发形霞水母触手提取物的心脏毒性及其作用机制探讨

发布时间：2018-05-01 07:24

本文选题：发形霞水母 + 触手提取物　；参考：《第二军医大学》2013年硕士论文

【摘要】：近年来，由于气候变暖与富营养化等因素的影响，全球近海海域水母爆发日益频繁[1,2,3,4,5]，水母蜇伤成为最常见的海洋生物伤[6,7]。水母蜇伤会产生剧烈疼痛、炎症与坏死等局部症状，严重者会出现肌肉痉挛、呼吸、循环系统抑制甚至休克等全身中毒症状，急性心力衰竭被认为是水母蜇伤致死的主要原因[4,8]。研究表明水母毒素对心血管、神经、肌肉、肝、肾等多器官系统具有毒性作用，其中心血管毒性被认为是水母毒素毒力强弱的主要判断依据[9]，但迄今水母毒素心血管毒性的作用机制尚不清楚。本文中，我们首先在离体心脏水平证明发形霞水母（Cyanea capillata）触手提取物（tentacle extract, TE）具有直接的心脏毒性作用；然后对TE心脏毒性的作用机理进行了研究，发现CICR（Ca~(2+)-induced Ca~(2+)release）信号通路在TE引起的急性心功能异常中起着重要的作用，β肾上腺素受体-cAMP-PKA-RyR2-Ca~(2+)信号通路也与TE引起的急性心功能异常有关。实验方法：第一部分：离体心脏水平TE直接心脏毒性作用的研究用Langendorff-Perfused离体心脏模型监测TE作用后各种心功能指标（心率（heart rate, HR）、左心室发展压（left ventricular developed pressure, Lvdp）、左心室压上升与下降的最大变化速率（the positive and negative derivatives of left ventricularpressure,±dP/dtmax）、冠脉流量（coronary flow, CF）、左心室舒张末压（left ventricularend diastolic pressure, Lvedp））以及心电图（electrocardiogram, ECG）的变化；采用自动生化分析仪测定TE作用后心脏损伤相关酶（乳酸脱氢酶（lactate dehydrogenase,LDH）、谷草转氨酶（aspartate aminotransferase, AST）、肌酸激酶（creatine kinase, CK））的变化；并观察TE作用后心脏出现的病理学变化。预先给予不同种类的药物（二氢吡啶类Ca~(2+)通道拮抗剂硝苯地平（0.25μM）与苯烷胺Ca~(2+)通道拮抗剂维拉帕米（0.1μM），非选择性β肾上腺素受体阻断剂普奈洛尔（1μM）与非选择性α肾上腺素受体阻断剂酚妥拉明（1μM），非选择性毒蕈碱受体拮抗剂阿托品（1.5μM）或可逆性胆碱酯酶药物新斯的明（3.3μM））后，分别观察离体心脏心功能指标在TE作用后的变化。第二部分：TE心脏毒性的作用机制研究在整体动物与离体心脏水平检测TE作用后各心功能指标的变化，在心肌细胞水平观察TE对乳鼠心肌细胞形态、存活率以及细胞内心肌酶的影响。用Ca~(2+)检测试剂盒检测TE作用后心脏组织中Ca~(2+)含量的变化，激光扫描共聚焦技术检测TE作用后乳鼠心肌细胞内Ca~(2+)含量及细胞形态的变化。观察不同阳离子通道阻断剂（13种）干预后，TE对乳鼠心肌细胞存活率的影响，筛选出有效拮抗TE心肌细胞毒性的药物，进一步在离体心脏与整体动物水平验证其对TE心脏毒性的拮抗作用。然后综合分析CICR信号通路在TE心脏毒性作用中的机制。我们还在心肌细胞水平，用western blot技术检测TE作用后细胞内β1-肾上腺素受体（β1-adrenergic receptor, β1-AR）蛋白表达量的变化。酶联免疫（enzyme linkedimmunosorbent assay, Elisa）法检测TE作用后乳鼠心肌细胞内cAMP浓度的变化，以及β肾上腺素受体阻断剂（普奈洛尔、阿替洛尔、艾司洛尔）干预后TE对细胞内cAMP浓度的影响。在细胞外不含Ca~(2+)的条件下用PKA（protein kinaseA, PKA）抑制剂H89干预后观察TE对乳鼠心肌细胞存活率的影响。