【作者】 陈国萍；

【导师】 胡申江；

【作者基本信息】 浙江大学 ， 临床医学， 2009， 博士

【摘要】 第一部分法尼基焦磷酸合酶的抑制对自发性高血压大鼠内皮功能的影响及其可能机制背景:法尼基焦磷酸合酶是甲羟戊酸途径的关键酶之一,能催化合成类异戊二烯中间产物,后者在蛋白异戊二烯化过程中是不可或缺的。而蛋白异戊二烯化过程又是RhoA等小GTP酶活化所必须的。活化的RhoA可负相调节内皮性一氧化氮合酶的表达和磷酸化,从而调控一氧化氮终产物。而一氧化氮减少被认为是多种心血管疾病(包括动脉粥样硬化和高血压等)的致病因子。既往研究发现:自发性高血压大鼠中甲羟戊酸途径的一些关键酶表达显著高于血压正常的Wistar-Kyoto大鼠,其中包括法尼基焦磷酸合酶。另有大量实验证据表明自发性高血压大鼠具有显著的内皮功能障碍。目的:明确用阿仑膦酸钠抑制法尼基焦磷酸合酶能否改善自发性高血压大鼠的内皮功能,并探索可能机制。方法:5周龄雄性自发性高血压大鼠和Wistar-Kyoto大鼠用阿仑膦酸钠(低剂量1mg/kg/day;高剂量10 mg/kg/day)干预12周。用离体血管环灌流技术检测内皮依赖性和非依赖性血管舒张作用。用pull-down法测定胸主动脉RhoA的活性。用Western blot法检测胸主动脉内皮性一氧化氮合酶的表达水平。用Griess法测定血浆一氧化氮水平。结果:高剂量阿仑膦酸钠(10 mg/kg/day)能部分改善自发性高血压大鼠受损的内皮依赖性血管舒张作用。阿仑膦酸钠干预可浓度依赖性的降低胸主动脉中RhoA的活性,上调内皮性一氧化氮合酶的表达水平,以及增加血清一氧化氮终产物的含量。结论:法尼基焦磷酸合酶的抑制能够改善自发性高血压大鼠的内皮功能,且其可能机制包括抑制RhoA活化,进而上调内皮性一氧化氮合酶的表达。第二部分自发性高血压大鼠组织胆固醇含量和HMG-CoA还原酶表达与活性的研究及阿托伐他汀对此的影响背景和目的:心血管重塑与胆固醇密切相关,HMG-CoA还原酶是胆固醇合成途径——甲羟戊酸途径的限速酶,该酶的抑制剂——他汀类药物可在一定程度上抑制心血管重塑。而自发性高血压大鼠,与血压正常的Wistar-Kyoto大鼠相比,具有显著的心血管重塑,但其血清胆固醇水平却较低。本实验的目的主要是为了明确自发性高血压大鼠肝脏和肝外组织(心脏、主动脉和肾脏)中胆固醇含量及HMG-CoA还原酶表达和活性的改变,并进一步研究阿托伐他汀干预对这些指标的影响。方法:8周龄雄性自发性高血压大鼠和Wistar-Kyoto大鼠用阿托伐他汀(50mg/kg/day)干预10周。用商业化酶法测定血清和组织(心脏、主动脉、肝脏和肾脏)的胆固醇水平。用Western blot法和高效液相色谱法检测这些组织中HMG-CoA还原酶的表达和活性。结果:自发性高血压大鼠肝脏组织总胆固醇含量明显低于Wistar-Kyoto大鼠,肝外组织(心脏、主动脉和肾脏)总胆固醇水平与Wistar-Kyoto大鼠相似,而四种组织中的HMG-CoA还原酶的表达和活性均明显高于Wistar-Kyoto大鼠。阿托伐他汀处理能显著降低自发性高血压大鼠四种组织心脏、主动脉、肝脏和肾脏组织总胆固醇含量以及HMG-CoA还原酶的表达和活性。然而,在Wistar-Kyoto大鼠,阿托伐他汀仅能影响肝脏HMG-CoA还原酶,使其蛋白表达上调而酶活性却下降。结论:阿托伐他汀对组织胆固醇以及HMG-CoA还原酶的作用存在种属和组织特异性。第三部分阿魏酸钠对大鼠离体胸主动脉环的作用及其可能机制背景:阿魏酸钠是阿魏酸的钠盐,阿魏酸是许多传统中草药(如当归和川芎等)的主要活性成分,具有强效抗氧化、抗炎症反应、抗血小板聚集和清除氧自由基等药理作用。目的:明确阿魏酸钠对大鼠离体胸主动脉环的作用,探讨其具体机制。方法:用血管环体外灌流技术观测阿魏酸钠对大鼠离体胸主动脉环的作用。培养的大鼠主动脉平滑肌细胞加载Fluo-3标记后,用荧光酶标仪检测不同处理下的细胞内游离钙离子浓度。结果:在内皮完整的胸主动脉环中,阿魏酸钠可浓度依赖性的舒张苯肾上腺素或氯化钾预收缩的血管环,并且去除内皮对该舒张作用无明显影响。在无钙灌流液条件下,阿魏酸钠能浓度依赖性的抑制由外源性钙离子引起的血管收缩,同时也能抑制由苯肾上腺素或咖啡因诱发内钙释放所致的血管收缩。钾离子通道抑制剂四乙铵、4-氨基吡啶、格列本脲或氯化钡的预处理不影响阿魏酸钠诱导的血管舒张作用。另外,在无钙液条件下,阿魏酸钠也能浓度依赖性的舒张由佛波酯预收缩的血管。在体外培养的大鼠主动脉平滑肌细胞中,在有钙或无钙培养液条件下,阿魏酸钠均不能阻断苯肾上腺素或氯化钾诱导的细胞内游离钙离子浓度的升高。结论:阿魏酸钠可直接作用于血管平滑肌,内皮非依赖性的舒张血管环。在钙离子依赖的收缩反应中,阿魏酸钠不影响细胞内钙离子浓度,其舒张血管的主要机制可能是作用于细胞内钙离子升高之后的环节;而在钙离子非依赖性收缩反应中,阻断蛋白激酶C介导的收缩过程可能是阿魏酸钠舒张血管主要机制。更多还原

