【作者】 郭韧；

【导师】 李元建；

【作者基本信息】 中南大学 ， 药理学， 2009， 博士

【摘要】 硝酸酯类药物是临床上治疗缺血性心肌病及充血性心力衰竭的一类主要药物。传统认为,有机硝酸酯类药物通过在体内代谢产生NO及NO类似物,激活血管平滑肌或内皮细胞中的鸟甘酸环化酶（soluble guanylate cyclase,sGC）,进而使第二信使环磷酸鸟苷浓度增加,引起动、静脉舒张而发挥药理学效应,NO是此类药物的最终效应分子。降钙素基因相关肽（calcitonin gene-related peptide,CGRP）是辣椒素敏感的感觉神经的主要递质,也是迄今发现的最强的舒血管物质之一。我们课题组的前期动物实验研究结果表明,CGRP介导硝酸甘油的心血管效应,且其合成和释放减少与硝酸甘油耐受性的形成有关。硝酸酯类在人体主要通过线粒体醛脱氢酶2（mitochrondrialaldehyde dehydrogenase-2,ALDH₂）、谷胱甘肽转硫酶（glutathione-S-transferases,GST）、细胞色素P450系统以及黄嘌呤氧化酶（xanthine oxidase,XOR）等生物转化酶代谢释放NO。本文在健康中国男性志愿者中探讨了CGRP在硝酸酯类药物心血管效应中的作用,并利用ALDH2和GSTM1基因的功能性遗传多态性或特异性抑制剂,研究了相关药物代谢酶对CGRP介导硝酸酯类药物药理作用的影响。第一章降钙素基因相关肽介导硝酸甘油的心血管效应与ALDH2基因型有关研究背景:硝酸甘油为抗心肌缺血的经典药物,广泛应用于治疗劳累性稳定型心绞痛,不稳定型心绞痛,急性心肌梗塞以及慢性充血性心力衰竭,主要通过通过舒张血管,降低心肌前后负荷,减少心肌耗氧,扩张冠状动脉而发挥作用。降钙素基因相关肽（CGRP）是人类用分子生物学方法发现的第一个多肽,为辣椒素敏感感觉神经的主要递质,广泛分布于中枢及外周血管组织,是迄今发现的最强的舒血管物质。动物实验证明,CGRP介导硝酸甘油的舒血管效应。线粒体醛脱氢酶ALDH₂是硝酸甘油的主要生物转化酶,催化硝酸甘油转化为1,2-二硝酸甘油和亚硝酸盐,抑制ALDH₂活性或去除细胞的线粒体可显著降低硝酸甘油的生物转化,削弱硝酸甘油的舒血管作用。硝酸甘油耐受与心血管线粒体ALDH₂酶活性降低有关。编码ALDH2基因的第12号外显子上存在功能性单核苷酸序列多态性（single nucleotide polymorphism,SNP）,该SNP使第504位的谷氨酸被赖氨酸取代（ALDH2*2多态）,这一多态性导致ALDH2酶活性降低。因此,本实验在ALDH2酶活性正常和低下的健康志愿者探讨了CGRP是否通过ALDH2依赖的方式介导硝酸甘油的心血管效应。方法:1:ALDH2*2对CGRP介导硝酸甘油药理效应的影响限制性片段长度多态性（PCR—RFLP）法80名健康中国汉族志愿者的ALDH2进行基因分型,随机选取ALDH2野生型个体及携带ALDH2*2等位的个体各9人,舌下含服硝酸甘油0.5mg,分别于服药前及服药后不同时间点测定坐位血压和心率,并于服药后第12min药理效应达到最大时抽取3ml外周静脉血检测血浆CGRP浓度（放射免疫法）。2:培养的外周血单个核细胞（peripheral blood mononuclear cell,PBMC）硝酸甘油对CGRP mRNA表达的影响抽取健康志愿者10ml外周静脉血,分离外周血单个核细胞并培养,实验分组:①ALDH2野生型个体对照组;②ALDH2野生型个体+硝酸甘油(10^-5M)组;③ALDH2功能缺失个体对照组;④ALDH2功能缺失个体+硝酸甘油(10^-5M)组。实时定量PCR测定CGRPⅠ和CGRPⅡmRNA的表达水平。3:ALDH2抑制剂水合氯醛对培养的外周血单个核细胞（PBMCs）硝酸甘油促CGRP合成与释放的影响抽取3名ALDH2野生型个体健康志愿者外周静脉血各10ml,分离外周血单个核细胞并培养,实验分组:①正常对照组;②10^-5M硝酸甘油组;③10^-5M硝酸甘油组+1mM水合氯醛组;④1mM水合氯醛组。时定量PCR测定CGRPⅠ和CGRPⅡmRNA的表达水平。结果:（1）与ALDH2酶功能缺失个体（ALDH2*1/*2+ALDH2*2/*2）相比,ALDH2野生型个体收缩压及心率在含服硝酸甘油后多个时间点呈显著增高。（2）志愿者舌下含服硝酸甘油12min后,血浆CGRP浓度显著升高,CGRP浓度上升的百分率在ALDH2野生型个体表现得更为显著,血浆CGRP浓度上升的百分率与△SBP及△HR呈明显正相关。（3）硝酸甘油(10^-5M)孵育分离的志愿者外周血PBMC 24h后,ALDH2野生型个体其CGRPⅠ和CGRPⅡmRNA的表达上调程度显著高于ALDH2酶功能缺失个体。（4）在ALDH2野生型个体PBMC,硝酸甘油孵育24h可显著上调CGRPⅠ和CGRPⅡmRNA的表达水平,这一效应可以被ALDH2抑制剂水合氯醛（1mM）部分阻断。结论:在健康志愿者体内,CGRP通过ALDH2依赖的方式介导硝酸甘油的心血管效应。第二章CGRP介导单硝酸异山梨酯的心血管效应与GSTM1基因型相关研究背景:硝酸酯类药物包括硝酸甘油、硝酸异山梨酯与单硝酸异山梨酯等,它们共同的药理作用基础是在体内释放NO,后者激活鸟苷酸环化酶,升高细胞内环磷酸鸟苷水平,进而激活cGMP依赖的激酶Ⅰ,使钙离子通道调节蛋白磷酸化而降低细胞内钙离子浓度,引起血管扩张。降钙素基因相关肽（CGRP）是辣椒素敏感的感觉神经的主要递质,由37个氨基酸残基组成,具有强烈的舒血管效应。CGRP可分为CGRPⅠ和CGRPⅡ两种亚型,分别由不同基因编码。人类CGRP的合成与释放受多种内源性活性物质包括NO的调节。硝酸酯类药物在体内代谢释放NO的过程涉及多种药物代谢酶,其中包括线粒体醛脱氢酶2（ALDH2）、谷胱甘肽-S-转移酶（GSTs）、黄嘌呤氧化酶（XOR）、细胞色素P450系统等。谷胱甘肽硫转移酶（GSTs）是机体一类重要的Ⅱ相代谢酶。人类GSTs由一个超基因家族编码,也与硝酸酯类在体内的代谢有密切关系,其中GSTM1存在基因多态性,表现为该基因部分或全部的缺失,导致其编码的酶蛋白活性丧失。