【作者】 杨波；

【导师】 夏强；

【作者基本信息】 浙江大学 ， 生理学， 2007， 硕士

【摘要】 背景据WHO 2004年世界卫生报告统计，由心血管疾病引起的病人死亡率占首位，其中主要是缺血性疾病。在心肌缺血/复灌等病理条件下，由于心肌氧供应减少、导致活性氧产生大大增加是造成细胞损伤的主要原因之一。雄激素是一类类固醇激素，主要有睾酮（testosterone）、双氢睾酮（dihydrotestosterone）、脱氢异雄酮（dehydroisoandrosterone）和雄烯二酮（androstenedione）。睾丸间质细胞分泌的雄激素主要是睾酮，可以直接作用于核内的雄激素受体，也可以转变为双氢睾酮（作用于雄激素受体的能力是睾酮的两倍）或雌二醇而间接起作用。绝经期前的女性比同龄男性患心血管疾病的几率要小得多，因而普遍认为，雄激素是导致心血管疾病的一种危险因素。但是，也有证据表明患心血管疾病的男性血中睾酮水平要比血管造影片正常人的低。睾酮作为雄激素的主要种类，能够提高性腺机能减退并患有心绞痛病人的心肌缺血阈值和生活质量，具有抗心肌缺血的保护作用。最重要的是，我们发现去势大鼠施行睾酮替代疗法后，提高心肌收缩和抗心肌缺血复灌损伤的功能。具有促合成代谢作用的雄性类固醇激素（anabolic androgenic steroids，AAS）由多种类固醇组成，由于每种成分药理学效应不同，因此作用比较复杂。至今临床上还没有确实的证据表明雄激素与心血管系统的关系。合成代谢的雄性类固醇激素与心肌缺血、急性心肌梗死、运动员的高血压有关，于是人们认为雄性激素对心血管系统是有害的。流行病学数据和干预学说则显示血液中的雄激素对心血管可能有中性或有益的作用。雄性激素对心血管的损伤作用主要表现在：（1）能够不可逆地改变了膜脂质和脂蛋白的构型；（2）产生血栓症；（3）心肌肥大；（4）可能会导致动脉粥样硬化。有报道发现，去势的大鼠补充睾酮后能够改善心肌抗缺血复灌损伤的功能。这些变化可能是通过雄激素受体介导的基因途径起作用的。据最近报道，存在一些急性的非雄激素受体介导的途径，如睾酮的短期效应是可以舒张全身的血管、冠状动脉或肺部的血管，从而增加心肌的抗缺血作用。有人在分离的心肌细胞和线粒体上用线粒体荧光和膜片钳技术研究发现，睾酮可以明显地去极化和氧化线粒体，而ATP、5-hydroxydecanoate（5-HD）、glibenclamide可以抑制这种作用，表明睾酮通过激活线粒体ATP敏感性钾通道（mitochondrial ATP-sensitive potassium channel）对心肌有直接的保护作用。因此，我们假设雄激素替代疗法不仅通过基因途径对心血管发生作用，可能还通过非基因途径对心血管产生保护作用。基因途径是指雄激素与核内受体结合后，调空基因表达，蛋白合成；非基因途径指雄激素穿透细胞膜后，作用于G蛋白偶联的受体，进一步激活PKA，PKC等下游途径，以线粒体上的相关通道或孔道为靶点发挥心血管保护作用。目的大鼠双侧输精管结扎，睾丸摘除，每天补充雄激素（200μg/100g体重），建立雄激素替代疗法模型。给药8周后，大鼠开胸取心脏，Langendorff装置上离体灌流，观察睾酮替代疗法的慢性心血管效应；在原代培养的乳鼠心肌细胞模型上，MTT法测定细胞存活率。在分离细胞模型上，台盼蓝拒染法测定心肌细胞存活率，视频跟踪系统观察单细胞收缩功能，测定线粒体膜电位和ROS生成情况；以线粒体上的多种通道/孔道为切入点，利用多种工具药（阻断剂/开放剂），深入研究睾酮急性心肌保护作用的具体机制。方法1．大鼠去势模型和雄激素替代疗法雄性Sprague-Dawley大鼠，体重（160～200）g，由浙江省医学科学研究院动物中心提供。大鼠麻醉后，用消毒过的刀片将阴囊处切开。轻轻拉出睾丸、附睾头、附睾尾后，摘除睾丸；结扎输精管并将其塞回阴囊。两侧相同处理。1周后，给药组每天补充丙酸睾酮（200μg/100g体重），去势组给予相应剂量溶媒。2．离体心脏Langendorff灌流雄性SD大鼠断头后开胸迅速取出心脏，置于4℃改良Krebs-Henseleit（K-H）液中洗净血液，迅速转移、固定于Langendorff灌流装置，以改良K-H液行常规恒压（76mmHg）灌流。各组心脏均稳定20min后，进行实验处理。结扎冠状动脉使局部心肌缺血30min，随后松开结扎线复灌120min。3．灌流心脏流出液乳酸脱氢酶活性的测定在复灌各时间点收集冠脉流出液，利用分光光度法测定乳酸脱氢酶（lactate dehydrogenase，LDH）的含量。4．心肌梗死面积测定离体心脏复灌120min后再结扎冠脉，取1％伊文思蓝经主动脉注入心脏，冷冻后与心脏纵轴垂直切成2mm均匀薄片，采用双染色法染色，使用Image软件（NIH）测量相关区域面积，计算梗死区占危险区的比值。5．乳鼠心肌细胞培养取生后1-2d的SD大鼠心室肌，剪成1mm³大小后，用含0.1％胰蛋白酶和0.05％胶原酶Ⅱ的溶液（37℃）消化10min，收集心肌细胞悬液，并用8ml含15％胎牛血清的DMEM（高糖）培养液中和胰酶作用。重复消化收集8-10次，直到心肌组织完全解离，以1200rpm离心6min，所得的沉淀用10ml DMEM（15％胎牛血清）重悬，然后置于二氧化碳培养箱（95％空气+5％CO₂，37℃）中预培养，1h后悬浮的心肌细胞用200目不锈钢网过滤。6．MTT法测定细胞存活率活细胞中的琥珀酸脱氢酶可使MTT（3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐）分解产生蓝色结晶状formazan沉淀于细胞内和细胞周围。其量与细胞数呈正比，也与细胞活力呈正比。7．心室肌细胞分离及存活率测定用酶解法分离成年大鼠心室肌细胞。大鼠处死后，迅速取出心脏，置于4℃氧饱和Tyrode氏液中洗净血液后固定于Langendorff灌流装置上进行恒流（10ml/min）灌流（37℃）。先以无钙Tyrode氏液灌流5min，再以含Ⅰ型胶原酶0.3g/L的无钙Tyrode氏液灌流10min。将心室肌剪碎，于含0.1％BSA的无钙Tyrode氏液中37℃孵育15min，细胞悬液用200μm的尼龙网过滤，滤液在无钙Tyrode氏液中40min内，每10min一次逐步复钙至Ca²⁺浓度为1.25mmol/L。台盼蓝拒染法测定心肌细胞存活率。0.4％台盼蓝染色3min后，死细胞蓝染，活细胞不染色，显微镜下分别计算死细胞和活细胞个数。8．线粒体膜电位和ROS生成的测定分离的心肌细胞中加入相应的荧光探针（TMRE测定膜电位，DCFH-DA测定ROS生成量），常温下避光孵育30min，然后用Tyrode氏液冲洗2～3次，酶标仪测定其荧光值改变量。将TMRE标记的细胞置于倒置荧光显微镜下选取合适的视野成像。9．单个心肌细胞收缩的测定将100μl细胞悬液滴于灌流槽中，以95％O₂+5％CO₂饱和的K-H液持续灌流，流速为2ml/min。在灌流槽中施加频率为0.2Hz、强度为50V的电场刺激，待细胞收缩稳定15min后，镜下选择横纹清晰、胞膜光整的杆状细胞，以计算机辅助的MedEase视频跟踪系统检测单个心室肌细胞收缩参数。结果1．丙酸睾酮替代疗法对大鼠体重及抗心肌缺血复灌损伤的慢性作用9周后，与正常对照组比较，去势组大鼠体重显著增加，丙酸睾酮（200μg/100g体重，8周）处理后大鼠体重明显下降。