【Abstract】 Part One Effect of farnesyl diphosphate synthase inhibition on endothelial function in spontaneously hypertensive rats and its mechanismsBackground:Famesyl diphosphate synthase（FDS）,an essential enzyme in the mevalonate pathway,catalyzes the synthesis of isoprenoid intermediates.The latter is needed for protein isoprenylation of RhoA for its function to negatively regulate expression and phosphorylation of endothelial nitric oxide synthase（eNOS）,thereby negatively regulating the production of nitric oxide（NO）.Up to date endothelial dysfunction characterized by an impairment of the production and release of the endothelial-derived NO is a strong predictor of cardiovascular disease.We previously reported that spontaneously hypertensive rat（SHR） in which endothelial dysfunction occurred,had higher FDS when compared with Wistar-Kyoto rat（WKY）.Aims:To determine whether chronic inhibition of FDS can improve the endothelial function in SHR and explore its mechanisms.Methods:After 12-week administration of alendronate（low dose of 1 mg/kg/day;high dose of 10 mg/kg/day）,endothelium-dependent and -independent vasorelaxation were measured in isolated aortic rings.Activation of RhoA in aorta was determined by pull-down assay.Aortic expression of eNOS was determined by Western blot.Serum nitric oxide（NO） end products were determined using Griess method.Results:High-dose of alendronate was able to improve the impaired endothelium-dependent vasodilation in SHR.Biochemically,alendronate was able to suppress the RhoA activation,but upregulate the eNOS expression in a dose-dependent manner.Conclusions:Our data suggested that chronic FDS inhibition could improve the endothelial function in SHR,and the upregulation of eNOS expression as a result of inhibition of RhoA activation might be an important mechanism. Part Two Studies of tissue cholesterol content and HMG-CoA reducatse expression/activity in spontaneously hypertensive rats and effects of atorvastatin on themBackground and Aim:Cardiovascular remodeling is closely associated with cholesterol and is attenuated by statins.The spontaneously hypertensive rat（SHR） has a low serum cholesterol level and evident cardiovascular remodeling.3-hydroxy-3-methylglutaryl- coenzyme A （HMG-CoA） reductase is the rate limiting enzyme of cholesterol synthesis pathway, which is also called mevalonate pathway.The aims of the present study were to characterize the effects of HMG-CoA reductase inhibitor,atorvastatin,on tissue cholesterol content and HMG-CoA reductase expression and activity in four tissues from SHR:liver and extrahepatic tissues（heart,aorta and kidney）.Methods:Eight-week old SHR and normotensive Wistar-Kyoto rats（WKY） were treated daily with atorvastatin（50 mg/kg） for 10 weeks.Cholesterol levels of serum and tissues （liver,heart,aorta and kidney） were determined by commercial enzymatic methods. Western blot analysis and high performance liquid chromatogram（HPLC） were used to assay the expression and activity of enzyme respectively.Results:The levels of total tissue cholesterol in SHR livers were significantly higer,but in SHR extrahepatic tissues were similar when compared with WKY rats.The expression and activity of HMG-CoA reductase in all these tissues were stringly higher in SHR than in WKY rats.Treatment with atorvastatin decreased cholesterol content and HMG-CoA reductase expression and activity in all four tissues of SHR.However,in WKY,atorvastatin only altered HMG-CoA reductase in liver,where the protein expression was upregulated but the enzyme activity was decreased.Conclusions:The present study demonstrates that the effects of atorvastatin on tissue cholesterol content and HMG-CoA reductase are strain-and tissue-specific. Part Three Effect of sodium ferulate on rat thoracic aorta and its mechanismsBackground:Sodium ferulate（SF） is a sodium salt of ferulic acid（FA）.FA is the active ingredient of the traditional Chinese herbal medicine such as Radix Angelicae Sinensis, Rhizoma Chuanxiong.In vitro and in vivo studies revealed that SF or FA possessed beneficial pharmacological effects including its strong antioxidant,antiinflammatory, platelet aggregation inhibitory,and free radical-scavenging activities.Aims:This study was designed to investigate the effects of SF on rat isolated aortas and the possible mechanisms.Methods:Isometric tension was recorded in response to drugs in organ bath.Cytosolic free Ca²⁺ concentration([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells（RASMC）.Results:SF relaxed the isolated aortic rings precontracted with phenylephrine（PE） or high-K⁺ in a concentration- dependent manner,and mechanical removal of endothelium did not significantly modify the SF-induced relaxation.In Ca²⁺-free solution,SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ or PE pre-challenged rings,and suppressed the transient contraction induced by PE or caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium,glibenclamide,4-aminopyridine,and barium chloride.In addition, SF concentration- dependently reduced the contraction induced by phorbol-12- myristate-13-acetate,an activator of protein kinase C（PKC）,in the absence of extracellular Ca²⁺.In RASMC,SF had no effect on PE- or KCl-induced[Ca²⁺]i increase either in the presence or in the absence of external Ca²⁺.Conclusions:These results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle.SF had no effect on[Ca²⁺]i,the direct inhibition of the common pathway after[Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction,while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.更多还原