初步探讨β肾上腺素-cAMP-PKA-RyR2-Ca~(2+)信号通路在TE心脏毒性作用中的机制。实验结果：第一部分：离体心脏水平TE直接心脏毒性作用的研究 TE剂量分别为60、120、180、240μg时，心功能指标HR、Lvdp、±dP/dtmax、CF呈明显的剂量依赖性降低，，Lvedp呈明显的剂量依赖性增加；ECG也出现明显的剂量与时间依赖性变化。当TE剂量大于240μg时，心脏会出现严重的心律不齐（心室颤动或者高度房室传导阻断），15min后骤停。在TE剂量为180μg时，心脏损伤相关酶LDH、AST、CK明显增加；组织病理学检查发现心脏出现多种损伤：波状纤维、心肌细胞直径不规则以及间质水肿。硝苯地平与维拉帕米可以明显改善TE引起的心脏损伤，普奈洛尔与酚妥拉明可部分改善TE引起的心脏损伤，但是阿托品与新斯的明对TE引起的心脏损伤没有明显作用。第二部分：TE心脏毒性作用机制的探讨在整体动物与离体心脏水平，TE均可导致心功能指标发生明显变化；在乳鼠心肌细胞水平，TE作用后细胞的存活率呈剂量与时间依赖性下降。激光扫描共聚焦检测发现在细胞外含Ca~(2+)（1.27mmol/L）与不含Ca~(2+)的条件下，TE都可引起心肌细胞内Ca~(2+)超载，并且细胞外含Ca~(2+)条件下比不含Ca~(2+)时Ca~(2+)增加更迅速（5min），达到Ca~(2+)峰值的时间更早（约10min），细胞形态变化更明显。利用多种离子通道阻断剂对TE的心肌细胞毒性作用进行干预，发现L-型Ca~(2+)通道（L-type Ca~(2+)channel,LTCC）阻断剂（如地尔硫卓）可明显提高心肌细胞的存活率，用RyR2拮抗剂（ryanodine）干预后心肌细胞的存活率也明显提高，提示TE的心脏毒性可能与CICR信号通路有关；地尔硫卓在离体心脏与整体动物水平也可以明显拮抗TE引起的急性心功能异常，进一步说明CICR信号通路在TE的心脏毒性中起着重要作用。 TE作用心肌细胞后，细胞内β1-AR的蛋白表达量与cAMP的浓度呈明显的剂量依赖性增加，提示TE的心脏毒性可能与过度激活β肾上腺素-cAMP-PKA-RyR2-Ca~(2+)信号通路有关；β肾上腺素受体阻断剂（普奈洛尔、阿替洛尔、艾司洛尔）干预后，可以明显拮抗TE引起的心肌细胞内cAMP浓度的增加；在细胞外不含Ca~(2+)的条件下用PKA抑制剂H89进行干预，可明显提高TE作用后乳鼠心肌细胞的存活率。进一步表明β肾上腺素受体-cAMP-PKA-RyR2-Ca~(2+)信号通路可能也是TE引起心功能异常的重要作用机制之一。结论： 1．在离体心脏水平通过对TE作用后各种心功能指标、ECG变化的监测以及对心肌损伤相关酶变化的测定，证明TE对心脏有直接的毒性作用。 2．在整体动物、离体心脏、心肌细胞以及分子水平证明CICR信号通路在TE引起的急性心功能异常中起着重要作用。 3．在细胞与分子水平初步证明β肾上腺素受体-cAMP-PKA-RyR2-Ca~(2+)信号通路与TE引起急性心功能异常有关。
[Abstract]:In recent years , due to the effects of climate warming and eutrophication , jellyfish sting has become the main cause of severe pain , inflammation and necrosis .
The effect of CICR ( Ca ~ ( 2 + ) - induced Ca ~ ( 2 + ) release signal pathway plays an important role in the acute cardiac dysfunction induced by TE , and 尾 - adrenergic receptor - cAMP - protein - RyR2 - Ca ~ ( 2 + ) signal pathway is also related to the abnormal cardiac function caused by TE .