与去势组相比，丙酸睾酮处理显著地减少缺血复灌后心肌梗死面积和冠脉流出液中LDH释放量。2．睾酮对H₂O₂应激引起的心肌细胞存活率的影响与正常组相比，100μmol/L H₂O₂处理6h后，心肌细胞MTT值显著降低；睾酮预处理(10^-5μmol/L，10min)能够显著增加心肌细胞MTT值。3．睾酮对H₂O₂应激引起的心肌细胞存活率的影响以及可能机制不同浓度的H₂O₂（10，100，1000μmol/L）均能引起心肌细胞存活率的降低，并呈现剂量依赖效应。与正常细胞相比，100μmol/L H₂O₂处理5min后，台盼蓝染色法测定的心肌细胞存活率约降低56％。睾酮对心肌细胞存活率的作用存在剂量依赖效应。与H₂O₂组相比，睾酮(10^-8，10^-7，10^-6，10^-5，10^-4μmol/L)预处理10min均显著增加心肌细胞的存活率，而浓度为10^-9μmol/L的睾酮无显著增加心肌细胞的存活率的作用。10^-5μmol/L睾酮组增加心肌细胞的存活率的作用与10^-6μmol/L睾酮组有显著差异，而与10^-4μmol/L睾酮组无显著差异。又根据文献报道，故后续实验我们采用浓度为10^-5μmol/L的睾酮处理。H₂O₂应激前加入线粒体通透性转换孔开放剂atractyloside（Atr，20μmol/L）预处理20min或加入5-HD（100μmol/L）预处理10min并于末10min给予睾酮(10^-5μmol/L)处理，均减小睾酮(10^-5μmol/L，10min)预处理增加心肌细胞存活率的作用。4．睾酮对H₂O₂应激的心肌细胞线粒体TMRE荧光值的影响以及可能机制与对照组相比，H₂O₂应激（100μmol/L）显著地增加心肌细胞荧光值，睾酮(10^-5μmol/L)预处理能够减少H₂O₂应激引起的心肌细胞荧光值△F增加。H₂O₂应激前加入Atr（20μmol/L）预处理20min或加入5-HD（100μmol/L）预处理10min并于末10min给予睾酮(10^-5μmol/L)处理，均减小睾酮(10^-5μmol/L，10min)预处理降低心肌细胞荧光值△F的作用。5．睾酮对H₂O₂应激导致的ROS生成量变化的影响及可能机制与对照组相比，H₂O₂应激组心肌细胞DCF荧光值显著增加，睾酮(10^-5μmol/L)或CsA（0.2μmol/L）预处理能够减少H₂O₂应激引起的心肌细胞DCF荧光值增加。H₂O₂应激前加入Atr（20μmol/L）预处理20min或加入5-HD（100μmol/L）预处理10min并于末10min给予睾酮(10^-5μmol/L)处理，均减小睾酮(10^-5μmol/L，10min)预处理降低心肌细胞DCF荧光值的作用。6．睾酮预处理对H₂O₂应激引起的心肌细胞收缩动力学变化的影响与对照组相比，H₂O₂组的+dL/dtmax，-dL/dtmax和dL显著降低。与H₂O₂组相比，睾酮预处理能显著增加+dL/dtmax和dL；第2min，显著增加心肌细胞舒张速度-dL/dtmax，第4，8min没有明显变化，第6，10min显著增加心肌细胞舒张速度-dL/dtmax。Atr（20μmol/L，20min）预处理减小睾酮增加心肌细胞收缩速度+dL/dtmax的作用。结论睾酮具有慢性和急性的抗心肌缺血复灌损伤的心血管保护效应。丙酸睾酮替代疗法显著减少缺血复灌后心肌梗死面积和冠脉流出液中LDH释放量；睾酮预处理显著抑制H₂O₂应激降低培养心肌细胞存活率的作用。在分离心肌细胞H₂O₂应激模型上，睾酮预处理显著增加心肌细胞存活率，抑制线粒体膜电位去极化和ROS生成，明显改善H₂O₂应激导致的±dL/dtmax和dL降低程度。睾酮的急性心肌保护作用可能与抑制线粒体通透性转换孔开放或/和促进线粒体ATP敏感性钾通道开放有关。更多还原

【Abstract】 BackgroundIt is well known that premenopausal women have lower incidence of coronary heart disease than age-matched men. This significant gender difference has been attributed at least in part to negative effects of testosterone on the cardiovascular system. Endogenous anabolic androgenic steroids （AAS） have been used by athletes to improve performance by increasing muscle mass and strength. Among the numerous documented toxic and hormonal effects of AAS, attention has been recently focused especially on the cardiovascular effects.It is reported that there are increases in blood pressure and peripheral arterial resistance, and there are also effects on the heart muscle, primarily left ventricular hypertrophy with restricted diastolic function. Severe cardiac complications （heart failure, ventricular fibrillation, ventricular thromboses, myocardial infarction or sudden cardiac death） in individual strength athletes with acute AAS abuse have also been reported. However, AAS consist of a variety of different steroids with differing pharmacological properties. No clinical study has yet demonstrated a conclusive link between physiological testosterone and fatal cardiovascular events. Epidemiological data and an intervention study rather suggest either a neutral or a beneficial effect ofnatural circulating androgens on coronary heart disease in men. Testosterone replacement in orchidectomized rats improved the recovery of myocardial function after ischemia/reperfusion