黄嘌呤氧化酶（XOR）介导的氧化还原反应是体内活性氧簇（ROS）产生的来源之一,与心血管疾病的发生有密切关系。XOR在NADH存在的条件下可以催化有机硝酸酯类、无机硝酸盐类和亚硝酸盐类生成NO。本研究拟在健康中国志愿者中观察GSTM1等代谢酶的基因多态性对单硝酸异山梨酯心血管效应的影响及其与CGRP的关系。方法:1:GSTM1基因多态性对CGRP介导单硝酸异山梨酯药理效应的影响PCR-RFLP对80名健康中国汉族志愿者的ALDH2和GSTM1进行基因分型,随机选取GSTM1野生型个体及GSTM1缺失个体各12人,其中也包括ALDH2正常个体与携带ALDH2突变个体各12人。口服单硝酸异山梨酯20mg,分别于服药前及服药后不同时间点测定坐位血压和心率,并于服药后不同时间点各抽取3ml外周静脉血检测血浆CGRP浓度（放射免疫法）和单硝酸异山梨酯的血药浓度（气象色谱—电子捕获法）。2:单硝酸异山梨酯对培养的外周血单个核细胞CGRP表达的影响抽取健康志愿者15ml外周静脉血,分离外周血单个核细胞并培养,单硝酸异山梨酯（50μM）处理PBMC后,检测CGRP mRNA表达;外周血单个核细胞经GST抑制剂依它尼酸（1μM）及黄嘌呤氧化酶抑制剂别嘌呤醇ALLopurinol（20μM）预处理30min后,加入单硝酸异山梨酯,观察这些酶抑制对单硝酸异山梨酯上调CGRP mRNA表达的影响。结果:（1）与GSTM1野生型个体相比,GSTM1缺失型个体SBP于服药后多个时间点均显著降低且恢复时间显著延长,GSTM1缺失个体服药后DBP显著低于GSTM1野生型个体且持续时间也显著延长。GSTM1缺失型与野生型个体间服药后心率变化存在显著差异;（2）GSTM1缺失型个体服药后单硝酸异山梨酯C_max和AUC_0→∝均明显低于GSTM1野生型个体;（3）服药后血浆CGRP浓度显著升高,GSTM1缺失型个体血浆CGRP浓度升高时间早于GSTM1野生型个体,且CGRP浓度升高持续时间显著长于GSTM1野生型个体,GSTM1缺失型个体血浆CGRP浓度升高的百分率也显著高于GSTM1野生型个体;（4）志愿者口服单硝酸异山梨酯后,在第10,11,12小时,其服药前XOR mRNA水平与△SBP呈明显的正相关;（5）服药后7小时GSTM1缺失型个体外周血单个核细胞CGRPⅠmRNA表达显著增高（p＜0.05）,而GSTM1野生型个体CGRPⅠmRNA表达变化不明显;（6）单硝酸异山梨酯可以明显地上调培养的PBMC CGRP 1和CGRPⅡmRNA的表达水平,这一效应可被黄嘌呤氧化酶抑制剂ALLopurinol（20μM）部分取消,GSTs抑制剂依它尼酸（1μM）对单硝酸异山梨酯诱导的CGRP mRNA表达无明显影响。结论:单硝酸异山梨酯的心血管作用部分通过CGRP所介导。GSTM1基因缺失可加速单硝酸异山梨酯代谢增强其心血管效应。更多还原

【Abstract】 Organic nitrates nitrates are widely used for myocardial ischemia and chronic congestive heart failure.It is widely accepted that nitrates are a group of parent drugs and act through release of NO,the latter can activate the soluble guanylate cyclase（sGC）,increase the the concentration of cyclic guanosine monophosphate（cGMP）,and finally lead to vasodilatation.No has been regarded as the only effector molecular of nitrates.Calcitonin gene-related peptide（CGRP）,the major transmitter in sensory nerves,is the most potent endogenous vasodilator identified so far.Previous studies in our group performed in animals have shown that CGRP is involved in the cardiovascular effects of nitroglycerin,and the decrease in synthesis and release of CGRP contribute to the development of nigroglycerin tolerance.Several drug metabolizing enzymes such as the mitochrondrial aldehyde dehydrogenase-2（ALDH2）,glutathione-S-transferases（GSTs）, xanthine oxidase and cytochrome P450 are involved in the biotransformation of nitrates.Therefore,the present study was designed to explore the involvement of CGRP in cardiovascular response to nitrates and association with enzymes involved in nitrates biotransformation in healthy Chinese male volunteers. Chapter 1 Evidence for involvement of calcitonin gene-related peptide in nitroglycerin response and association