【作者】 李茜；

【导师】 张翼；

【作者基本信息】 河北医科大学 ， 生理学， 2010， 博士

【摘要】 内源性大麻素物质来源于细胞膜上的脂类前体物质,具有脂类物质的特征,包括氨基化合物和长链脂肪酸。Anandamide(N-花生四烯酸氨基乙醇,AEA)是一种内源性大麻素物质,为花生四烯酸的衍生物。内源性大麻素物质可以参与机体内许多生理和病理生理过程,如神经行为、胃肠功能、应激和焦虑、心血管功能。到目前为止根据药理学效应和基因序列,发现了两种内源性大麻素受体,1型受体(CB1)和2型受体(CB2)。这两种受体广泛分布于机体各种组织细胞,其中包括心肌细胞。据报道内源性大麻素物质对心血管功能的调节发挥重要作用。如:内源性大麻素物质可以抑制自发性高血压大鼠的心肌收缩力;内源性大麻素物质anandamide可以减轻由肾上腺素或缺血/再灌所导致的心律失常;anandamide可以减轻大鼠离体心脏缺血再灌注损伤。然而,有关anandamide抗心律失常和抗缺血再灌注损伤的机制尚未明瞭。本研究旨在应用电生理学、功能学、免疫组化和流式细胞等方法探讨内源性大麻素物质anandamide的心脏保护作用及其作用机制。研究分为两部分:(1)应用冠脉结扎方法、细胞内记录方法、全细胞膜片钳技术,观察anandamide对大鼠心肌缺血再灌注所致心律失常的影响,并探讨其电生理学机制。(2)应用Langendorff灌注技术,观察心功能的变化;通过TUNEL和流式细胞技术检测细胞凋亡;利用激光共聚焦技术,观察心室肌细胞内游离钙浓度。观察内源性大麻素物质anandamide对大鼠心肌缺血/再灌注损伤(I/R)的作用,并探讨其机制。Ⅰ内源性大麻素物质Anandamide抗大鼠心律失常的电生理学研究目的: Anandamide是一种内源性大麻素物质,具有抗心肌缺血、抗心律失常等心脏保护作用,但其机制尚未完全明确。本研究旨在探讨Anandamide的抗心律失常作用及其电生理学机制。方法:利用大鼠冠脉结扎方法,观察anandamide对在体心脏的抗心律失常作用;应用细胞内记录方法,观察anandamide对离体心室乳头肌动作电位的影响;利用全细胞膜片钳技术,观察anandamide对心室肌细胞L-型钙电流以及各种钾电流的影响,包括瞬时外向钾电流(Ito)、缓慢延迟整流钾电流(Iss)、内向整流钾电流(IK1)和ATP敏感钾电流(IKATP)。结果:(1) Anandamide (1μM.kg-1)显著抑制I/R诱发的心律失常。(2) Anandamide (1、10、100 nM)浓度依赖性的缩短乳头肌动作电位复极化50%(APD50)和90% (APD90)时程;对部分去极化乳头肌, Anandamide(100 nM)不仅缩短APD50和APD90,同时降低动作电位幅度、超射值和0期最大去极化速度。上述效应可被内源性大麻素1型受体阻断剂AM251 (100 nM)所阻断,而内源性大麻素2型受体阻断剂AM630 (100 nM)对Anandamide的效应无影响;L型钙通道开放剂Bay K8644 (0.5μM)、钾通道阻断剂氯化四乙铵(TEA, 20mM)和NO合酶抑制剂L-NAME (1 mM)对Anandamide的效应无影响。(3) Anandamide (1、10、100 nM)浓度依赖性降低心肌细胞L型钙电流,使电流-电压关系曲线上移。Anandamide ( 100 nM)不影响L型钙通道的激活过程,但可以加速稳态失活过程,并且减慢通道失活后的复活。上述效应可以被AM251 (100 nM)所阻断,而AM630 (100 nM)对Anandamide的效应无影响。(4) Anandamide (1、10、100 nM)浓度依赖性地降低Ito、增加IKATP,而对Iss和IK1没有影响。Anandamide使Ito的稳态失活曲线左移,而使其失活后的复活曲线右移。内源性大麻素1型受体阻断剂AM251和2型受体阻断剂AM630都不能阻断anandamide对Ito的抑制效应。内源性大麻素2型受体阻断剂AM630可以阻断anandamide增加IKATP的效应,而内源性大麻素1型受体阻断剂AM251没有此阻断效应。小结: Anandamide具有明显的抗I/R心律失常作用。Anandamide的抗心律失常作用可能通过内源性大麻素1型受体,导致心肌细胞L型钙通道失活加速,失活后复活减慢,降低L型钙电流,缩短动作电位时程而实现。Anandamide亦可通过内源性大麻素2型受体途径增加IKATP;通过非受体途径加速Ito通道失活,减慢失活后复活,从而抑制Ito。Ⅱ内源性大麻素物质anandamide对大鼠心肌缺血再灌注损伤的影响及机制目的:本实验旨在观察内源性大麻素物质anandamide(AEA)对大鼠心肌缺血再灌注(I-R)所致心功能损伤,梗死面积和凋亡的影响,并探讨其机制。方法:应用Langendorff灌注技术,给予离体心脏30 min缺血/180 min再灌注处理,期间观察左心室功能的变化;再灌注结束后,通过氯化三苯基四氮唑染色法测定心肌梗死面积;通过TUNEL和流式细胞技术检测细胞凋亡;利用激光共聚焦技术观察心室肌细胞内游离钙浓度,[Ca2+]i用相对荧光强度((FI-FI0) /FI0, %)表示。结果:(1)应用anandamide (1、10、100 nM)可浓度依赖性地促进大鼠I-R后心功能的恢复,减少梗死面积。线粒体通透性转换孔开放剂苍术苷(Atr,20μM)、内源性大麻素1型受体阻断剂AM251(1μM)、内源性大麻素2型受体阻断剂AM630(1μM)都可以完全阻断anandamide对I-R所致心功能损伤的保护作用。(2)应用anandamide后TUNEL染色的心肌凋亡细胞数量以及流式细胞术测定的心肌细胞凋亡率都明显降低。与缺血再灌注组比较,应用anandamide后心肌Bax蛋白表达明显降低,而心肌Bcl-2蛋白表达和Bcl-2/Bax比率明显升高。线粒体通透性转换孔开放剂苍术苷(Atr,20μM)、内源性大麻素1型受体阻断剂AM251(1μM)、内源性大麻素2型受体阻断剂AM630(1μM)都可以完全阻断anandamide对I-R所致凋亡的减少作用。(3)在正常台式液、无钙台式液以及模拟缺血液中,anandamide(1、10、100 nM)浓度依赖性地降低细胞内游离钙浓度。在正常台式液中,内源性大麻素1型受体阻断剂AM251(100 nM)可抑制anandamide(100 nM)降低细胞内钙浓度的效应,而内源性大麻素2型受体阻断剂AM630(100 nM)对anandamide(100 nM)降低细胞内钙效应无影响。