【作者】 杨彩红；

【导师】 吴博威； 李青山；

【作者基本信息】 山西医科大学 ， 生理学， 2011， 博士

【摘要】 研究背景抗肿瘤药物的心脏毒性已经严重影响患者的远期生存质量,许多癌症幸存者发生心脏病的危险与癌症复发危险持平。一些恶性肿瘤幸存者有可能因心血管的并发症而影响疗效,甚至危及生命。因此,探讨抗肿瘤药物对心脑血管的影响及其作用机制无疑具有重要意义。本课题组以芳香异羟肟酸等螯合配体合成筛选得到的新型有机锡化合物二-(4-氯苯甲酰异羟肟酸)二正丁基合锡(DBDCT),具有高效广潜抗癌活性,但DBDCT对心血管系统的毒性作用日前尚未得到较全面的评估,更没有进一步的毒性作用机制分析的研究。因此,本课题的主要内容为研究DBDCT在心血管系统中的作用及其可能机制。心血管系统的毒性损伤是由多素共同作用的结果。这些因素包括自由基损伤、钙超载、线粒体损伤等,它们之间互相联系,互为因果,形成心血管毒性的恶性网络,共同引导毒性的发生和发展。细胞胞内钙离子(Ca2+)浓度变化可作为对外源性化合物侵袭的敏感信号。而钙稳态失衡被认为是所有细胞死亡的最后通路。心功能的改变和血管舒缩的变化通常是与肌细胞胞内钙浓度的变化紧密相关的,是反映细胞钙转运异常的敏感指标。我们在研究有机锡类化合物DBDCT对大鼠心脏作用的预实验过程中发现,DBDCT主要对心脏的舒张功能产生抑制,同时伴有冠脉流量的异常增加,而且在灌流冲洗后心功能继续恶化,直至心脏停跳,呈现一种不可逆的变化。这表明,在DBDCT作用下心肌和冠脉平滑肌细胞的钙转运发生了显著变化,这些变化涉及到DBDCT毒性作用的关键,值得我们进一步深入研究。本课题拟围绕细胞的钙转运问题,通过研究DBDCT对在体和离体大鼠心功能、相关的生化指标、组织形态结构、心肌细胞离子电流和细胞内钙浓度变化的影响,观测DBDCT对心脏的毒性作用及作用机制；通过观测DBDCT对冠脉流量和大鼠胸主动脉环张力的影响,以及离子通道阻断剂的干预效应,探讨DBDCT对血管毒性的作用及其机制。目的研究不同浓度DBDCT对在体大鼠心脏心功能的急性和慢性作用、相关的生化指标及组织形态结构的影响。方法1.急性给药模型制备健康成年SD大鼠,麻醉,行左心室插管手术,腹腔注射给药,给药后记录2 h。记录药前和药后左心室收缩压(LVSP)、左室舒张末压(LVEDP)、左心室主动收缩压(LVSP-LVEDP)、左室压力最大上升和下降速率(±dp/dtmax),Ⅱ导心电图,并采集血清作酶学指标测定。实验分组：1)正常对照组：腹腔注射5.0 mg/kg的同体积生理盐水。2)溶剂对照组：腹腔注射5.0 mg/kg的同体积溶剂。3)DBDCT2.5 mg/kg组：腹腔注射2.5 mg/kg DBDCT。4)DBDCT5.0 mg/kg组：腹腔注射5.0 mg/kg DBDCT。2.慢性给药模型制备健康成年SD大鼠,随机分为实验组和对照组,隔天腹腔注射给药,于给药后的第10天和第20天两个时间点,测量左室收缩压(LVSP)、左室舒张末压(LVEDP)、左心室主动收缩压(LVSP-LVEDP)及左室压力最大上升及下降速度(±dp/dtmax)、Ⅱ导心电图、体重(BW)、心室重量(VW),并作酶学指标测定和心肌组织病理切片观察其组织学变化。实验分组：1)正常对照组：隔天腹腔注射5.0 mg/kg同体积生理盐水饲养10、20天。2)溶剂对照组：隔天腹腔注射5.0 mg/kg同体积溶剂饲养10、20天。3)DBDCT2.5mg/kg 10天组：隔天腹腔注射2.5mg/kg DBDCT饲养10天。4)DBDCT5.0mg/kg 10天组：隔天腹腔注射5.0mg/kg DBDCT饲养10天。5)DBDCT2.5mg/kg 20天组：隔天腹腔注射2.5mg/kg DBDCT饲养20天。6)DBDCT5.0mg/kg 20天组：隔天腹腔注射5.0mg/kg DBDCT饲养20天。结果1. DBDCT对在体心脏的急性作用1)在小剂量(2.5mg kg)作用下左室收缩压(LVSP)、左室主动收缩压(LVSP-LVEDP)和左室最大收缩速率(+dp dtmax)均有升高,显示心功能略有增强：而大剂量(5.0mg/kg)时，上述指标均较溶剂对照组降低，表现出对心功能的抑制，两组动物的左室舒张末压(LVEDP)均有升高趋势,但无统计学意义。2)大鼠腹腔注射2.5 mg/kgf15.0mg/kg DBDCT可使血清中乳酸脱氢酶(LDH)含量由溶剂组的(707±50)U/L分别升高至(722±86)U/L和(872±106)U/L,其中后者的变化有显著性差异(P<0.01);肌酸激酶(CK)、丙二醛(MDA)含量略有升高,超氧化物歧化酶(SOD)略有降低,但与对照组比较均无统计学差异(P>0.05)。3)一次性腹腔注射DBDCT 5.0mg/kg对大鼠心电图的影响主要表现为偶发的室性早搏。2. DBDCT对在体心脏的慢性作用1)低剂量(2.5 mg/kg)的DBDCT可使心功能的各项指标普遍升高,但反映舒张功能的LVEDP在第10天和第20天均无明显变化；高剂量(5.0 mg/kg)的DBDCT对心功能则有明显的抑制作用,且随时间的延长而加重,全部指标都有显著差异,仅LVSP第10天一项例外,表明左心室主动收缩压(LVSP-LVEDP)的下降主要与LVEDP升高有关。2) DBDCT可引起血清乳酸脱氢(LDH)持续升高,2.5mg/kg 10天组和5.0mg/kg 10天组分别由溶剂组的(564±153)U/L升高到(900±103)U/L和(912±88)U/L,均有显著差异(P<0.01)；两组LDH的升高持续至第20天均无明显变化。DBDCT引起组织丙二醛(MDA)含量升高主要表在给药后的早期阶段。2.5mg/kg 10天和5.0mg/kg 10天给药后MDA含量分别由溶剂组的(59±13)nmol/mgprot明显升高至(110±37)nmol/mgprot和(112±33)nmol/mgprot,均有统计学差异(P<0.05);但给药20天后均下降至给药前水平。3)在2.5mg/kg 20天组动物可见Ⅱ导心电图电压明显增加,或者心率加快,其余各组动物心电图无明显异常。4)2.5mg/kg和5.0mg/kg 10天、20天组均未见到心重体重比与对照组比较有显著性变化。病理组织学检查未发现心脏明显病理改变,5.0mg/kg20天组偶可见有炎性细胞浸润,但细胞形态正常,纤维排列整齐,结构清楚,着色均匀,细胞核形态正常,未出现坏死破裂。目的观测DBDCT对离体心功能、酶学指标、心肌细胞离子电流和细胞内钙浓度变化的影响，探讨DBDCT对心肌毒性的作用及其机制。方法1.离体灌流心脏模型的制备健康成年SD大鼠,麻醉,开胸取出心脏,经主动脉逆行灌流台氏液,左室内置入水囊并与Powerlab及Chart6.0生物信号数据记录及分析系统连接,维持左心室舒张末期压力(LVEDP)2-10mmHg。心脏稳定搏动的标志为：左室主动收缩压(LVSP-LVEDP)大于60mmHg, HR大于200次/分。平衡30min后,持续给药30min,然后恢复正常台氏液灌流30 min。测量左室收缩压(LVSP)、左室舒张末压(LVEDP)、左心室主动收缩压(LVSP-LVEDP)及左室压力最大上升及下降速率(±dp/dtmax)、冠脉流量,并采集灌流液作酶学指标测定和心肌组织病理切片观察其组织学变化。实验分组：正常对照组：台氏液灌流持续灌流1.5小时。2)溶剂对照组：台氏液灌流稳定30 min,然后灌注已加入1×10-5M同体积溶剂的台氏液30 min,恢复正常台氏液灌流30 min。3) DBDCT5×10-6M组：台氏液灌流稳定30 min,然后灌注已加入DBDCT终浓度为5×10-6M的台氏液30 min,恢复正常台氏液灌流30 min。