用透射电镜技术(TEM)观察右心室超微结构;用TdT介导的缺口末端标记法(TNNEL)检测右心室心肌细胞凋亡的变化,用免疫组织化学技术观察肺动脉α-平滑肌肌动蛋白(α-SMA抗原),增殖细胞核抗原(PCNA),第八因子抗原表达的变化,以及右心室Bcl-2抗原和Bax抗原表达的变化。另外,针对造模后第14d-21d给予GCIP-27或Res处置的动物、正常对照组动物和单纯MCT模型组动物,我们用CDFA和PW技术观察了这些动物在造模前、造模第14d、造模第21d的PAAT、RVFWT等指标的动态变化。结果:1.MCT模型组有33%动物死亡,尽管这组动物开始时数目为12只,但在实验期间分别于13d、14d、19d和20d各死亡1只动物,故该组仅剩8只存活动物进入超声及血流动力学检测。其余存活MCT模型组大鼠一般状况差,体重降低明显;而GCIP-27或Res处理组无一只动物死亡,一般状况较好,并且显著改善MCT所引起的体重下降。2.与造模前相比,MCT显著缩短大鼠的第14 d和21 d的PAAT(P&0.01);与MCT组第21d相比,GCIP-27或Res明显改善MCT所诱导的PAAT的缩短(P&0.05或P&0.01);在造模第14-21d,给予GCIP-27或Res处置的MCT造模的大鼠,其第21天PAAT值比第14天的PAAT值延长(P&0.05)。3.与正常对照组相比,MCT明显升高PASP、PADP、MPAP、RVSP(P&0.01);与MCT组相比,GCIP-27显著抑制MCT所诱导的PASP、RVSP的升高(P&0.05,P&0.01),但仅GCIP-27-P-H处理组显著抑制MCT所诱导的MPAP的升高(P&0.05);与MCT组相比,除了对PADP没有显著水平的降低外(P&0.05),Res明显改善上述所有指标的变化(P&0.05,P&0.01)。4.偏振光学显微镜观察左肺组织HE染色的病理结构揭示:MCT引起明显的肺间质炎症,与正常对照相比,显著增加LI、肺小动脉中膜相对厚度(P&0.01),上调肺动脉α-SMA抗原的表达(P&0.01),增加PCNA抗原核阳性的PASMCs数目(P&0.01);与MCT组相比,GCIP-27和Res均一定程度减轻肺间质炎症,并且二者均不同程度显著地改善上述指标的变化(P&0.05,P&0.01)。5.MCT明显减少肺小动脉第八因子抗原的表达,减少肺毛细血管数量(P&0.01);与MCT组相比,Res不同程度地增加肺动脉第八因子抗原的表达,增加肺毛细血管数量(P&0.05,P&0.01)。6.与造模前相比,MCT显著升高大鼠第14 d和21 d的RVFWT(P&0.01),在造模21d,MCT组出现75%三尖瓣返流;与MCT组相比,GCIP-27或Res明显改善MCT所诱导的RVFWT的增加(P&0.05,P&0.01),并且GCIP-27或Res处理的动物均无三尖瓣返流;在造模第14-21d,给予GCIP-27或Res处置的MCT造模的大鼠,其第21天RVFWT值比第14天的RVFWT值明显减少(P&0.01)。7.与正常对照组相比,MCT明显升高RVHI、RVMI(P&0.01);与MCT组相比,GCIP-27显著抑制MCT所诱导的RVHI和RVMI的增高(P&0.01);与MCT组相比,Res也显著抑制MCT所诱导的RVHI,RVMI的增高(P&0.05,P&0.01)。8.偏振光学显微镜观察右心室组织HE染色的病理结构揭示:MCT组心肌细胞排列紊乱,心肌细胞横截面积和细胞长度均显著增加,胞浆肿胀,心肌细胞间质增生等。与MCT组相比,GCIP-27及Res均不同程度地明显抑制MCT所引起的右心室组织病理结构改变。9.TEM观察右心室心肌细胞超微结构显示:右心室心肌细胞线粒体肿胀、内有空泡形成、呈髓鞘样退行性改变、肌浆网扩张、横纹、Z线不清、肌丝排列紊乱及部分肌丝局灶性坏死;与MCT组相比,GCIP-27及Res均不同程度地明显改善MCT所引起的右心室心肌细胞超微结构的改变。10.MCT明显增加右心室心肌细胞TUNEL阳性心肌细胞的数量(P&0.01),下调右心室心肌细胞的Bcl-2抗原的表达(P&0.01)和上调心肌细胞的Bax抗原的表达(P&0.01);与MCT组相比,GCIP-27显著改善MCT所引起上述指标的变化(P&0.01);Res也改善MCT所引起上述改变(P&0.05,P&0.01)。结论:1.GCIP-27特异性抑制MCT诱导的右心室重构效应,与抗心肌细胞凋亡及一定程度地降低肺动脉高压作用相关。2.GCIP-27通过上调心肌细胞Bcl-2抗原、下调Bax抗原的表达、保护心肌细胞的线粒体及一定程度降低右心室后负荷而抑制右心室心肌细胞凋亡。3.GCIP-27抑制PASMCs增殖、改善肺动脉重构,一定程度地抑制MCT诱导的肺动脉高压。4.Res抑制MCT诱导的右心室重构,与抗心肌细胞凋亡及降低肺动脉高压作用相关。5.Res通过上调Bcl-2抗原、下调Bax抗原表达、保护心肌细胞线粒体及降低右心室后负荷而抑制右心室心肌细胞凋亡。6.Res通过增加肺毛细血管数量,抑制PASMCs的增殖以及改善肺动脉重构,从而较明显地抑制MCT诱导的肺动脉高压。
&&&&BACKGROUD: Pulmonary hypertension is a serious illness, with multiple potential causes that may progressively worsen and eventually prove fatal, which was classified into two categories: primary pulmonary hypertension and secondary pulmonary hypertension. Pulmonary hypertension is characterized by progressive remodeling of the small pulmonary arteries, causing increased resistance to blood flow in the lung, which, in turn, can raise the pulmonary arterial pressure. As the resistance rises, the afterload on the right ventricle increases, which leads to right ventricle remodeling and ultimately failure. Available treatments for pulmonary hypertension are limited, and often associated with significant side effects. Other deseases are associated with right ventricle remodeling such as left ventricular pathology, Chagas’ disease, and arrhythmogenic right ventricle cardiomyopathy. The