injury. These processes involve changes in gene-expression controlled by intracellular androgen receptor mediated pathways.Recent studies have, however, demonstrated alternative, rapid intracellular androgen receptor-independent mode of testosterone action. For example, administration of testosterone acutely induces vasodilatation in the systemic, coronary, and pulmonary vascular beds. Acting in this way, testosterone might increase myocardial tolerance to ischemia. Er et al. have recently shown that testosterone is directly cytoprotective in the myocardium, through activation of ATP-sensitive K⁺ channels in the mitochondrial inner membrane. It has been proposed that these rapid androgen actions may be exerted through membrane receptors that stimulate early intracellular signaling pathways through interaction with G proteins. Jose Miguel Vicencio found that in rat neonatal cardiac myocytes, testosterone increases intracellular Ca²⁺ levels by a non-genomic mechanism, which involves a membrane androgen receptor, a PTX sensitive G protein, PLC, IP₃ and IP₃R as signaling pathway. Androgens act acutely to stimulate contractility of cardiac myocytes independent of other factors which can influence myocyte function in vivo.In this study, we examined the chronic and acute effect of testosterone treatment on rat myocardium. Furthermore, we hypothesize that testosterone exert its acute cardioprotective effects by a mitochondrial pathway, specifically via inhibition of mPTP opening. Also we tested the role of mPTP modulators in the effect of testosterone.ObjectivesThe aim of the present study was to investigate the chronic and acute effect of androgen replacement therapy on rat hearts against ischemia/reperfusion injury. Eight weeks after gonadectomy, all the heart were mounted on a Langendorff apparatus to assess contractile function. In the isolated myocytes model, cell viability was measured by trypan blue exclusion and cell contraction were observed using vediotracking system. We hypothesize that mitochondrial channel or pores are involved in the mechanisms of acute cardioprotective effect of testosterone.Methods1. Gonadectomy model and hormonal replacement therapyFor the gonadectomized （GDX） group, the cauda epididymidis, caput epididymidis, vas deferens, and testis were extracted. The vas deferens were ligated bilaterally and the testes removed. The vas deferens were reinserted into the scrotal sac and underlying membrane. The replacement regimen of testosterone propionate （TP） to TP group and vehicle to GDX group was begun 1 week after gonadectomy, in order to provide sufficient period to clear the endogenous steroids.2. Isolated Perfused Heart PreparationAfter 8 weeks all the rats were anesthetized and the hearts were excised rapidly, placed in ice-cold Krebs-Henseleit buffer, mounted on a Langendorff apparatus. In the present study, the isolated heart was subjected to 30 min of ischemia followed by 120 min of reperfusion, which induced myocardial injury.3. Lactate dehydrogenase measurementTo assess the extent of myocardial tissue injury, the effluent from the