4) DBDCT1×10-5M组：台氏液灌流稳定30 min,然后灌注已加入DBDCT终浓度为1×10-5M台氏液30 min,恢复正常台氏液灌流30 min。5) L-NAME组：台氏液灌流稳定30 min,然后灌注已加入L-NAME (100μmol/L)的台氏液30 min,恢复正常台氏液灌流30 min。6) L-NAME组+DBDCT 5×10-6M组：台氏液灌流稳定30 min,然后灌注已加入L-NAME (100μmol/L)和5×10-6M DBDCT的台氏液30 min,恢复正常台氏液灌流30 min。7) L-NAME组+DBDCT 1×10-5M组：台氏液灌流稳定30 min,然后灌注已加入L-NAME (100μmol/L)和1×10-5M DBDCT的台氏液30 min,恢复正常台氏液灌流30 min。2.单细胞离子电流的测定选健康成年雄性SD大鼠,用胶原酶法急性分离大鼠左心室肌细胞,采用全细胞膜片钳技术,在电压钳模式下记录心室肌细胞ICa、Ito、INa和INa/Ca,分别观测10-7M、1×10-6M、3×10-6M、5×10-6M、8×10-6M、10-5M六个浓度浓度DBDCT对上述离子电流的影响。3.大鼠心室肌细胞钙瞬变和静息钙浓度的测定用胶原酶法急性分离大鼠心室肌细胞，Fluo-3 AM或Fluo-4 AM染色，采用IonOptix单细胞动缘同步检测系统同步监测细胞收缩舒张功能和钙瞬变的变化：在激光共聚焦显微镜下选择细胞状态良好的视野,在有钙和无钙细胞外液的条件下,观测DBDCT 1×10-5M,引起的细胞荧光强度的变化，以及肌浆网RyR组断剂ryanodine 100μmol/L和肌浆网钙泵抑制剂thapsigargin 5μmol/L干预后的效应结果1. DBDCT对心功能具有显著的抑制作用，特别是对舒张功能的抑制更为明显，LVEDP的升高最敏感也最显著,且随剂量的增加而加重。值得注意的是台氏液灌流冲洗期间药物继续发挥作用,心功能继续下降。高浓度组出现心脏停跳在收缩期的现象。2.低浓度(5×10-6M)和高浓度(1×10-5M)DBDCT灌流组漏出液中LDH含量均明显升高,由正常的(43±5)U/L分别升高至(127±20)U/L和(11 1±11)U/L;台氏液复灌冲洗30min,分别下降至(100±19)U/L和(129±12)U/L,但仍显著高于给药前的基础值(P<0.01)3.病理组织学检查发现,DBDCT引起离体灌流心肌组织明显的病理改变。DBDCT5×10-6M和1×10-5M给药组心肌纤维排列尚整齐,但局部着色不均匀,细胞核脱失。4.在离体灌流心脏中,DBDCT可明显增加冠脉流量。在恒压灌注下,DBDCT 5×10-6M和1×10-5M分别使冠脉流量由给药前的(12.2±0.7)ml/min升高至(22.9±1.5)ml/min和(24.0±1.5)ml/min,台氏液灌流冲洗后冠脉流量继续升高,分别达(24.8±1.1)ml/min和(24.6±1.1)ml/min,上述升高的流量值与给药前比较均有显著性差异(P<0.01)。一氧化氮合酶抑制剂L-NAME与DBDCT 1×10-5M同时应用时,冠脉流量增加至(25.4±1.1)ml/min,与DBDCT单独应用增加流量的作用持平。表明DBDCT增加冠脉流量的效应主要是通过药物直接舒张血管平滑肌的作用产生的。DBDCT对血管平滑肌的作用在灌流冲洗的30min内持续存在5.5×10-6M、8×10-6M和10-5M的DBDCT对ICa-L电流产生剂量依赖性地抑制作用,使ICa-L电流密度从给药前的41.75±4.60(pA/pF)分别减少到27.57±4.83(pA/pF)(33.75%)、21.82±4.88 (48.54%)(pA/pF)和17.56±4.34 (58.71%)(pA/pF)(P＜0.05)。冲洗后3min电流无恢复的趋势。DBDCT对Ito电流产生剂量依赖性地抑制作用。DBDCT 5×10-6M、8×10-6M和10-5M可分别使Ito电流密度减少49.56%、72.42%和73.15%(P<0.05)。冲洗后3min,电流无恢复的趋势。DBDCT对INa电流产生轻度抑制作用,但无统计学差异。DBDCT对对内向和外向的INa／Ca均无明显影响。6. DBDCT无论在胞外灌流液有钙和无钙条件下,均可明显增加静息心肌细胞胞内钙浓度,表明DBDCT增加心肌细胞胞静息钙浓度的作用不依赖于胞外Ca2+。与静息状态下胞内钙的基础值相比,在含钙液条件下,DBDCT 10-5M作用5 min时钙浓度增加(16.39±2.71)%(P<0.05),15 min后进一步增加至(23.55±1.29)%;在无钙条件下,DBDCT 10-5M 5min和15 min则分别增加胞内钙浓度(4.83±2.02)%和(23.55±2.20)%(P<0.05)。RyR阻断剂ryanodine 100μmol/L完全阻止了上述变化，提示DBDCT使胞内钙度增加的作用有赖于ryanodine受体。进一步的研究表明，无钙胞外液中加入肌浆网钙泵抑制剂thapsigargin 5μmol/L进一步增强了DBDCT增加胞内钙浓度的效应，5min和15min时分别增加(9.52±2.13)%和( 29.37±3.36)%,特别是在作用5 min时,DBDCT与thapsigargin合用较DBDCT单独应用的效应增加了一倍( 9.52%对4.83%)。这提示DBDCT对肌浆网钙泵亦有激动作用，而且在早期(5 min)的激动作用比晚期(15 min)强。无钙胞外液中同时加入ryanodine和thapsigargin,DBDCT 5 min后使胞内钙浓度降低(14.38±5.34)%,15min后又使钙浓度升高(15.71±4.81)%,同样支持上述推论。7.DBDCT明显抑制搏动心肌细胞的钙瞬变和收缩。DBDCT 10-5M使心肌细胞钙瞬变峰值减小到基础值的(90.64±2.97)%,细胞收缩幅度下降到基础值的(80.42±2.52)%,与基础值比较均具有显著性差异。目的通过观测DBDCT对大鼠胸主动脉环张力的影响,以及离子通道阻断剂的干预效应,探讨DBDCT对血管毒性的作用及其机制。方法采用离体血管张力实验方法。观察DBDCT在5×10-6M,1×10-5M浓度时,对氯化钾(KC1,60 mmol/L)诱发大鼠离体胸主动脉环收缩的影响以及在5×10-6M浓度时,对去甲肾上腺素(NE,1×10-6 mol/L)诱发大鼠离体胸主动脉环收缩的影响。观察内皮、一氧化氮合酶(eNOS)抑制剂N-硝基-L-精氨酸甲酯(L-NAME.10-4 mol/L).环氧合酶抑制剂吲哚美辛(indometacin,10-5 mol/L)、KV通道阻断剂四胺基吡啶(4-AP,1×10-3 mol/L)、KATP通道阻断剂格列苯脲(Gli,1×10-5 mol/L)、KCa通道阻断剂四乙胺(TEA,1×10-2 mol/L)、KiR通道阻断剂氯化钡(BaCl2,1×10-3 mol/L)对DBDCT作用的影响,以及DBDCT对血管环外钙依赖性收缩和内钙依赖性收缩的影响。结果1.DBDCT在5×10-6M,1×10-5M浓度时对KCl(60mmol/L)、NE(1×10-6 mol/L)预收缩的离体胸主动脉环均产生显著的舒张作用，与溶剂对照组相比,张力变化有显著统计学差异(P<0.01)。同浓度DBDCT对KCl(60mmol/L)、NE(1×10-6 mol/L)预收缩的去内皮离体胸主动脉环也产生显著的舒张作用,与内皮完整组相比，张力变化无统计学差异(P>0.05)。2.