pathophysiology of right ventricular remodeling is a complex process. Due to the structure and function between the two chambers are very different, the right ventricular remodeling may include unique elements not observed in the left ventricular remodeling. Therefore, further understanding of these issues is of pivotal importance. The available treatments for right ventricle remodeling are also quite limited. Therefore, it is necessary to seek for some materials for treatment of pulmonary hypertension and right ventricle remodeling.Huilixinkang （GCIP-27） is a Gαq protein carboxyl terminus imitation polypeptide, which was cloned and constructed in our laboratory previously, prevent the hypertrophyic responses in cultured rat cardiomyocyte by noradrenaline （NA） and angiotensin II （AngII） and inhibited the left ventricular hypertrophy induced by NA or AngII in rats and mice, and also inhibited the left ventricular remodeling in spontaneously hypertensive rats （SHR）. Interestingly, GCIP-27 inhibiting left ventricular remodeling is almost not depended on the effect of decreasing blood pressure in SHR. In addition, GCIP-27 inhibited the remodeling of aorta in SHR and prevented the proliferation responses responses in cultured vascular smooth muscle cell （VSMCs） by AngII. The polyphenolic compound resveratrol （Res, trans-3, 4’, 5-trihydroxystilbene）, is a naturally occurring phytochemical, which has been found in many plants. Relatively high quantity of Res is found in grapes. Res possesses a wide range of biological and pharmacological properties such as anti-oxidant, anti-inflammatory, anti-proliferation properties and the cardio-protective effects. Res inhibits the cardiomyocyte hypertrophy induced by AngII or PE in rat neonatal myocyte cultures, and the cardiac fibroblast proliferation. Res shows the protective effect on pulmonary endothelial cell, and represses the proliferation responses of in cultured VSMCs. In addition, the glycoside of Res was reported to prevent the pulmonary hypertension in the acute and chronic hypoxia animal model.AIM: The study is designed to study the potential effects of GCIP-27 on pulmonary hypertension and right ventricle remodeling using monocrotaline （MCT）-treated rats, and primarily analyze the mechanism.METHODs: Male Sprague-Dawley rats were given a single injection of MCT （50 mg/kg, sc） and then received treatment with vehicle or GCIP-27 （30, 90μg/kg, ip bid） or Res （10 and 30 mg/kg, ig, bid） from 121 days or 1421days, respectively. The general state was observed. At the end of treatments, all survival rats received echocardiography to observe the RV free wall thickness （RVFWT）, pulmonary arterial acceleration time （PAAT） and tricuspid regurgitation. All survival rats also received right catheterization to observe pulmonary arterial systolic pressure （PASP）, pulmonary artery diastolic pressure （PADP）, mean pulmonary arterial pressure （MPAP） and right ventricular systolic pressure （RVSP）. In addition, right ventricular hypertrophy index （RVHI）, right ventricular mass index （RVMI）, lung