【作者】 李岱；

【导师】 邓汉武；

【作者基本信息】 中南大学 ， 心血管药理， 2008， 博士

【摘要】 第一章高血压时花生四烯酸乙醇胺转运体活性变化及其对CGRP释放的影响研究背景降钙素基因相关肽（CGRP）是辣椒素敏感感觉神经的主要递质,在外周神经系统,背根神经节（DGR）是合成CGRP的主要部位。CGRP随外周感觉神经广泛分布于心血管组织,是目前已知最强的舒血管物质,在维持循环稳定中起重要作用。新近研究发现,非神经组织细胞如淋巴细胞、内皮细胞也能合成CGRP,通过自分泌或旁分泌而发挥作用。CGRP的合成与释放主要受辣椒素受体（VR1）调节。CGRP在高血压的发生发展中起重要作用。原发性高血压患者及自发性高血压大鼠（SHR）血浆CGRP浓度降低,被认为是血压升高的重要原因;在继发性高血压患者（如醛固酮增多症）或两肾一夹高血压大鼠（2K1C）,CGRP血浆浓度增高,可能是对抗血压升高的有益代偿。在不同原因引起的高血压中CGRP浓度差异的原因尚未阐明。N-花生四烯酸乙醇胺（anandamide,AEA）是VR1的内源性配体,体内多种细胞可合成AEA,包括内皮细胞、巨噬细胞和单核细胞等。VR1定位于细胞膜内侧,因此其相应配体需要进入细胞膜内才能发挥作用。血浆或组织液中的AEA进入靶细胞依赖于细胞膜上AEA转运体,现已证明转运体途径是细胞外AEA进入细胞内的主要方式。因此,血浆中AEA水平及AEA转运体活性可能是影响机体CGRP合成与释放的重要因素。AEA转运体活性可受一氧化氮（NO）及外源性一氧化氮合酶抑制物（L-NAME）影响。本实验通过比较分析原发性高血压患者与自发性高血压大鼠（SHR）及两肾一夹高血压大鼠（2K1C）AEA与CGRP水平变化,并观察高血压动物淋巴细胞AEA转运体活性对CGRP合成与释放的影响及ADMA对AEA转运体活性的影响,以探讨AEA转运体活性变化在高血压发生发展中的作用。方法临床实验在未经治疗原发性高血压患者及正常人,测量血压,测定血浆CGRP及AEA浓度。动物实验在SHR与WKY大鼠（正常对照）,两肾一夹高血压大鼠（2K1C）与正常大鼠（假手术处理）比较实验中,分别测量血压（尾动脉测压法）,检测血浆中CGRP（放射免疫分析法）、AEA（液质联用法）、非对称二甲基精氨酸（ADMA）（高效液相色谱法）及一氧化氮（NO）（硝基还原法）;取大鼠背根神经节检测α-CGRP mRNA及β-CGRPmRNA表达量（荧光定量实时PCR）;检测AEA转运体摄取活性（同位素标记法）。细胞实验取正常Wistar大鼠外周血淋巴细胞,以荧光定量PCR法检测AEA对细胞CGRP mRNA合成的影响,并观察AEA转运体抑制剂AM404对其影响;以同位素标记法检测ADMA对淋巴细胞转运体活性影响。结果原发性高血压患者血浆中AEA水平升高,CGRP浓度降低。同样,SHR血浆AEA水平升高,CGRP浓度降低,DRG中α-CGRP mRNA及β-CGRP mRNA表达量降低;而2K1C血浆AEA水平升高,CGRP浓度升高,DRG中α-CGRP mRNA及β-CGRP mRNA表达量升高。SHR的淋巴细胞AEA转运体摄取活性降低,血浆中ADMA水平升高,NO水平降低;2K1C大鼠AEA转运体摄取活性、ADMA及NO水平无明显变化。AEA可上调淋巴细胞α-CGRP mRNA及β-CGRP mRNA表达,预先给予AEA转运体抑制剂AM404可抑制这种效应。外源性ADMA能抑制AEA转运体活性。结论原发性高血压患者与SHR血中CGRP浓度合成与释放减少可能与AEA转运体活性降低有关,ADMA升高可能是降低AEA转运体活性的原因。第二章CGRP介导吴茱萸次碱降压与抗血小板聚集作用研究背景高血压是严重危害人类健康的常见病,高血压的病理过程涉及神经内分泌功能紊乱。已知中枢与外周血管组织主要受交感缩血管神经和感觉舒血管神经支配,共同调节心血管功能,以维持循环稳定。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,具有强效降压作用。在自发性高血压大鼠与原发性高血压患者,血中CGRP浓度显著降低,可能是高血压患者发展的原因之一。高血压患者血小板易激活,导致血小板血栓形成,诱发严重的血栓性疾病;血小板活化也是急性心血管事件的重要诱因。在高血压时,血小板的高活性状态和高聚集性与循环中内源性物质水平异常有关。内源性舒血管物质CGRP在凝血系统中表现为抑制血小板聚集的作用。已知辣椒素受体（VR1）是体内调节CGRP合成与释放的关键受体。吴茱萸是一种具有降压效应的传统中药,吴茱萸次碱（rutaecarpine,Rut）是吴茱萸的重要药理活性成分,具有广泛的心血管作用,通过激动VR1促进感觉神经递质CGRP合成与释放而发挥作用。文献报道,Rut能抑制血小板聚集,但其机制未明。本实验在SHR,研究了Rut对血压影响及其与CGRP的关系;因为TF是血小板活化并引起聚集的重要因子,我们也探讨CGRP介导Rut抑制血小板聚集是否与抑制TF活性有关。方法在体实验在SHR大鼠连续灌胃给予不同剂量Rut（10、20、40mg/kg固体分散体）18天,并连续监测血压（鼠尾动脉测压法）。实验结束后麻醉动物,收集血液。检测血浆中CGRP含量（放射免疫法）,取背根神经节检测CGRP mRNA表达量（荧光定量实时PCR）;收集血液,检测血小板活性及血浆中TF的含量（ELISA）及活性（一期凝固法）。实验中设WKY大鼠为正常对照组。离体实验分离Wistar大鼠外周血血小板与离体胸主动脉共同孵育（95%O₂,5%CO₂,37℃,pH=7.4 Krebs-Henseleit溶液）,观察Rut对血小板聚集性（ADP诱导法）影响。分离Wistar大鼠血小板,观察外源性CGRP对血小板聚集及血小板源性TF（ELISA）释放的影响。结果Rut能剂量依赖性降低SHR血压,并伴随血浆中CGRP浓度增加,DRG中CGRP mRNA表达上调。Rut能剂量依赖性抑制SHR血小板聚集（ADP诱导）,并伴随血浆中TF水平及活性的降低。离体实验中,Rut也能抑制血小板的聚集,VR1阻断剂Capsazepine能减弱这种作用。CGRP能显著抑制血小板聚集与血小板源性TF释放,其作用可被CGRP受体阻断剂CGRP（8-37）减弱。结论在SHR,Rut能产生显著的降压及抑制血小板聚集效应,其作用机制与促进CGRP合成与释放有关。CGRP抑制血小板聚集主要是通过抑制血小板源性的TF释放而发挥作用。第三章CGRP介导吴茱萸次碱对高血压大鼠心脏缺血再灌损伤的保护作用研究背景基础与临床研究证明,心脏缺血一定的时间后再灌可加剧缺血心肌损伤。而某些心血管系统疾病患者（如高血压、糖尿病等）心肌缺血再灌注引起的损伤更为严重。心肌缺血的病理生理过程涉及内源性活性物质变化,因此抑制有害物质和/或促进有益物质的生成与释放是治疗药物保护缺血心肌的重要途径。降钙素基因相关肽（calcitonin gene-related peptide CGRP）由37个氨基酸组成,是辣椒素敏感感觉神经的主要递质,广泛存在于心血管组织。CGRP除了舒张血管调节血压外,局部组织可通过轴突反射释放而参与局部组织功能的调节。在心脏,CGRP被认为是一种内源性保护物质。吴茱萸次碱（rutaecarpine,Rut）是传统中药吴茱萸中重要药理活性成分,具有广泛而复杂的心血管作用。Rut的药理学效应是通过激动辣椒素（VR1）促进CGRP释放而发挥作用。我们实验室以前的工作证明,Rut对多种损伤因素所致心肌缺血具有保护作用。