用L-NAME、Indo、Gli、4-AP预处理的血管环对DBDCT的舒张反应与未经处理时比较无显著性差异(P＞0.05)TEA、BaCl2预处理可减弱DBDCT对血管环的舒张作用(P<0.05)。3.在无钙PSS液中，分别加入DBDCT 5×10-6 mol/L及溶剂，孵浴20min后，加入NE10-6mol/L,血管环产生迅速而短暂的收缩,溶剂组和DBDCT 5×10-6M组的NE的收缩百分比分别为(20.44±1.60)%、(1.89±0.32)%,DBDCT 5×10-6M组与溶剂组比较有统计学差异(P<0.01)。待其舒张并平稳后,加入CaCl22.5 mmol/L,血管环再次收缩,溶剂组和DBDCT5×10-6M组的CaCl2的收缩百分比分别为(91.87±2.16)%、(1.60±0.48)%, DBDCT 5×10-6M组与溶剂组比较有统计学差异(P<0.01)。结果显示DBDCT(5×10-6M)对NE诱发的内钙释放和外钙内流引起的收缩均有明显的抑制作用。结论1.二-(4-氯苯甲酰异羟肟酸)二正丁基合锡(DBDCT)对大鼠心脏具有与剂量和时间相关的毒性作用。表现为心功能抑制并逐渐加重,特别是对舒张功能的抑制,血清LDH和组织MDA升高,以及心肌组织的炎性浸润。与已有的文献结果相比,其心脏毒性作用相对阿霉素较弱。2. DBDCT可剂量依赖性地抑制大鼠心肌ICa-L,同时对肌浆网ryanodine受体有很强的激动作用,后者可能是产生胞内钙持续升高和心肌舒张功能减退的主要原因。3. DBDCT可显著增加冠脉流量,这一作用不被一氧化氮合酶抑制剂L-NAME阻断,表明该药物对冠脉平滑肌可能具有直接的舒张作用。4. DBDCT对KCl和NE诱发的大鼠胸主动脉环的收缩均具有舒张作用,其舒张反应无内皮依赖性,与内皮生成的NO和PGI2均无关,其舒张作用可能是直接作用于血管平滑肌,与激活KCa和KiR通道,抑制钙内流和肌浆网钙释放有关。更多还原

【Abstract】 BackgroundThe cardiovascular toxicity of anticancer agents can lead to serious complications in tumor patients and impact their long-term survival quality. Many cancer survivors will actually be at as great a risk from the involved cardiac disease as from the recurrent of cancer. Some necessary therapies for them may be encumbered with their cardiovascular complications. Therefore, the antitumor diorganotin compounds with relative low toxicity have been paid more attentions. Dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) (DBDCT) is a new dioganotin (Ⅳ) arylhydroamate complex with 4-chlorobenzohydroxamic acid as a ligand which shows high antitumor activity in vivo and in vitro. However, it still does not get into clinical trail due to the remaining necessary researches and its toxicity on cardiovascular system which have ever not evaluated yet. In this research, the toxic effects of dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) as a organotin compounds on cardiovascular system and the mechanisms underlying them were studied.Various factors were implicated in the cardiovascular toxicity of antitumor agents. In general, free radical induced by oxidative stress, calcium overload and mitochondrial damage were to be the crucial events and they formed a network.with action each other, which aggravated further the damage to heart and vessels. Among them, the intracellular calcium overload was considered as a pivotal and sensitive event to exogenous compounds invasion, being the common pathway of cell damage and death.The changes of cardiac function and vascular tone were related closely with intracellular calcium concentration and could be as the sensitive index for abnormal calcium handling. During our pilot experiment, we found that DBDCT inhibited cardiac function and deteriorated particularly cardiac diastolic function and aggravated continuously after washing that, showing an irreversible change. In the meantime, DBDCT caused evident increase of the coronary flow, suggesting its relaxed action on coronary. The mentioned results mean that DBDCT induced salient changes of calcium handling in myocardium and vascular smooth muscle, which may be the pivotal step in its toxic effects on heart and vessels. In this study, our work would focus in calcium handling and relative events underlying it in order to explore the toxic effects of DBDCT on cardiovascular system and analysis their mechanisms. The cardiac function in vivo and