【作者】 王秀琴；

【导师】 李晓辉；

【作者基本信息】 第三军医大学 ， 药理学， 2008， 博士

【摘要】 高血压病是遗传和环境等多种因素所致的全球性常见病和多发病。近20多年来,随着社会经济状况的改善和人民生活方式的转变,高血压的发病率呈逐年增加趋势;如果高血压长期不治疗,会引起心、脑、肾等重要脏器的损害,如脑卒中,心肌梗死,充血性心衰及肾衰等。左室肥厚是高血压主要的靶器官损害之一,可以引起心脏顺应性以及收缩和舒张功能的下降,并最终导致心力衰竭的发生。心肌肥厚与重构是引起心血管疾病发生率和死亡率显著升高的独立的危险因素。心室重构特点为心肌细胞肥大,间质纤维化及左室重量、形状、结构及功能的改变。高血压导致的左心室肥厚、心肌纤维化及冠脉结构异常即心室重构,是高血压患者心血管事件发生与死亡的主要病理基础。抗高血压治疗不仅应有效控制血压,更应有效抑制和逆转其心血管结构和功能异常,以减少心血管事件的发生率。G蛋白在高血压及心血管重构的形成发展过程中扮演着非常重要的角色,在多种因素诱发心肌肥大的细胞信号转导过程中,G蛋白处于枢纽地位,针对G蛋白设计特异性阻止心肌肥大发生发展的药物可能具有更强的作用。GCIP(G protein competitive inhibitory peptide)是由我室针对在心肌肥厚发生发展过程中具有重要作用的G蛋白α亚单位克隆制备出的一种G蛋白竞争性抑制肽,对体外培养的去甲肾上腺素诱导的大鼠心肌细胞肥大具有较好的抑制作用。GCIP-27是在GCIP基础上经分子优化制备出的一种GCIP的衍生肽,能够抑制由AngⅡ、NE等诱导的心肌细胞的肥大和增生;在多种心肌肥厚模型上具有良好的抗心肌肥大作用;并对自发性高血压大鼠(spontaneous hypertensive rat,SHR)的血管重构有良好的抑制作用。本实验以自发性高血压大鼠(SHR)为研究对象,采用多普勒超声心动图及无创尾套法,系统评价GCIP-27对SHR心室重构及血压的影响;采用RT-PCR法从分子水平初步探究其抗心室重构的作用机制;并用同位素标记示踪法观察了GCIP-27在动物体内的分布、排泄、代谢过程,获得药代动力学参数,为其以后研发及应用奠定基础。方法:1.用SHR作为实验动物模型,给予GCIP-27(10、30、90μg.kg-1,i.p.,bid)或氯沙坦(6mg.kg-1,ig,o.d.)治疗8周,并设立Wistar大鼠正常对照。通过如下方法评价GCIP-27对大鼠心室重构及血压的影响:①大鼠血压的测定采用无创尾套法;②超声心动图评价大鼠的左心室结构和心功能;③心肥厚指数及心肌间质胶原含量的测定评价大鼠的心室肥厚程度;④HE染色,光镜下观察大鼠心肌组织形态学改变;VG染色,对大鼠心肌胶原进行定性及半定量分析;透射电镜观察大鼠心肌细胞超微结构的改变。2.采用RT-PCR法检测大鼠心肌ANPmRNA、BNPmRNA、α-MHCmRNA、β-MHC mRNA的表达;放射免疫分析法测定心肌细胞磷脂酶C(PLC)活性。3.采用同位素标记示踪法,结合三氯乙酸(TCA)沉淀法及低分子量SDS-PAGE法,对GCIP-27进行了药代动力学研究。结果:1. GCIP-27对SHR血压的影响SHR的收缩压显著高于正常组,10、30、90μg.kg-1的GCIP-27治疗8周后均能明显降低SHR的收缩压(p<0.01),但其降压效果远不如6mg.kg-1的氯沙坦,90μg.kg-1的GCIP-27降压效果仅为后者的60.6%。2. GCIP-27对SHR心室重构的影响(1) SHR模型组大鼠有明显的心室重构现象,与正常组相比,模型组大鼠左室后壁厚度(PWT)、室间隔厚度(IVST)、心肌肥厚指数(LVMI)、心肌胶原含量、心肌间质胶原面积(CA)、及胶原容积分数(CVF)明显增高(p<0.01);经治疗8周后,GCIP-27 (10、30、90μg.kg-1)均能显著减少(p<0.05)大鼠的PWT、IVST、LVMI、CA、CVF及胶原含量;氯沙坦也能降低除LVMI之外的上述指标(p<0.05),但降低程度不及GCIP-27显著。(2)病理及电镜:①SHR模型组大鼠心肌组织形态及心肌细胞超微结构严重受损,光镜下可见心肌细胞明显浊肿,空泡样变化;细胞核肥大或固缩;细胞内容物成颗粒状,断裂融合;可见到坏死灶。VG染色可见模型组SHR大鼠心肌胶原分布较多。电镜下可见心肌细胞肿大,核肥大、畸形、溶解等呈不规则变化;肌浆网扩张;线粒体增生明显,排列紊乱,不同程度(轻、中、重)肿胀,内有空泡形成。②GCIP-27(90μg.kg-1)能明显改善上述病理改变及心肌细胞超微结构,心肌组织形态及心肌细胞超微结构基本正常。③GCIP-27(10、30μg.kg-1)和氯沙坦组大鼠心肌病理改变及超微结构有所改善,但改善不明显,个别视野仍可见到坏死灶。3. GCIP-27抗心室重构作用机制的研究(1) SHR模型组大鼠心肌ANPmRNA、BNPmRNA、β-MHCmRNA的表达均显著增加及α-MHCmRNA表达减少;治疗8周后,GCIP-27(10、30、90μg.kg-1)及氯沙坦均能降低ANPmRNA、BNPmRNA、β-MHCmRNA的表达及增加α-MHC mRNA的表达。(2)模型组大鼠左心室心肌细胞PLC活性显著增加(p<0.01);治疗8周后,GCIP-27(10、30、90μg.kg-1)大鼠心肌细胞PLC活性显著降低(p<0.01);氯沙坦也显著降低SHR大鼠左室心肌细胞PLC活性(p<0.01),相当于GCIP-27低剂量(10μg.kg-1)的疗效。4. GCIP-27在动物体内的药代动力学研究(1)血药浓度的测定血药浓度-时间曲线数据经DAS2.0药代动力学统计程序拟合,求出有关药代动力学参数为:在3.75-480 ng·ml-1剂量范围内,GCIP-27在小鼠体内按一级动力学代谢,并呈三室开放模型。静注125 I-GCIP-27 90μg·kg-1 ,电泳法和酸沉法测得t1/2α、t1/2β、t1/2γ分别为0.009h、0.245h、2.054h和0.025h、0.306h、2.323h;达峰时间tmax均为0.0333 h,平均血浆清除率(cl)分别为0.295 L·h-1·kg-1、0.322 L·h-1·kg-1,表观分布容积(Vd)分别为0.559 L·kg-1、1.29 L·kg-1 ,体内平均滞留时间(MRT)分别为2.353 h、2.515h。(2) GCIP-27在小鼠体内的分布GCIP-27在小鼠体内分布广泛,其中心、血管、肾、胃、肺、小肠等组织分布较高,肌肉、脂肪等组织相对较低,脑最低。(3) GCIP-27在动物体内的排泄大鼠72h尿、粪、胆汁原形药物排泄量分别为给药量的26.13%,0.95%和4.12%,72h总排泄量为31.21%。小鼠72h尿、粪原形药物排泄量分别为给药量的27.92%,0.84%,72h总排泄量为28.76%。GCIP-27主要经尿液排泄,少量经胆汁排泄,少许经粪便排泄,提示肾是GCIP-27的主要排泄器官。结论:1.GCIP-27具有良好的抗SHR心室重构作用,及较明显降低SHR血压的效应,且呈剂量依赖关系。2. GCIP-27改善心室重构作用明显优于氯沙坦,90μg/kg的GCIP-27,剂量仅为氯沙坦(6mg/kg)的1/67,其降低大鼠的PWT、IVST、LVMI分别为氯沙坦的1.6倍、2.7倍、3.2倍;但其降压效果仅为后者的60.6%。因此,GCIP-27改善心室重构的作用并非完全依赖于血压的降低,而有较高的选择性和特异性。3. GCIP-27改善心室重构的作用机制与降低心肌细胞磷脂酶C(PLC)活性,以及抑制大鼠心肌ANP mRNA、BNP mRNA、β-MHC mRNA及增加α-MHC mRNA的表达有关。4. GCIP-27在动物体内按一级动力学代谢,并呈三室开放模型;GCIP-27在动物体内广泛分布,尤其在心、血管、肾、胃、肺等组织分布较高;肾为其主要排泄器官。更多还原