本实验在自发性高血压大鼠（SHR）,探讨了高血压加剧心肌缺血损伤与CGRP的关系;在此基础上,观察了Rut（促进内源性CGRP释放）对心肌缺血再灌损伤的保护作用。方法SHR大鼠连续灌胃给予不同剂量Rut（20、40mg/kg固体分散体）18天,测量实验前后血压（鼠尾动脉测压法）。实验结束后麻醉动物,收集血液,检测血浆中CGRP含量（放射免疫法）;取出心脏,以langendorff法灌流离体心脏,给予缺血再灌注处理（缺血20min,再灌30min）,观察心功能变化。收集冠脉流出液检测肌酸激酶（CK）活性及定时冠脉流量。取胸主动脉检测内皮依赖性舒张功能。取Wistar胸主动脉,Rut孵育后检测孵育液中CGRP含量（放射免疫法）。实验中,WKY作为正常对照,SHR大鼠给予氯沙坦作为阳性药物对照。结果不同剂量的Rut能降低SHR血压,并伴有血浆CGRP浓度升高。离体心脏缺血20min再灌30min能引起明显心肌损伤（心功能下降,冠脉流量减少,CK释放增加）,SHR组损伤显著重于WKY组。Rut处理组能显著改善心功能,增加冠脉流量,减少CK释放。氯沙坦也能改善缺血心脏心功能。SHR血管内皮依赖性舒张功能减弱,不同剂量Rut能改善内皮舒张功能,氯沙坦也具有同样效应。离体实验显示,Rut能增加胸主动脉孵育液中CGRP含量,而这种效应被VR1阻断药Capsazepine取消。结论高血压能进一步加重缺血—再灌注损伤,Rut对缺血心肌的保护与其促进CGRP释放,改善血管内皮功能有关。更多还原

【Abstract】 BACKGROUNDCalcitonin gene-related peptide （CGRP）, the principal neurotransmitter of capsaicin-sensitive sensory nerves, is predominately synthesized in dorsal root ganglia （DRG） in peripheral nervous system. CGRP is widely distributed in cardiovascular system and it is thought as the most powerful vasodilator so far. It is well established that CGRP plays an important role in regulation of blood pressure. There is growing reports to show that, in addition to DRG, many of other type of cells such as leukomonocyte and endothelial cells are also able to synthesize CGRP, which exerts multiple functions through autocrine or paracrine secretion. It has been shown that the synthesis and release of CGRP are regulated by Vanilloid receptor subtype 1（VR1） or capsaicin receptor, which exists in all above mentioned cells.There is increasing evidence to suggest that CGRP plays an important role in the development of hypertensive disease. The decreased level of plasma CGRP is thought as one of the main factors involved in the pathogenesis of essential or spontaneous hypertension. Whereas in acquired hypertension patients （such as hyperadrenocortism） or 2 kidneys 1 clip hypertensive rats （2K1C）, the level of plasma CGRP was increased, which was considered as beneficial compensation against high blood pressure. It is unclear, however, the mechanisms underlying the difference in the level of plasma CGRP in different type of hypertensions.Anandamide （AEA）, an endogenous ligand of VR1, could be synthesized by multiple cells, such as endothelial cell, macrophage and monocyte. It has been shown that VR1 was located in superficies interna of membrane, so its specific ligands （such as AEA） need to across membrane before binding to it. The AEA uptake of cells is mediated by the so-called AEA transporter which is across the membrane and is the major channel for AEA to enter the cells. Therefore, the plasma level of AEA and the activity of AEA transporter are the two key factors to determine the efficiency of AEA uptake, and in turn to regulate the synthesis and release of CGRP via VR1. It was reported that the activity of AEA transporter was affected by nitric oxide （NO） or exogenous NO synthase inhibitor （L-NAME）.To elucidate the mechanisms underlying the possible role of CGRP in the development of essential or spontaneous hypertension, in the present study, we first examined plasma levels of AEA and CGRP in essential hypertension patients, SHR and 2K1C, and then collected the