in vitro, biochemical index, tissue morphology, myocardial ionic currents and intracellular calcium concentrations were observsed. We also observsed the changes of the coronary flow and the tone of aortic rings induced by DBDCT, combinating with the ion channel blockers to search for mechanism of them.The study is divided into three parts as below.Objective:In order to confirm the effects of DBDCT at different concentrations on rat heart we observsing the changes of cardiac function, biochemical index and morphology structure.Methods:1. Preparation of acute administrationAfter the adult Sprague-Dawley rats were anesthetized, left ventricular cannulation was performed to detected the cardio funtion indexes such as left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVSP-LVEDP),±dp/dtmax, and the standard limbⅡ-leads of the ECG were recorded. All of the above indexes were recorded for 2 h before and after administrated (intraperitoneal injection) DBDCT or vehicle or saline. Serum samples were collected for detection of enzyme targets.The experimental groups were divided as follows:1) The control group:intraperitoneal injection at 5.0 mg/kg dose of the same volume of saline.2) The vehicle group:intraperitoneal injection at 5.0mg/kg dose of the same volume of vehicle.3) DBDCT 2.5 mg/kg group:intraperitoneal injection of 2.5 mg/kg DBDCT.4) DBDCT 5.0 mg/kg group:intraperitoneal injection of 5.0 mg/kg DBDCT.2. Preparation of chronic administrationHealthy adult SD rats were randomly divided into experimental and control group, intraperitoneal injection every other day. Left ventricular systolic pressure (LVSP). left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVSP-LVEDP),±dp/dtmax. the standard limbⅡ-leads of the ECG. body weight (BW), ventricular weight (VW), and zymologic index of myocardial tissue, histological changes were observed at the first 10 days and the first 20 days after administration of drugs.The rats were randomly divided into 6 groups as follows:1) The control group:intraperitoneal injection the same volume saline of 5.0 mg/kg 5 times and 10 times every other day.2) The vehicle group:intraperitoneal injection the same volume vehicle of 5.0 mg/kg 5 times and 10 times every other day.3) DBDCT 2.5 mg/kg 10 days group:intraperitoneal injection 2.5 mg/kg DBDCT 5 times every other day.4) DBDCT 5.0 mg/kg 10 days group:intraperitoneal injection 5.0 mg/kg DBDCT 5 times every other day.5) DBDCT 2.5 mg/kg 20 days group:intraperitoneal injection 2.5 mg/kg DBDCT 10 times every other day.6) DBDCT 5.0 mg/kg 20 days group:intraperitoneal injection 5.0 mg/kg DBDCT 10 times every other day.Results:1. The acute effect of DBDCT on heart in vivo1) A slightly enhanced cardiac function was observed during administrating DBDCT at the low dose (2.5mg/kg), showing a slight increase in left ventricular systolic pressure (LVSP), left ventricular developed pressure (LVSP-LVEDP) and+dp/dtmax. In contrast, above parameters were lower significantly than vehicle group in high dose (5.0mg/kg) group, showing obvious decrease of cardiac function. Noteworthy is the left ventricular end diastolic pressure (LVEDP) in both two groups were increased though have no statistics significant vs vehicle group.2) After administrating 2.5 mg/kg and 5.0 