with mitochondrial aldehyde dehydrogenase-2（ALDH2） Glu504Lys polymorphismBACKGROUND:Nitroglycerin（GTN） has been one of most widely used anti-ischemic drugs for more than a century.Organic nitrates are excellent agents for the treatment of stable-effort angina,unstable angina,in patients with myocardial infarction and in patients with chronic congestive heart failure, the therapeutic effect of organic nitrates is due to venous and arterial dilation,decreased oxygen consumption and relaxation of coronary artery.Calcitonin gene-related peptide（CGRP）,the major transmitter in sensory nerves,is widely distributed in the cardiovascular system.CGRP is the most potent endogenous vasodilator identified thus far.It has been confirmed in animal experiment that the CGRP plays an important role in the GTN response.It has recently been reported that the mitochondrial aldehyde dehydrogenase-2（ALDH2,mtALDH） catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from GTN,inhibition of ALDH2 or deleted the cellular mitochondria leads to the impaired biotransformation of GTN.Naitrate tolerance is also partly due to the decreased bioactivity of ALDH2.A common Glu504Lys polymorphism（also called ALDH2^*2） of ALDH2,which accounts for decreased ALDH2 activity.In the present study,we explored the invlvement of CGRP in healthy Chinese volunteers and association with ALDH2 polymorphism.METHODS:1.Effect of ALDH2^*2 on CGRP mediated GTN responseEighty unrelated male volunteers were enrolled and screened for their ALDH2 Glu504Lys genotypes.Nine wild-type homozygotes （ALDH2^*1/^*1） and nine subjects with the ALDH2^*2 allele respectively, were randomly selected.All subjects received a sublingual administration of 0.5 mg nitroglycerin after the first blood samples were drawn.Blood pressure（BP） and heart rate（HR） were measured at 0,2,5,8,10,15,20, 25,30 and 35 min,respectively,after GTN administration in a sitting position.A second venous blood sample of 3 ml was taken 12 min after GTN administration for CGRP analysis.2 Effect of GTN on the mRNA expression of CGRPⅠ和CGRPⅡin cultured PBMCs10 ml of venous blood sample was drawn to separate and cultivate PBMCs.Experiments were divided into 4 groups:（1） ALDH2^*1/^*1 homozygotes control;（2） ALDH2^*1/^*1 homozygotes+GTN(10^-5M);（3） carriers of the ALDH2^*2 allele control;（4） carriers of the ALDH2^*2 allele+GTN(10^-5M)3 Effect of ALDH2 inhibitor on stimulation of CGRP mRNA expression by GTNThe PBMCs from 3 ALDH2^*1/^*1 homozygotes were obtained from venous blood,experiments were divided into 4 groups:（1） control;（2） GTN(10^-5M);（3） GTN+ 1mM chloral hydrate（CH）;（4） chloral hydrate （CH）.RESULTS:（1） In contrast with carriers of the ALDH2^*2 allele,ALDH2^*1/^*1 homozygotes showed significantly higher extent of absolute changes in both systolic blood pressure（ΔSBP） and HR（ΔHR） at several time points after GTN administration.