leukomonocytes from hypertensive rats for analyzing the activity of AEA transporter to determine its role in regulating the synthesis and release of CGRP.METHODSClinical studyBlood pressure, plasma CGRP （RIA） and AEA levels （HPLC） were determined in essential hypertension patients （before taking medicine for therapy） and healthy volunteers.Animal experimentBlood pressure （tail-cuff method）, plasma levels of CGRP, AEA, endogenous NOS inhibitor ADMA and NO （nitroreduction method） were measured in SHR and age-matched WKY, or 2K1C and control （Sham operation group）. The expression ofα-CGRP andβ-CGRP mRNA in DRG were detected （Real-Time PCR）. AEA transporter activity was also measured （isotope labeling method）.Cell experimentThe peripheral blood lymphocytes were collected from Wistar rats, and the effects of AEA on the expression of CGRP mRNA were determined by Real-Time PCR. AM404, the AEA transporter inhibitor, was selected for confirming the effect of AEA. The effect of ADMA on AEA transporter activity was evaluated by the isotope labeling method. RESULTSIn essential hypertension patients, the plasma levels of CGRP were decreased, whereas the AEA levels were decreased. Similar results were obtained in SHR, plus the reduced expression ofα-CGRP andβ-CGRP mRNA; In 2K1C, both AEA and CGRP levels were elevated, accompanying by the increased expression ofα-CGRP andβ-CGRP mRNA. The activity of AEA transporter in lymphocytes from SHR was decreased; and the ADMA levels were elevated concomitantly with an increased NO level in SHR. There is no significant differences in AEA activity, ADMA and NO levels in 2K1C. AEA was able to stimulate the expression ofα-CGRP andβ-CGRP mRNA in lymphocytes, which was blocked by pretreatment with AEA transporter inhibitor. Treatment with exogenous ADMA inhibited AEA transporter activityCONCLUSIONSThe decrease in plasma levels of CGRP whereas the increase in AEA levels in essential hypertension patients and SHR is likely due to the reduced activity of AEA transporter. The increased level of ADMA may account for, at least in part, the reduced AEA transporter activity. BACKGROUNDHypertension is a common global disease that severely threatens the health of human being. Neuroendocrine dysfunction is one of the key factors that contribute to the pathological process of hypertension. It is well known that central and peripheral vascular system is under control by both sympathetic vasoconstrictor and sensory vasodilator, which plays an important role in regulating the cardiovascular function and maintaining the stability of blood pressure. Calcitonin gene-related peptide （CGRP）, the principal transmitter of capsaicin-sensitive nerves, is thought as the most powerful vasodilator to date. It has been reported the plasma level of CGRP was decreased in essential hypertension patients as well as in spontaneous hypertensive rat （SHR）. It is possible that dysregulation of CGRP synthesis and release might be a key factor contributed to the development of hypertension.Since high blood pressure is prone to activating the platelet and resulting in thrombus formation, so the occurrence of thrombus diseases is more