在模拟缺血液中,AM630(100 nM)可抑制anandamide降低细胞内钙效应,而AM251(100 nM)对anandamide(100 nM)降低细胞内钙效应无影响。在正常台式液中,anandamide(100 nM)可抑制由L型Ca2+通道激动剂Bay K8644所致细胞内钙浓度的升高。在无钙台式液中,anandamide(100nM)可抑制由ryanodine所致的细胞内钙浓度的升高。小结: Anandamide可通过激动内源性大麻素1型和2型受体,降低心室肌细胞内游离钙浓度,抑制线粒体通透转换孔开放而有效促进I-R后左心室功能的恢复,减少梗死面积,抑制I-R诱导的心肌细胞凋亡。Anandamide降低心室肌细胞内游离钙效应与其抑制心室肌细胞L型钙通道和肌浆网内钙释放有关。Anandamide抑制心肌细胞凋亡效应与增加心肌Bcl-2/Bax蛋白表达比率有关。更多还原

【Abstract】 Endogenous cannabinoids (endocannabinoids), which are synthesized from lipid precursors in plasma membranes, are signaling lipids consisting of amides and esters of long chain fatty acids. Anandamide (N-arachidonoylethanolamide, AEA) is one of endocannabinoids that are derivatives of arachidonicacid, which are involved in many physiological and patho-physiological processes such as neurobehavior, gastrointestinal function, stress and anxiety, and cardiovascular functions. At least two types of cannabinoid receptors, the CB1 and CB2 has been found and cloned depending on their pharmacological effect and gene sequences. These two receptors are widespread in many tissues including cardiac myocyte. It has been shown that cannabinoids (CBs) are importantly involved in regulating cardiovascular function. For example, endocannabinoids have been shown to tonically suppress cardiac contractility in spontaneously hypertensive rat. Anandamide has been proved to protect the heart from adrenaline-induced arrhythmias or arrhythmias induced by ischaemia/reperfusion. Similarly, anandamide can limit the damage induced by ischaemia/reperfusion in rat isolated hearts through different mechanisms. However, the mechanism underlying for the anti-arrhythmias effects and anti-ischaemia–reperfusion injury of anandamide are not clear.The purpose of this study was to explore the protective effects of anandamide on rat heart and underlying mechanisms using electrophysiological, functional, immune histochemistry and flow cytometric techniques. Our study consisted of two parts: (1) To investigate the antiarrhythmic effect of anandamide in rat and the electrophysiological mechanism. (2) To investigate the cardiac protection of anandamide against ischemia/reperfusion injury in rat and the underlying mechanism. ⅠElectrophysiological mechanism for the antiarrhythmic effect of anandamide in ratObjective: Anandamide, one of endocannabinoids, has been reported to have anti-arrhythmia effect. The aim of present study was to study the electrophysiological mechanism for antiarrhythmic effect of anandmide.Methods: Arrhythmias were observed by using heart ischaemia/reperfusion experiment in vivo. Action potential (AP) (evoked) was recorded by using intracellular recording technique in rat cardiac papillary muscles. Also, L-type Ca2+ current and different K+ currents (including transient outward potassium current (Ito), steady-state outward potassium current (Iss), inward rectifier potassium current (IK1), ATP sensitive potassium current (IKATP)) were measured and analysised by using whole-cell patch-clamp recording technique in isolated rat ventricular myocytes.Results:(1) Anandamide (1μMkg-1) suppressed the ischaemia/reperfusion-induced ventricular arrhythmias in vivo heart.(2) In ventricular papillary muscles, anandamide (1, 10, 100 nM) decreased the AP duration (APD) in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased action potential amplitude (APA), action potential overshoot (OS), and maximal rising velocity of depolarization in phase 0 (Vmax) in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB1 receptor, but not AM630 (100 nM), a CB2 receptor antagonist. Also, a agonist of L-type Ca2+ channel Bay K8644 (0.5μM), a K+ channel blocker tetraethylammonium chloride (TEA, 20mM) and the nitric oxide synthase inhibitor L-NAME (1 mM) had no effect on anandamide-induced shorten of AP duration.(3) In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca2+ current concentration-dependently and shifted the current-voltage relationship curve of Ca2+ current upword. Anandamide (100 nM) shifted steady-state inactivation curve to the left while shifted the recovery curve to the right. Blockade of CB1 receptors with AM251 (100 nM), but not CB2 receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca2+ current.(4) In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased Ito and increased IKATP in a concentration-dependent manner, but had no effects on Iss and IK1. Anandamide shifted steady-state inactivation curve of Ito to the left, and shifted the recovery curve of Ito to the right. Blockade of CB1 receptors with AM251 and CB2 receptors with AM630, did not eliminate the inhibition effect of anandamide on Ito. Blockade of CB2 receptors, but not CB1 receptors, eliminated the augmentation effect of anandamide on IKATP.Conclusion: These data suggest that anandamide has a significant antiarrhythmic effect through activation of CB1 receptor, acceleration of inactivation and slowing of recovery from inactivation in L-type Ca2+ channel as well as decrease of L-type Ca2+ current, shorten of APD. Also anandamide augments IKATP through CB2 receptor, suppresses Ito through a non-CB1 and non-CB2 receptors pathway, acceleration of inactivation and slowing of recovery from inactivation in ventricular myocytes.