（2） Plasma concentrations of CGRP were increased significantly 12 min after GTN administration,and the percentage increase in plasma concentrations of CGRP correlated positively with bothΔSBP andΔHR, and percentage increase in plasma concentrations of CGRP was significantly higher in ALDH2^*1/^*1 homozygotes. （3） PBMCs from ALDH2^*1/^*1 homozygotes showed higher fold increase in both CGRPⅠand CGRPⅡmRNA after GTN stimulation.（4） The GTN-induced increase in CGRP mRNA expression in PBMCs from ALDH2^*1/^*1 homozygotes was inhibited by ALDH2 inhibitor chloral hydrate.CONCLUSION:CGRP is involved in the cardiovascular effect of GTN through an ALDH2-dependent pathway in human. Chapter 2 Evidence for involvement of calcitonin gene-related peptide in 5-ISMN response and association with GSTM1 polymorphismBackground:Organic nitrates nitrates such as nitroglycerin,isosorbide dinitrate, isosorbide-5-mononitrate（5-ISMN） can release NO to activate the guanylate cyclase,increase the the concentration of cyclic guanosine monophosphate,then activate the cGMP-dependent kinaseⅠ,decrease the concentration of Ca²⁺ in cells,finally induce vasodilatation.Calcitonine gene-related peptide（CGRP）,the major neural transmitter in capsaicin sensitive nerves,comprised of 37 amino-acid residues,and is a potent vasodilator.Two isoforms of human CGRP,namely CGRP-1 and CGRP-2,have been identified,the two isoforms encoded by different gene.It has been reported that the synthesis and release of CGRP is regulated by multiple endogenous substances,incuding NO.There are many metabolic enzymes participate the biotransformation of nitrate drugs,such as mitochondrial aldehyde dehydrogenase-2 （ALDH2）,glutathione- S-transferase（GSTs）,xanthine oxidase, cytochrome P450.Glutathione-S-transferase is an importantⅡphase metabolic enzyme,encoded by a supergene family,it has been reported that Glutathione-S-transferase is involved in the metabolism of nitrates in vivo,there is a polymorphism in GSTM1,leads to decreased activity of the enzyme.The redox reaction mediated by xanthine oxidase is a source of reactive oxygen species in the cardiovascular system.XOR catalyzed organic nitrates,nitrates,and nitrites to NO though a NADH-dependent way.In this study,we explored the invlvement of CGRP in healthy Chinese volunteers and