index （LI） were obtained by weighting. Lung and right ventricles tissue were embedded in paraffin and stained with haematoxylin–eosin （HE）, then examined under an optical microscope. Furthermore, the right ventricles were subjected to untrastructural analysis by transmission electron microscopy （TEM）. The expression ofα-smooth muscle actin （α-SMA）, proliferating cell nuclear antigen （PCNA） orⅧfactor antigen in pulmonary arteries and the expression of Bcl-2 and Bax antigen in right ventricles were done through immunohistochemical technique. The TdT mediated nick dUTP end labeling （TUNEL） technique was used to observe the apoptosis of cardiomyocytes in right ventricles. In addition, we used chocardiography to observed dynamic changes for PAAT, RVFVT and tricuspid regurgitation in some animals on 0 day, 14 day and 21day, respectively, which include animals treatment with GCIP-27 or Res from 1421days, control animals and MCT model animals.RESULTs1. MCT produced 33% mortality in model rats, while no mortality occurs in animal treatment with Res or GCIP-272. As compared with 0 d, MCT significantly decreased PAAT on 14day and 21day, respectively （P&0.01）. As compared with MCT group, both Res and GCIP-27 improved the decreased PAAT on 21day （P&0.05, P&0.01）. As compared with 14day, the decreased PAAT could be ameliorated on 21day （P&0.05） in animals treatment with GCIP-27 or Res from 1421days.3. As compared with the control group, MCT increased RVSP, PASP, PADP, MPAP （P&0.01）. Except for PADP, Res improved all the above changes （P&0.05, P&0.01）. GCIP-27 could improve the increased RVSP, PASP induced by MCT （P&0.05, P&0.01）, but only the treatment with high dose of GCIP-27 （90μg/kg） from 121 day improved the increased MPAP induced by MCT （P&0.05）.4. As compared with the control group, MCT effectively increased LI, the pulmonary arterial medial thickness and the expression ofα-SMA antigen and PCNA antigen in pulmonary arterial smooth muscle cell （PASMCs）. Both Res and GCIP-27 improved all these changes to some extent （P&0.05, P&0.01）.5. As compared with the control group, MCT also effectively decreased the expression ofⅧfactor antigen in the PAs and the number of pulmonary capillary （P&0.01）, however, Res improved these changes （P&0.05, P&0.01）.6. As compared with 0 d, MCT increased RVFWT on 14day and 21day （P&0.01）, respectively, and induced 75% tricuspid regurgitation on 21day. As compared with in MCT group, both Res and GCIP-27 could improve the increased RVFWT induced by MCT （P&0.05, P&0.01）. No tricuspid regurgitation occured in animal treatment with Res or GCIP-27. As compared with 14day, the increased RVFWT could be ameliorated on 21day in animals treatment with GCIP-27 or Res from 1421days （P&0.01）.7. As compared with the control group, MCT increased the RVHI, RVMI （P&0.01）. However, GCIP-27 and Res improved all these changes （P&0.05, P&0.01）.8. In MCT-treated rats, the microstructure of right ventricles was as follows: the cardiomyocytes hypertrophy, interstitium fibrosis and myofibrilla lined up in disorder. However, both GCIP-27 and Res improved these morphological changes to some extent. 