ⅡCardiac protection of anandamide against ischemia/reperfusion injury and underlying mechanism in ratObjective: The aim of this study was to investigate the effects of anandamide (AEA) on cardiac function injury, infarct size and apoptosis induced by ischemia-reperfusion (I-R) in rat myocardium and underlying mechanism.Methods: In Langendorff isolated rat heart, cardiac function was recorded before and after 30 min global ischemia followed by 180 min reperfusion. At the end of reperfusion, the infarct size of the heart was measured by triphenyltetrazolium chloride (TTC) staining. TUNEL labeling and flow cytometric techniques were used for the measurement of apoptosis in cardiomyocytes of rat. Intracellular calcium ([Ca2+]i) was detected by confocal microscopy and represented by relative fluorescent intensity ((FI-FI0) /FI0, %).Results: (1) Anandamide (1, 10, 100 nM) displayed a better recovery of cardiac function and reduction of infarct size during reperfusion after ischemia in a concentration-dependent manner. The protective effects of anandamide were completely abolished by mitochondrial permeability transition pore (MPTP ) opener atractyloside (20μM), CB1 receptor antagonist AM251 (1μM) and CB2 receptor antagonist AM630 (1μM).(2) Anandamide significantly decreased the TUNEL-positive cells and apoptosis rate detected by flow cytometry. Compared with I/R group, Bax protein expressions of the myocardium decreased after administration of anandamide, while Bcl-2 protein expressions and Bcl-2/Bax ratio increased. The effects of anandamide on apoptosis were completely abolished by MPTP opener atractyloside (20μM), CB1 receptor antagonist AM251 (1μM) and CB2 receptor antagonist AM630 (1μM).(3) Anandamide (1, 10 and 100 nM) reduced [Ca2+]i of cardiomyocytes in normal Tyrode’s solution, Ca2+-free Tyrode’s solution and simulated ischemic solution in a concentration-dependent manner. In normal Tyrode’s solution, inhibition of anandamide (100 nM) on [Ca2+]i was canceled by pretreatment with CB1 receptor antagonist AM251 (100nM) , but not by CB2 receptor antagonist AM630 (100nM). While in simulated ischemic solution, pretreatment of cells with AM630, not AM251, abolished the inhibitory effect of anandamide (100 nM) on [Ca2+]i. The augment of [Ca2+]i induced by L-type Ca2+ channel agonist Bay K8644 was inhibited by anandamide (100nM) in normal Tyrode’s solution. The ryanodine-induced [Ca2+]i increase was markedly inhibited by anandamide (100nM) in Ca2+-free Tyrode’s solution.Conclusion: Anandamide can promote the recovery of left ventricular function, reduce myocardial infarct size and inhibit the apoptosis induced by I-R through activation of both CB1 receptor and CB2 receptor, decrease of [Ca2+]i, and inhibition of MPTP open of rat ventricular myocytes. The decreasing effect of anandamide on [Ca2+]i may be related with inhibition of Ca2+ influx from L-type Ca2+ channel and releasing of Ca2+ from sarcoplasmic reticulum. The depression effect of anandamide on apoptosis may be related with increase of Bcl-2/Bax proteins expression in myocardium.更多还原