association with GSTM1 polymorphism.METHODS:2.Effect of GSTM1 polymorphism and on CGRP mediated 5-ISMN responseEighty unrelated male volunteers were enrolled and screened for their GSTM1 and ALDH2 Glu504Lys genotypes.Twelve GSTM1 wildtype genetype,twelve GSTM1 null genetype respectively,were randomly selected,included twelve wild-type homozygotes（ALDH2^*1/^*1） and twelve subjects with the ALDH2^*2 allele.All subjects received a oral administration of 20 mg 5-ISMN after the first blood samples were drawn. Blood pressure（BP） and heart rate（HR） were measured at several time points after 5-ISMN administration in a sitting position.Each 3 ml venous blood sample was taken at several time points after 5-ISMN administration for CGRP and blood drug level analysis.2.Effect of 5-ISMN on the mRNA expression of CGRPⅠ和CGRPⅡin cultured PBMCs15 ml of venous blood sample was drawn to separate and cultivate PBMCs.PBMC were treated with 5-ISMN（50μM） 24 hour to detcted the mRNA expression of CGRPⅠ和CGRPⅡ.Cells were pretreated with ethacrvnic acid（1μM） or ALLopurinol（20μM） 30 min before 5-ISMN treatment to explore the effects of these enzymes inhibitor on up-regulated CGRP expression induced by 5-ISMN.RESULTS:（1） As compared with GSTM1 wildtype genetype,GSTM1 null genetype showed significant lower SBP and DBP at several time points after 5-ISMN administration.There is a significant deviation of changes in HR between GSTM1 wildtype genetype,GSTM1 null genetype.（2） As compared with GSTM1 wildtype genetype,GSTM1 null genetype showed significant lower 5-ISMN C_max and AUC_0→∝.（3） Plsma concentration of CGRP were increased in all volunteers,as compared with GSTM1 wildtype genetype,plsma concentration of CGRP in GSTM1 null genetype increased earlier and last for a longer time,percentage increase in plasma concentrations of CGRP was significantly higher in GSTM1 null genetype.（4） The XOR mRNA expression in volunteers before 5-ISMN administration correlated positively withΔSBP at 10,11,12 hour after 5-ISMN administration.（5） Treatment with 5-ISMN for 7 hour significantly up-regulated CGRPⅠmRNA expression in PBMC from GSTM1 null genetype,there is no significant deviation in PBMC from GSTM1 wildtype genetype.（6） Treatment with 5-ISMN significantly up-regulated the expression of CGRP 1和CGRPⅡmRNA in PBMC,which was abolished by xanthine oxidase inhibitor ALLopurinol（20μM）,but not affected by GSTs inhibitor ethacrvnic acid（1μM）. CONCLUSION:CGRP is involved in the cardiovascular effect of 5-ISMN.GSTM null genetype can accelerate the metabolism of 5-ISMN and improve its cardiovascular effect.更多还原