9. In MCT-treated rats, the ultrastructure of right ventricular cadiocytes was observed as the mitochondria swell and medullary sheath-like degeneration, the sarcoplasmic reticulum enlarged and the myofilaments dissolved and the Z lines broken. However, GCIP-27 and Res improved these morphological changes.10. As compared with the control group, MCT induced the apoptosis of cardiomyocytes and the over-expression of the Bax antigen and the low-expression of Bcl-2 antigen in the right ventricle tissues, respectively （P&0.01）. However, both Res and GCIP-27 markedly attenuated the above changes （P&0.05, P&0.01）.CONCLUSION1. Huilixinkang （GCIP-27） significantly inhibits right ventricle remodeling induced by MCT. The mechanism is associated with the anti-apoptosis effect in right ventricular cardiomyocytes and the effect of reducing pulmonary hypertension to some extent.2. GCIP-27 significantly attenuates the apoptosis of right ventricular cardiomyocytes in MCT-treated rats. The mechanism is relative to the overexpression of Bcl-2 antigen, low expression of Bax antigen, the protective effect of mitochondrium in right ventricular cardiomyocytes as well as the effect of reducing pulmonary hypertension to some extent.3. GCIP-27 inhibits the pulmonary hypertension to some extent, which is relative to the impressing effect on the proliferation of PASMCs and pulmonary artery remodeling.4. Res inhibits right ventricle remodeling induced by MCT. The mechanism is associated with the anti-apoptosis effect in right ventricular cardiomyocytes and the reducing pulmonary hypertension effect significantly.5. Res significantly attenuates the apoptosis of right ventricular cardiomyocytes in MCT-treated rats. The anti-apoptotic effects is relative to the overexpression of Bcl-2 antigen, low expression of Bax antigen, the protective effect of mitochondria in right ventricular cardiomyocytes as well as the significant effect of reducing pulmonary hypertension.6. Res significantly inhibits the pulmonary hypertension, which is relative to the impressing effect on the proliferation of PASMCs, pulmonary artery remodeling, and the increased number of pulmonary capillary in MCT- treated rats.
&&&&&&&&&&汇利心康及白藜芦醇对野百合碱诱导的肺动脉高压及右心室重构的影响及机制研究英文缩写词表6-8英文摘要8-11中文摘要12-16正文 汇利心康及白藜芦醇对野百合碱诱导的肺动脉高压及右心室重构的影响及机制研究16-105&&&&前言16-20&&&&第一部分 汇利心康对MCT 诱导的肺动脉高压及右心室重构的影响及机制研究20-58&&&&&&&&分题一 汇利心康对MCT 诱导的肺动脉高压及右心室重构的影响20-43&&&&&&&&&&&&材料和方法20-27&&&&&&&&&&&&结果27-36&&&&&&&&&&&&讨论36-43&&&&&&&&分题二 汇利心康抑制MCT 诱导的肺动脉高压及右心室重构的机制研究43-57&&&&&&&&&&&&材料和方法43-48&&&&&&&&&&&&结果48-53&&&&&&&&&&&&讨论53-57&&&&&&&&小结57-58&&&&第二部分 Res 对MCT 诱导的肺动脉高压和右心室重构的影响和机制的研究58-94&&&&&&&&分题一 Res 对MCT 诱导的肺动脉高压和右心室重构的影响58-78&&&&&&&&&&&&材料和方法58-65&&&&&&&&&&&&结果65-73&&&&&&&&&&&&讨论73-78&&&&&&&&分题二 Res 抑制MCT 诱导的肺动脉高压和右心室重构的机制研究78-93&&&&&&&&&&&&材料和方法78-82&&&&&&&&&&&&结果82-88&&&&&&&&&&&&讨论88-93&&&&&&&&小结93-94&&&&全文结论94-95&&&&致谢95-96&&&&参考文献96-105文献综述 右心室重构的研究进展及评价105-123&&&&参考文献116-123博士在读期间发表的论文123-124
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