severe and frequent in essential hypertension patients. The activation of platelet is also the important motivation for acute cardiovascular accidents. The activation and aggregation of platelet are closely associated with abnormal concentration or/and activity of endogenous substance in the essential hypertension. It has been indicated that CGRP, an endogenous vasodilator, also exerts the effects on inhibiting platelet aggregation.It is well documented that activation of Vanilloid receptor subtype 1 （VR1） will lead to the synthesis and release of CGRP. Rutaecarpine is a major component isolated from Evodia Fruit, which is a traditional Chinese medicine with the property of anti-hypertension. Recent studies have shown that rutaecarpine is able to produce multiple cardiovascular effects by stimulating CGRP release via activation of VR1. There are reports to show that rutaecarpine has inhibitory effect on platelet aggregation, but the underlying mechanisms have not been fully elucidated.In the present study, we tested the role of CGRP in the effect of anti-hypertension and anti-platelet exerted by rutaecarpine. Since tissue factor （TF） is the most important initiator of intravascular coagulation, the effect of rutaecarpine or CGRP on TF activity was also explored. METHODSIn vivo experimentsSHR were treated with Rut （10> 20 or 40 mg/kg solid dispersant） by intragastric administration and the blood pressure was monitored （tail-cuff method） timely for 18 consecutive days. After experiment, the blood was collected for the measurement of CGRP concentration （radioimmunity）, TF concentration （ELISA）, TF activity and platelet aggregation （ADP-induced method）; and the dorsal root ganglia （DRG） were saved for CGRP mRNA analysis （Real-Time PCR）.In vitro experimentsPlatelet and thoracic aorta isolated from healthy Wistar rats were co-incubated in Krebs-Henseleit solution （95%O₂, 5%CO₂, 37℃） to assess the effect of rutaecarpine on platelet aggregation; The isolated platelet was also used to determine the effects of exogenous CGRP on platelet aggregation and the release of TF.RESULTSRutacarpine decreased blood pressure in a dose-dependent manner, concomitantly with the increased levels of plasma CGRP and CGRP mRNA in DRGRutacarpine inhibited ADP-induced platelet aggregation, concomitantly with the decreased TF concentration and activity in plasma. In vitro experiment, rutacarpine was also able to inhibit platelet aggregation which was blocked by VR1 antagonist capsazepine. Exogenous CGRP inhibited platelet aggregation and platelet-derived TF release, which were blocked by CGRP receptor antagonist CGRP （8-37）.CONCLUSIONRutacarpine exerts effects on both anti-hypertension and anti-platelet in SHR, which is related to the synthesis and release of CGRP, and the anti-platelet effect of rutacarpine is mediated by CGRP through inhibition of platelet-derived TF release. BACKGROUNDThe results from experimental and clinical studies have shown that the heart suffers severe damage when blood supply returns to the tissue after a long time ischemia. This reperfusion injury could be exacerbated by certain diseases such as hypertension and