isolated perfused heart was collected at 5 min of reperfusion and lactate dehydrogenase （LDH） was spectrophotometrically assayed. LDH activity was expressed as units per liter.4. Measurement of the Area of RiskFor determination of infarct size, the coronary artery was reoccluded at the end of reperfusion, and a solution with 2.5% Evans blue was perfused. Hearts were then frozen and cut into slices, which were then incubated in a sodium phosphate buffer containing 1% （w/v） 2, 3, 5-triphenyl-tetrazolium chloride for 15 min to visualize the unstained infarcted region. Infarct and risk zone areas were determined by planimetry. The infarct size was expressed as a percentage of the risk zone.5. Primary culture of neonatal rat cardiomyocytesCardiac ventricular myocytes were prepared from neonatal （1- to 2-d old） Sprague-Dawley rats as follows. Hearts were excised and the ventricles trisected andincubated with collagenase type II （0.05%） and pancreatin （0.1%）. Cardiac myocytes were plated in DMEM culture media supplemented with 15% fetal bovine serum.6. Determination of MTT formazan formationTo determine the viability of cardiac myocytes, we carried out the MTT （3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide） assay as follows. The MTT assay is based on the ability of mitochondria to reduce MTT （a yellow tetrazolium dye） to MTT formazan （a blue mitochondrial by-product）, the reduction is mediated by mitochondrial dehydrogenases in living but not in dead cells. The amount of MTT formazan in DMSO was then measured at a wavelength of 570 nm, using a spectrophotometer.7. Preparation of isolated ventricular myocytesIsolated adult ventricular myocytes from male Sprague-Dawley rats were obtained by enzymatic dissociation. Trypan blue exclusion was used as an index of the viability of the ventricular myocytes.8. Measurement of the mitochondrial membrane potential and reactive oxygen species productionMitochondrial membrane potential （Δψm） was measured using the fluorescent dye tetramethyl-rhodamine ethyl ester （TMRE）. TMRE fluorescence was excited at 514 nm and collected at 590 nm. Reactive oxygen species （ROS） production was measured by loading with fluorescent probe DCFH-DA. Levels of DCF in cell lysates or in solutions were measured using 488 nm excitation/530 nm emission settings.9. Measurement of myocyte contractionMyocytes were placed in a chamber mounted on an inverted microscope. The chamber was perfused continuously at 2 ml/min with KH buffer, with 1% BSA and a gas phase of 95% O₂/5% CO₂ at room temperature. A video-tracking system was used to measure the peak velocity of cell shortening （+dL/dtmax）, the peak velocity of cell relengthening （-dL/dtmax）, the amplitude of contraction （dL） and the end-diastolic length of the isolated myocytes.Results1. Chronic effect of testosterone propionate on rat body weight and heart against ischemia/reperfusion injuryTP replacement inhibited gonadectomy induced increase of body weights.Gonadectomy significantly increased the infarct size and LDH release at the time of 5 min reperfusion in the isolated perfused rat heart