【Abstract】 BACKGROUND: A major advance in understanding the pathophysiology of heart failure has been gaining an understanding of the process of left ventricular (LV) remodelling. Hypertension is always accompanied with left ventricular hypertrophy, cardiac fibrosis and artery wall thickening. These structural changes in heart and blood vessels are known as cardiovascular remodeling. Cardiovascular remodeling mainly caused by hypertension is the major pathogeny of cardiovascular diseases, and it also increases morbidity and mortality significantly. Left ventricular hypertrophy and remodling is one of the major forms of cardiovascular remodeling, which consists of cardiac cell proliferation, hypertrophy, interstitial fibrosis and necrosis, and then affects ventricle functions. Left ventricular (LV) remodeling , which is characterized by the alteration of LV size, shape, and function, occurs during several clinical conditions, such as hypertension, chronic heart failure, valvular heart disease, and myocardial infarction, etc. Therefore, the aim of treatment hypertension is not only to control the blood pressure effectively, but also to prevent the cardiovascular remodeling, and then to protect target organ.Various factors, including stretch stimulation, AngII, NE, neuropeptide Y and endothelin (ET)-1, among others, can induce ventricular hypertrophy and remodelling through their respective receptor and G-protein signalling pathways. The G-protein is located at the convergent point in the signal transduction pathway that leads to cardiovascular remodelling. It has been shown that the Gα-protein carboxyl terminus imitation polypeptide (GCIP), cloned and expressed in our laboratory, can inhibit cardiomyocyte hypertrophy induced by NE in vitro. GCIP-27, the optimised form of GCIP, has been shown to effectively prevent cardiac hypertrophy in vitro and in vivo.AIM: This study was to explore the effects of G protein inhibitory polypeptide GCIP-27 on the left ventricular remodeling and blood pressure in spontaneous hypertensive rats (SHR), the potential underlying mechanisms and its pharmacokinetics. METHODS:1. In the present study,10, 30 or 90μg/kg, i.p., GCIP-27 was administered for 8 weeks to SHR. In addition, another two groups of SHR were treated with either 6 mg/kg losartan or vehicle (saline). Wistar-Kyoto rats were used as controls. Systolic blood pressure (SBP) was measured using the standard tail-cuff method once every 2 weeks. At the end of the experiment, the LV mass index (LVMI) was evaluated. In addition, LV structure and function, collagen content, microstructure and ultrastructure were examined using echocardiography, the hydroxyproline assay, routine light microscopy and transmission electron microscopy, respectively.2. The expression of ANP, BNP,α-MHC,β-MHC were measured with RT-PCR method. The phosphslipase C activity was determined with radioimmunoassay technique. 