diabetes. The pathological process of myocardium ischemia is related to alteration of some endogenous substances level and activity. Therefore, to protect the organs or tissues against reperfusion injury, one important avenue is to seek novel drugs which could increase the beneficial substances but decrease the harmful substances to the body.Calcitonin gene-related peptide （CGRP）, the predominate neurotransmitter of capsaicin-sensitive sensory nerves, is widely distributed in the cardiovascular system. Besides being as a systemic depressor, CGRP is also considered as a local functional regulator through axon reflex-controlled release. It has been shown that CGRP is an endogenous substance which is able to exert protective effects on the heart. Rutaecarpine （Rut）, a major component of Chinese traditional herb Evodia Fruit, has wide and complicate cardiovascular effects. The pharmacological activity of Rut is mediated by endogenous CGRP via activation of VR1..Our previous works has been demonstrated that Rut exerted cardioprotective effect on ischemic myocardium induced by multiple detrimental factors. In the present study, therefore, we explored the correlation between CGRP and exacerbated reperfusion injury by hypertension, and in turn to explore whether Rut is able to exert the protective effects on reperfusion injury by promoting the release of CGRP.METHODSSHR were treated with rutacarpine （20 or 40 mg/kg solid dispersant） by intragastric administration and blood pressure was monitored （tail-cuff method） for 18 consecutive days. At the end of experiment, the blood was collected for measurement of CGRP concentration （radioimmunity）. Hearts were isolated and subjected to 20- min global ischemia and 30-min reperfusion; cardiac functions were recorded; coronary flow rate and creatine kinase （CK） activity were measured. Thoracic aortas were separated for determining endothelium-depended relaxation. In this study, WKY and losartan-treated SHR were used as normal and positive drug control, respectively. Thoracic aortas were isolated from healthy Wistar rats and Rut-induced CGRP release was examined after incubation in K-H solution.RESULTSRut decreased blood pressure in dose-dependent manner, concomitantly with the increased plasma CGRP in SHR. The ischemia reperfusion caused a dramatic decrease in myocardial function and coronary flow, and a significant increase in the release of CK. The degree of reperfusion injury in SHR was more severe than that in WKY. Pretreatment with Rut dramatically improved the recovery of cardiac function and coronary flow, and reduced the release of CK. Similar results were observed in Losartan-treated animals.The endothelium-dependent vasodilatation was impaired in SHR, which could be reversed by Rut. Similar results were observed in Losartan group. In vitro, Rut stimulated CGRP release after incubation with thoracic aorta, which was blocked by capsazepine, the VR1 antagonist.CONCLUSIONHypertension exacerbated the reperfusion injury. The protective effect exerted by Rut on reperfusion injury is related to the increased release of CGRP and the improved the vascular endothelium function.更多还原