mg/kg DBDCT intraperitoneally, the changes of zymologic index are following:lactate dehydrogenase (LDH) content in serum were increased to (722±86) U/L and (872±106) U/L(P<0.01), respectively; creatine kinase (CK) and malondial-dehyde (MDA) increased slightly; superoxide dismutase (SOD) decreased slightly (P>0.05).3) Change of ECG showed occasionally the premature ventricular contractions after administrating 5.0 mg/kg DBDCT trapentoneally.2. Chronic effect of DBDCT on heart in vivo1) The index of cardiac function generally increased by low dose (2.5 mg/kg) DBDCT, but the LVEDP in 10 days and 20 days have no significant change. In contrast, the cardiac function is significantly inhibited and is getting deterioration with time by high dose (5.0 mg/kg) DBDCT. All index have significant differences comparing with vehicle group besides the LVSP at the first 10 days, indicating the decrease of left ventricular developed pressure (LVSP-LVEDP) mainly related to the increasing LVEDP.2) Serum lactate dehydrogenase (LDH) caused by DBDCT 2.5 mg/kg 10 days and 5.0 mg/kg 10 days were significantly rised from (564±153) U/L to (900±103) U/L(P<0.01) and (912±88) U/L (P<0.01), respectively, and keeped at this level in the twentieth day. DBDCT caused increase of tissue malondialdehyde (MDA) mainly in the early stages after administrating DBDCT from (59±13) nmol/mgprot to (110±37) nmol/mgprot (2.5 mg/kg 10 days) (P<0.05) and (112±33) nmol/mgprot (5.0 mg/kg 10 days)(P<0.05). But it could recovery to the level before administration at the twentieth days.3) Increase of the voltage and the heart rate inⅡlead ECG can be observed at 2.5 mg/kg 20 day group, and the remaining groups had no significant change in ECG.4) There were no significant changes of VW/BW in all groups of animal. Pathological examination demonstrated that the myocardial tissue have also no obvious abnormal. Occasionally we can see infiltration of inflammatory cells at 5.0 mg/kg 20 days group.Objective:To explore the mechanisms of myocardial toxicity by DBDCT, we observed the changes of cardiac function in vitro, myocardial ion currents and intracellular calcium concentration after administration of DBDCT.Methods:1. Preparation of perfused heart of rat in vitroAfter heparinization and anesthetization, the rat hearts were quickly removed and mounted on Langendorff aortic retrograde perfusion system. Water balloon that connectioned with the biological signaling recording and analysis system of Powerlab and Chart 6.0 Data was put into the left ventricular to detect the cardio funtion indexes such as left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP). left ventricular developed pressure (LVSP-LVEDP),±dp/dtmax and coronary flow was recorded. Water balloon maintained left ventricular end diastolic pressure (LVEDP) at 2-10mmHg. Left ventricular developed pressure (LVSP-LVEDP) greater than 60mmHg and HR>200 times/min were considered the steady condition of the heart. After 30 min equilibrium, administrated drugs for 30 min, and then perfusion Tyrode’s solution 30 min. Perfusate and heart tissue were collected to observe zymologic index and histological changes.The rats were randomly divided into 7 groups as follows:1) The control group:perfusion with Tyrode’s solution for 1.5 