【作者】 杨大春；

【导师】 祝之明；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 背景和目的:高血压是临床常见病和多发病,其主要并发症有中风、心肌梗死及肾功能衰竭,严重危害人们的身心健康。原发性高血压发生机制目前尚不十分清楚,目前认为高血压病的病因为多因素,可分为遗传因素和环境因素两个方面,是遗传因素和环境因素相互作用的结果。原发性高血压发病率呈上升趋势,其防治具有重要意义。流行病学研究表明,多种饮食因素可影响血压,在过去的十余年中,通过减少钠盐的摄入、增加钾盐的摄入和增加以蔬菜和水果为基础的“清淡饮食”的摄入,可有效的降低血压。红辣椒的主要成分-辣椒素,常常作为一种食品添加剂,是短暂受体电位通道-香草醛亚型1(TRPV1)的高选择性激动剂。TRPV1主要分布在神经系统中,激活TRPV1可引起细胞内钙升高。研究发现,急性激活TRPV1可引起血管舒张,血压一过性下降;也有研究表明,急性激活TRPV1后,血管收缩,血压升高。而长期激活TRPV1后,对血管和血压的影响未见报道。我们既往还发现,辣椒素可激活脂肪细胞TRPV1导致细胞内钙增加,减少脂质在细胞内的堆积,辣椒素长期干预可预防高脂饮食诱导的肥胖。据报道,TRPV1在血管内皮细胞也有表达。我们推测,长期激活TRPV1可能有助于改善血管功能和预防高血压。eNOS和PKA是一氧化氮(NO)的合成和释放的关键信号转导蛋白,都受钙信号的调控。因此我们推测,激活TRPV1可能通过细胞内钙变化上调上述信号转导蛋白的表达,促进内皮细胞NO的合成和释放,从而改善血管功能和预防高血压。为了验证上述假设,本研究分为三部分进行。第一部分通过RT-PCR、蛋白免疫印迹和免疫荧光染色验证TRPV1在小鼠血管内皮细胞和小鼠及自发性高血压大鼠(SHR)肠系膜动脉中表达和分布情况。第二部分分题一通过应用辣椒素急性激活TRPV1后,观察其对小鼠肠系膜动脉舒张功能的影响。分题二通过长期辣椒素饮食干预C57/BL6J野生型小鼠和TRPV1基因敲除型小鼠,观察TRPV1激活后对血压正常者血管功能和血压的影响;分题三通过长期辣椒素饮食干预SHR大鼠,观察TRPV1激活后对高血压者的血管功能和血压的影响。分题四通过长期辣椒素干预,观察其对SHR内源性大麻素系统的影响。第三部分通过检测辣椒素刺激后血管内皮细胞内钙离子浓度([Ca2+]i)变化,明确内皮细胞上的TRPV1通道的功能变化。通过检测不同干预组内皮细胞及各组动物肠系膜动脉组织NO合成相关信号蛋白的表达,检测辣椒素刺激后血管和内皮细胞NO合成和释放的变化情况,明确TRPV1激活后对血管功能和血压作用的机制。材料与方法:整个研究由离体实验和在体实验两部分组成。离体实验以培养的小鼠胸主动脉内皮细胞以及肠系膜动脉为研究对象。在体试验采用了多种动物模型。首先以C57/BL6J野生型和TRPV1基因敲除(TRPV1-/-)小鼠及SHR为模型,分为辣椒素组(含辣椒素饲料喂养)和对照组(普通饲料喂养)。另外还以TRPV1过表达的转基因(TRPV1-tg)小鼠、TRPV1杂合子小鼠(TRPV1+/-)及其同窝野生型小鼠为动物模型,均以普通饲料喂养。1.分别用RT-PCR、蛋白免疫印迹检测TRPV1蛋白和mRNA在血管内皮细胞和肠系膜动脉中的表达;用免疫荧光染色法观察TRPV1在血管内皮细胞和肠系膜动脉中的分布。2.应用小血管张力测定技术检测辣椒素急性刺激后或长期干预后小鼠和SHR肠系膜动脉舒缩功能的变化。3.采用鼠尾测压方法测定小鼠及SHR鼠尾血压变化情况;颈动脉插管测定小鼠平均动脉压及急性静脉推注乙酰胆碱和硝酸甘油后的血压变化情况;应用无线遥测信号系统检测辣椒素干预后大鼠血压变化情况。4.比率荧光成像系统检测辣椒素刺激后血管内皮细胞[Ca2+]i的变化,观察TRPV1特异性拮抗剂辣椒卓平或TRPV1基因敲除后对钙信号变化的影响。5.蛋白免疫印迹法检测血管内皮细胞和肠系膜动脉组织NO合成相关信号蛋白(PKA、phospho-PKA、eNOS、phospho-eNOS)的表达。6.荧光显像技术检测辣椒素刺激对内皮细胞NO合成和释放的影响,及应用TRPV1受体拮抗剂、eNOS抑制剂和PKA抑制剂对内皮细胞NO生成的影响;比率荧光成像系统检测辣椒素刺激后小鼠肠系膜动脉NO的合成、释放情况,应用TRPV1受体拮抗剂、eNOS抑制剂对肠系膜动脉NO生成的影响及辣椒素刺激后不同基因型小鼠肠系膜动脉NO的生成情况。结果:1.TRPV1在血管内皮细胞和小鼠及SHR大鼠肠系膜动脉中均有表达。2.急性激活TRPV1后,呈浓度依赖性方式舒张苯肾上腺素预收缩的小鼠肠系膜动脉,TRPV1基因敲除后、去除内皮、应用eNOS抑制剂或PKA抑制剂明显减弱血管的舒张反应。长期激活TRPV1后可显著改善阻力血管-肠系膜动脉的内皮依赖性舒张功能,降低小鼠胸主动脉对血管紧张素Ⅱ诱导的收缩。3.长期激活TRPV1或TRPV1过表达后,可增强小鼠对乙酰胆碱诱导的降压反应。长期激活TRPV1后,可降低SHR的血压。4.辣椒素呈浓度依赖性诱导血管内皮细胞[Ca2+]i的升高, TRPV1基因敲除或辣椒卓平预处理可抑制[Ca2+]i的升高。5.辣椒素刺激可显著上调野生型小鼠血管内皮细胞phospho-PKA和phospho-eNOS蛋白的表达,PKA抑制剂可降低phospho-eNOS蛋白的表达;辣椒素刺激对TRPV1-/-小鼠血管内皮细胞phospho-PKA和phospho-eNOS蛋白的表达无明显影响。长期激活TRPV1后,野生型小鼠和SHR肠系膜动脉phospho-PKA和phospho-eNOS蛋白的表达增强,在TRPV1-/-小鼠中变化不明显。6.辣椒素刺激可增加血管内皮细胞NO的合成、释放,应用TRPV1受体拮抗剂、eNOS抑制剂和PKA抑制剂可降低内皮细胞NO的生成。辣椒素呈浓度依赖性方式增加小鼠肠系膜动脉NO的生成,应用TRPV1受体拮抗剂、eNOS抑制剂可减弱NO的合成、释放;辣椒素刺激后,TRPV1 tg小鼠肠系膜动脉NO生成最强,WT次之,TRPV1-/-最弱。结论:1.TRPV1在小鼠血管内皮细胞和小鼠及SHR肠系膜动脉中均有表达。2.急性或长期激活TRPV1后,能改善阻力血管的内皮依赖性舒张功能,降低SHR血压。3.激活TRPV1可引起[Ca2+]i的升高,上调phospho-PKA和phospho-eNOS蛋白的表达,促进血管内皮细胞和肠系膜动脉NO的生成。提示长期激活TRPV1后,通过cAMP/PKA-eNOS信号通路促进NO的合成、释放,是其改善血管功能、预防高血压的机制之一。更多还原

【Abstract】 Background and Objectives: Hypertension is currently one of the most common diseases in the world. Its main complications include stroke, myocardial infarction and renal failure, which are associated with substantial morbidity and mortality. The pathogenesis of primary hypertension is due to both the genetic susceptibility and environmental factors. However, the detail underlying mechanism has not been clarified. Epidemiologic studies demonstrated that arterial blood pressure can be regulated by a number of dietary factors. Over the past decade, reduced salt diet, increased potassium intake and consumption of fruits and vegetables based on the“DASH diet”have emerged as effective strategies for lowering blood pressure. Capsaicin is a major pungent