3. The pharmacokinetics of GCIP-27 were measured by isotope labeling tracer method.RESULTS:1. In the losartan- and GCIP-27 (10, 30 and 90μg.kg-1)-treated groups, SBP decreased significantly compared with that of the vehicle group. However, even at the highest concentration used, the hypotensive effect of GCIP-27 was weaker than that of losartan.2. GCIP-27 (10, 30 and 90μg.kg-1) significantly reduced LV posterior wall thickness(PWT), the thickness of the interventricular septum(IVST), LVMI, collagen content, collagen areas(CA), and collagen volume fractin(CVF) compared with vehicle group(p<0.01), and Losartan (6 mg.kg-1) also obviously reduced PWT, IVST, collagen content, CA and CVF(p<0.05),and reduced LVMI(p>0.05). And the effects of GCIP-27 at all three concentrations tested being greater than that of losartan.3. GCIP-27 was more obvious in improving myocardial ultrastructure and pathology such as inflammation, hypertrophy, fibrosis, degeneration and necrosis, simultaneously. And the effects of GCIP-27 was superior to Losartan.4. Both GCIP-27 (10, 30 and 90μg.kg-1) and Losartan (6mg.kg-1) significantly inhibited Phospholipase C activity(p<0.01), and ANPmRNA, BNPmRNA,β-MHCmRNA levels expression in SHR(p<0.01), and increasedα-MHCmRNA levels, respectively.5. The plasma concentration-time curves of GCIP-27 in mice conformed to three-compartment open model with first order kinetics pattern.After intravenous injection of 125I GCIP-27 at a dose of 90μg.kg-1, t1/2α, t1/2β, t1/2γdetermined by trichloroacetic acid (TCA) method were 0.009h, 0.245h and 2.054h, respectively, and by SDS-PAGE method were 0.025h, 0.306h and 2.323h, respectively. The time to peak ( Tmax) for both methods was 0.0333h, with mean plasma clearance (CL) of 0.295 L·h -1·kg-1 and 0.322 L·h -1·kg-1, apparent volume of distribution ( Vd) of 0.559 L·kg-1 and 1.29L·kg-1 and mean residence time( MRT) of 2.353 h and 2.515h in mice.6. GCIP-27 was shown to be widely distributed to the various tissues. There was a relatively higher in heart, blood vessel, kidney, stomach, lung, mall intestine,etc, and relatively lower in fat and muscle, and the lowest in brain.7. GCIP-27 excreted in urine, feces and bile within 72h was 26.13% , 0.95% and 4.12% in rats, and urine, feces within 72h was 27.92%, 0.84% in mice respectively.The parent compound of GCIP-27 was mainly excreted by urine. And kidney is main emunctory organ.CONCLUSION:1. GCIP-27 could effectively attenuate left ventricular remodelling and decreases the SBP in SHR, and the effect was dose-dependent.2. GCIP-27 treatment of SHR is superior to treatment with losartan in terms of suppressing the development of LV remodelling, although the hypotensive effect of GCIP-27 is weaker than that of losartan. Therefore, the anti-remodelling effect of GCIP-27 in SHR is not entirely dependent on reductions in BP.3. The potential underlying mechanisms of GCIP-27 is the inhibition of G protein, it could inhibite the decrease of phospholipase C activity and ANPmRNA, BNPmRNA,β-MHC mRNA expression, and increaseα-MHCmRNA levels in SHR, respectively.4. The Plasma concentration-time curves of GCIP-27 in mice conformed to three-compartment open model of first order kinetics.5. GCIP-27 was shown to be widely distributed to the various tissues. There was a relatively higher in heart, blood vessel, kidney, stomach, lung, mall intestine, etc. The parent compound of GCIP-27 was mainly excreted by urine, and, kidney is major emunctory organ.更多还原