hours.2) The vehicle group:administerate the same volume of vehicle as 1×10-5M in the Tyrode’s solution.3) DBDCT 5×10-6M group:administerate 5×10-6M DBDCT in the Tyrode’s solution.4) DBDCT 1 x 10-5M group:administerate 1×10-5M DBDCT in the Tyrode’s solution.5) L-NAME group:administerate L-NAME (100μmol/L) in the Tyrode’s solution.6) L-NAME group+DBDCT 5×10-6M group:administerate L-NAME (100μmol/L) and 5×10-6M DBDCT in the Tyrode’s solution.7) L-NAME group+DBDCT 1×10-5M group:administerate L-NAME (100μmol/L) and 1×10-5M DBDCT in the Tyrode’s solution.2. Record the effects of ion channel and transporter.Single rat ventricular myocyte was obtained by enzymatic dissociation procedure with collagenase. Using whole cell recording and voltage-clamp mode, the effects of different dose DBDCT (10-7M、1×10-6M、3×10-6M、5×10-6M、8×10-6M、10-5M)on ICa、Ito、INa and INa/Ca, were recorded.3. Calcium transient and resting calcium concentration determination in rat ventricular myocytesSingle rat ventricular myocyte was obtained by enzymatic dissociation procedure with collagenase, then staining with Fluo-3 AM or Fluo-4 AM. Using IonOptix single cell synchronous motion edge detection system to monitor cell synchronization systolic and diastolic function and calcium transient; At a condition of calcium Tyrode’s solution and calcium-free Tyrode’s solution, we observed the changes in fluorescence intensity caused by DBDCT 1×10-5 and with the intervention of sarcoplasmic reticulum RyR blocker ryanodine 100μmol/L and sarcoplasmic reticulum calcium pump inhibitor thapsigargin 5μmol/L in the laser confocal microscope view.Results:1. In the isoladed and perfused heart, DBDCT inhibited cardiac function significantly with a concentration dependence, especially on diastolic function. The rise of LVEDP is the most salient and sensitive, so that the arrest in contracture could be observed in high concentration groups. In addition, during the period of washout of drug, the cardiac function was deteriorated continuously rather than recovered.2. LDH in perfusate of low concentration (5×10-6M) and high concentration (1×10-5M) DBDCT were significantly increased from (43±5) U/L to (127±20) U/L and (111±11) U/L, respectively. After washout of drug, LDH was reduced, but still significantly higher than that before administering (P<0.01).3. Histopathological examination revealed that DBDCT caused obvious pathological changes of myocardium, including nuclear loss and color inequality.4. DBDCT could increase coronary blood flow in vitro. Under constant pressure perfusion, DBDCT 5×10-6M and 1×10-5M increased coronary flow before the administration from (12.2±0.7) ml/min to (22.9±1.5) ml/min and (24.0±1.5) ml/min, respectively. It continues to increase coronary flow to (24.8±1.1) ml/min and (24.6±1.1) ml/min after washing drug with Tyrode’s solution.Combining application of nitric oxide synthase inhibitor L-NAME and DBDCT 1×10-5M caused the coronary blood flow increased to (25.4±1.1) ml/min that is the same level as the DBDCT did alone. It suggested that the increase in coronary flow induced by DBDCT was mainly result from the directly relaxing effect of drug on vascular smooth muscle.5. DBDCT at 5×10-6M,8×10-6M and 10-5M could concentration-dependently decreased ICa-L and