ingredient in red pepper and is used as a food additive. Capsaicin is a highly selective agonist for the transient receptor potential vanilloid receptor 1 (TRPV1). TRPV1 as a nonselective cation channel is expressed in sensory neurons and brain but is although present in various non-neuronal tissues including the vasculature. The effect of capsaicin on vascular tone and blood pressure is somewhat of a paradox. Acute, short-term administration of capsaicin either increases or lowers blood pressure transiently in human and rodents. However, the long-term effect of TRPV1 activation on blood pressure and vascular reactivity were unknown. Our previous study demonstrated that activation of TRPV1 with capsaicin can reduce the adipogenesis of 3T3-L1 preadipocytes and prevent high fat diet induced obesity in mouse through increasing calcium entry. Considering endothelial nitric oxide synthase and PKA implicated in the regulation of blood pressure is dependant on calcium signal, we hypothesized that long-term activation of TRPV1 would improve vascular reactivity and prevent hypertension through PKA/eNOS/NO pathway.To test the above hypothesis, the present study was divided into three parts. Part one: Protein and mRNA expression and the distribution of TRPV1 in vascular endothelial cells and mesenteric arteries from mice and spontaneous hypertensive rats were evaluated by immunoblotting, RT-PCR and immunofluorescence, respectively. Part two, section 1: The vasodilation response of mice mesenteric arteries to acute administration with capsaicin was detected. Part two, section 2: Arterial blood pressure and vascular reactivity in normotensive mice (TRPV1 null mice and wild type littermates) after long-term activation of TRPV1 by oral administration with capsaicin were evaluated. Part two, section 3: Arterial blood pressure and vascular reactivity in spontaneous hypertensive rats after long-term activation of TRPV1 by oral administration with capsaicin were identified. Part two, section 4: The effect of long-term TRPV1 activation by oral administration with capsaicin on the endocannabinoid system. Part three: Calcium image in vascular endothelial cells with capsaicin intervention was detected to confirm the functional expression of TRPV1 channel. Nitric oxide bioavailability, total and phospho-PKA/eNOS were evaluated to identify the underlying molecular mechanisms involved in the effects of TRPV1 on the regulation of arterial blood pressure and vascular reactivity.Material and methods: The whole research includes in vitro and in vivo experiments. In vitro tests were conducted on cultured vascular endothelial cells from mice thoracic aorta and mesenteric arteries. When investigating in vivo, TRPV1 transgenic mice, TRPV1 deficient mice and wild type littermates as well as spontaneous hypertensive rats were assigned into normal chow diet and chow plus capsaicin diet group.1. TRPV1 protein and mRNA expression in vascular endothelial cells and mesenteric arteries were detected using RT-PCR and immunoblotting, respectively. While the distributions of TRPV1 in vascular endothelial cells and mesenteric arteries were evaluated by immunofluorescence.2. Vascular reactivity of mesenteric artery from mice and rats after acute or long-term activation with capsaicin was identified using isotonic myograph.3. Arterial blood pressure was obtained using tail-cuff plethysmography every month; Invasive mean blood pressure from carotid artery response to intravenous acetylcholine and nitroglycerine was evaluated; Arterial blood