subjected to 30 min ischemia and 120 min reperfusion compared with untreated control hearts, and TP replacement attenuated the injury of gonadectomy.2. Testosterone treatment on cultured cell viabilityTestosterone also increases cell viability in primary cultured cardiomyocytes against H₂O₂-stress.3. Testosterone acutely prevents H₂O₂-induced loss of cardiomyocytes cell viabilityApplication of H₂O₂ induced a dose dependent viability loss in cardiomyocytes measured by trypan blue exclusion. Cell death was detected following application of H₂O₂ （10, 100, 1000 μmol/L） for 10 min. Treatment with 100 μmol/L H₂O₂ for 10 min has been shown to cause nearly 60% viability loss in cardiomyocytes.Pretreatment of cardiomyocytes with testosterone （T） (10^-8, 10^-7, 10^-6, 10^-5, 10^-4 μmol/L) protected the cells from the toxicity of H₂O₂, while T at final concentration of 10^-9 has no effect. It also showed that T had a dose-dependent cardioprotective effect.Application of 20 μmol/L Atr, a known opener of the mitochondrial permeability transition pore, for 20 min before H₂O₂-stress, significantly attenuated the effects of T on cell viability. Blockade of the mitoK_ATP channel with a selective inhibitor, 5-HD （100 μmol/L） for 10 min before H₂O₂-stress also abolished the effects of T on cell viability.4. Testosterone prevented H₂O₂ induced depolarization of mitochondrial membrane potentialWe observed that H₂O₂-stress significantly increased the fluorescence intensity, which indicated that the Δψm was depolarized. Pretreatment with T beforeH₂O₂-stress prevented the depolarization of Δψm, and co-treatment with Atr or 5-HD attenuated the effect of T.5. Testosterone inhibited H₂O₂ induced ROS productionWe observed that H₂O₂-stress significantly increased the fluorescence intensity, which indicated that ROS were producted largely. Pretreatment with T or CsA before H₂O₂-stress prevented the production of ROS, and co-treatment with Atr or 5-HD attenuated the effect of T.6. Effect of testosterone on contraction of isolated ventricular myocytes H₂O₂-stress （100 μmol/L） produced significant decreases of ±dL/dtmax and dL.T attenuated the alterations of cell contraction induced by H₂O₂-stress. Perfusion with 10^-5 μmol/L T 10 min prior to H₂O₂-stress significantly attenuated the inhibitory effect of H₂O₂-stress on +dL/dtmax and dL, and on -dL/dtmax at 2,6,10 min afterH₂O₂-stress. Application of 20 μmol/L Atr, a known opener of the mitochondrial permeability transition pore, for 20 min before H₂O₂-stress, significantly attenuated the effects of T on +dL/dtmax, while it had effect on -dL/dtmax only at 6 min and on dL at 8,10 min.ConclusionsIn conclusion, this study shows the chronic cardioprotection of testosterone against ischemia/reperfusion injury and acute effect against H₂O₂-stress injury. Gonadectomy significantly increases the infarct size and LDH release at the time of 5 min reperfusion. Furthermore, the acute cardioprotective effect conferred by testosterone is mediated by inhibiting opening of the mitochondrial permeability transition pore and activating the mitoKATP channel.更多还原