Ito. DBDCT at 5×10-6M,8×10-6M and 10-5M decreased the current density of ICa-L before the administration from 41.75±4.60 (pA/pF) to 27.57±4.83 (pA/pF) (33.75%),21.82±4.88 (48.54%) (pA/pF) and 17.56±4.34 (58.71%) (pA/pF) (P<0.05), and made the based current density of Ito decrease of 49.56%、72.42%和73.15%(P<0.05). Both of ICa-L and Ito were not recovered after washing 3 min.Meanwhile, DBDCT at different concentration have no effect on INa and INa/Ca.6. DBDCT can significantly increase the resting intracellular calcium concentration of cardiac myocytes in both calcium-containing and calcium-free perfused solution. DBDCT 10-5M in the calcium-containing solution 5 min and 15 min could increase calcium concentration of (16.39±2.71)% and (23.55±1.29)%(P<0.05), respectively; DBDCT 10-5 M in the calcium-free conditions 5 min and 15 min could increased intracellular calcium concentration of (4.83±2.02)% and (23.55±2.20)%(P<0.05). This results suggesting that the increase intracellular resting calcium concentration by DBDCT was independent of extracellular Ca2+ Meanwhile, the increase of intracellular calcium concentration induced by DBDCT could be preventing completely by RyR blocker ryanodine 100μmol/L, which suggesting the increase of intracellular calcium concentration by DBDCT depends on the role of ryanodine receptors. Further investigation showed that in calcium-free extracellular solution, adding thapsigargin, a sarcoplasmic reticulum (SR) calcium pump inhibitor, and 15 min could enhance the increased effects of DBDCT on intracellular calcium concentration. Combining applycation of 10-5M DBDCT and 5μM thapsigargin at 5 min after administering could double the effects of DBDCT alone (9.52% vs.4.83%). This result suggests that SR calcium pump might be excited by DBDCT as well. Furthermore, the exciting effect of DBDCT on SR calcium pump showed a time phase that is stronger in the early time (5 min) but weaker in the late time (15 min). This conclusion was supported by the fact that DBDCT in the calcium-free solution containing both ryanodine and thapsigargin could decline the intracellular calcium concentration (-14.38±5.34)% at 5 min after administering while elevate it (15.71±4.81)% at 15min after administering.7. DBDCT significantly inhibited the calcium transient and contraction of myocardial cells. The peak of calcium transient in myocardial cells is reduced to (90.64±2.97)% of baseline and the amplitude of cell contraction was decreased to (80.42±2.52)% of baseline after administering DBDCT 10-5M.Objective:The study was designed to observe the impact of dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) (DBDCT) to aortic rings to investigate the effects and the possible mechanism(s) of DBDCT on isolated thoracic aorta rings.Methods:The rat was sacrificed by cervical vertebra luxation and the thoracic aorta was quickly isolated from each animal and placed in cold physiological salt solution (PSS) to be used in vascular tone of isolated thoracic aortic experiments. Isotonic tension of thoracic aortic rings pre-contracted by KCl (30 mmol/L) or NE (10-6 mol/L) was recorded. The relaxant effects of DBDCT in 5×10-6 mol/L,1×10-5 mol/L and effects of various drugs include endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME.10-4 