pressure of rats after oral administration was detected by telemetry.4. Calcium image in vascular endothelial cells from TRPV1 deficient mice and wild type littermates with capsaicin intervention in the presence and absence of TRPV1 specific blocker capsazepine was detected by fluorescence.5. Protein expressions of PKA, phospho-PKA, eNOS and phospho-eNOS in vascular endothelial cells and mesenteric arteries were detected by immunoblotting.6. NO bioavailability in vascular endothelial cells and mesenteric arteries from TRPV1 deficient mice, TRPV1 transgenic mice and wild type littermates with capsaicin intervention in the presence or absence of TRPV1 specific blocker, eNOS inhibitor or PKA inhibitor was detected by fluorescence.Results:1. Protein and mRNA expressions of TRPV1 were detected in vascular endothelial cells and mesenteric arteries from mice and spontaneous hypertensive rats.2. Acute activation of TRPV1 caused concentration-dependent vasodilation in PE-induced precontraction of mice mesenteric arterial rings. TRPV1 gene knockout, endothelium denudation, eNOS inhibitor or PKA inhibitor significantly attenuated the vasodilation response induced by TRPV1 activation. Long-term activation of TRPV1 significantly improved endothelium-dependent vasodilation in mesenteric resistance artery, and markedly blunted angiotensin II-induced vasocontraction in mouse aorta.3. Long-term activation of TRPV1 or TRPV1 overexpression markedly enhanced acetylcholine-induced hypotensive response; moreover, long-term activation of TRPV1 can reduce the arterial blood pressure of spontaneous hypertensive rats.4. Capsaicin dose-dependently increased intracellular free calcium concentration in vascular endothelial cells, while this effect can be inhibited in TRPV1-deficient mice or in the presence of capsazepine.5. After intervention with capsaicin, the protein expression of phospho-PKA and phospho-eNOS were significantly upregulated in endothelial cells from wild type mice, but not from TRPV1 null mice. After long-term activation of TRPV1, the protein expression of phospho-PKA and phospho-eNOS were markedly upregulated in mesenteric arteries from wild type mice and spontaneous hypertensive rats, but not from TRPV1 mutant mice.6. Intervention with capsaicin significantly increased the NO bioavailability, while TRPV1 blocker, eNOS inhibitor or PKA inhibitor markedly attenuated these effects. Similarly, capsaicin markedly increased the NO production in a dose-dependent manner, while TRPV1 blocker or eNOS inhibitor significantly blunted these effects. Capsaicin induced highest amount of NO in mesenteric artery from TRPV1 transgenic mice, wild type mice took the second place, while TRPV1 null mice was the lowest one.Conclusion:1. TRPV1 was expressed in mice vascular endothelial cells and mesenteric arteries from mice and spontaneous hypertensive rats.2. Acute or long-term activation of TRPV1 would improve endothelium-dependent vasodilation in resistance arteries, and reduce arterial blood pressure of spontaneous hypertensive rats.3. Activation of TRPV1 can increase intracellular calcium concentration in vascular endothelial cells, upregulate the protein expression of phospho-PKA and phospho-eNOS, and increase the bioavailability of nitric oxide in vascular endothelial cells and mesenteric arteries. The present study suggest that long-term activation of TRPV1 induced nitric oxide increasing mediated by cAMP/PKA-eNOS signal pathway would be a underlying mechanism for improving vascular function and preventing hypertension.更多还原