mol/L), cyclooxygenase inhibitor indomethacin (Indo, 10-5mol/L), KCa channel blocker tetraethylammonium (TEA, 1×10-2 mol/L). KA channel blockers 4-aminopyridine (4-AP, 10-5mol L), were observed in the rings. The influence of KATP channel blocker glibenclamide (Gli,1×10-5 mol L), barium chloride (BaCl2,10-5 mol/L) on the effects of DBDCT were also studied. Results:1. DBDCT (5×10-6 M,1×10-5 M) concentration-dependently attenuated the contraction induced by KC1 (60 mmol/L) and NE (1×10-6 mol/L) in intact endothelium and denuded endothelium of isolated thoracic aorta. There was significant difference in the results campared with vehicle group (P<0.01), but in that between the rings with intact and denuded endothelium at the concentrations of DBDCT (P>0.05).2. The relaxant effects of DBDCT have no significant difference between pretreated and untreated with L-NAME, Indo, Gli, and 4-AP (P>0.05). The relaxant effects of DBDCT on vascular rings were attenuated by pretreated with TEA, BaCl2(P<0.05).3. DBDCT 5X 10-6mol/L and the vehicle were administrate in the calcium-free PSS solution, after 20min incubation bath, a rapid and short-term contraction of vascular ring were produced by NE 10-6 mol/L, the percentage of NE contraction in vehicle group and DBDCT 5×10-6M group was (20.44±1.60)% and (1.89±0.32)%, respectively. The amplitude inhibited by DBDCT 5×10-6mol/L group was significantly larger comparing with that by vehicle group (P<0.01). The contraction of vascular ring was produced again by adding CaCl2 2.5 mmol/L in the same condition, the percentage of contraction produced by CaCl2 in vehicle group and DBDCT 5×10-6 mol/L group were (91.87±2.16)% and (1.60±0.48)%, respectively. The amplitude inhibited by DBDCT 5×10-6 mol/L group was significantly larger comparing with that by vehicle group (P< 0.01). The results showed that DBDCT (5×10-6 mol/L) inhibited the contractions induced both by NE-induced calcium release and calcium influx.Conclusion:1. dibutyldi-(4-chlorobenzohydroxamato) tin (Ⅳ) (DBDCT) displays a dose and time-dependent toxicity on the rat heart, including gradually aggravating deterioration of the cardiac function, especially in diastolic function, the increase of LDH and MDA, and inflammatory infiltration of myocardial tissue. However, its toxicity is still lighter comparing with that of doxorubicin in the existing document.2. ICa-L was inhibited by DBDCT dose-dependently, while the sarcoplasmic reticulum ryanodine receptor can be intensely excited, which may be responsible for the persistent elevation of intracellular calcium and geting deterioration of diastole.3. DBDCT can significantly increase the coronary flow. This effect was not inhibited by L-NAME. indicating that the drugs may have a direct relaxation on coronary artery smooth muscle.4. DBDCT attenuated the contraction induced by KCl and NE in intact endothelium and denuded endothelium of isolated thoracic aorta. The relaxtion of thoracic aortic rings by DBDCT were independent of endothelium as well as the NO and PGI2 released by it. This effect may be a result from a direct action on vascular smooth muscle and implicate the activation of KiR and KCa currents and the inhibition of calcium influx and sarcoplasmic reticulum calcium release.更多还原