Test Method :

Part I : Study on the direct cardiotoxicity of isolated rat heart

The changes of left ventricular pressure ( 卤 dP / dtmax ) , left ventricular pressure ( 卤 dP / dtmax ) , left ventricular pressure ( 卤 dP / dtmax ) , left ventricular diastolic pressure ( Lvedp )) , and electrocardiogram ( ECG ) were monitored by using Langenstein - Perfused isolated heart model to monitor the changes of heart rate ( HR ) , left ventricular developed pressure ( LVdp ) , left ventricular pressure rise and left ventricular developed pressure ( LVdp ) , left ventricular pressure rise and left ventricular pressure ( 卤 dP / dtmax ) , coronary flow ( CF ) , left ventricular diastolic pressure ( Lvedp )) , and electrocardiogram ( ECG ) ;
The changes of cardiac injury related enzymes ( lactate dehydrogenase , LDH ) , aspartate aminotransferase ( AST ) , creatine kinase ( CK )) were measured by automated biochemistry analyzer .
The changes of cardiac function in isolated heart after TE action were observed after administration of nifedipine ( 0.25 渭M ) and phenylalkylamine Ca ~ ( 2 + ) channel antagonist verapamil ( 0.1 渭M ) , non - selective 尾 - adrenergic receptor blocker ( 1 渭M ) and non - selective 伪 - adrenergic receptor blocker phentolamine ( 1 渭M ) , non - selective muscarinic receptor antagonist atropine ( 1.5 渭M ) or reversible cholinesterase ( 3.3 渭M ) .

Part Two : Study on the Mechanism of TE Cardiac Toxicity

The effects of TE on myocardial cell morphology , survival rate and intracellular myocardial enzymes were observed after TE action . The effects of TE on the survival rate of cardiac muscle cells were detected by Ca ~ ( 2 + ) detection kit . The effect of TE on the survival rate of rat cardiomyocytes was detected by using Ca ~ ( 2 + ) detection kit .

The effect of TE on the intracellular cAMP concentration in rat cardiomyocytes was detected by Western blot . The effect of TE on the intracellular cAMP concentration was observed after the intervention of 尾 1 - adrenergic receptor ( 尾 1 - AR ) . The effect of TE on the survival rate of rat cardiomyocytes was observed after the intervention of 尾 - adrenergic receptor blocker H89 . The mechanism of 尾 - adrenergic - cAMP - protein - RyR2 - Ca ~ ( 2 + ) signaling pathway in the toxicity of TE cardiotoxicity was investigated .

Experimental results :

Part I : Study on the direct cardiotoxicity of isolated rat heart

When the doses of TE were 60 , 120 , 180 and 240 渭g , HR , LVdp , 卤 dP / dtmax , CF decreased significantly and Lvedp increased significantly .
There was also a significant dose and time - dependent change in ECG . When the TE dose was greater than 240 渭g , severe arrhythmia ( ventricular fibrillation or high AV block ) occurred in the heart . After 15 min , the cardiac injury related enzyme LDH , AST and CK were significantly increased .
It was found that there were a number of lesions in the heart : wave - like fibers , irregular diameter of myocardial cells , and interstitial edema . nifedipine and verapamil could significantly improve the cardiac injury induced by TE , but both propofol and phentolamine could improve TE - induced cardiac injury , but atropine and neostigmine had no significant effect on TE - induced cardiac injury .

Part Two : Discussion on the Mechanism of TE Cardiac Toxicity

In the whole animal and in vitro cardiac level , TE could lead to a marked change in cardiac function index .
In the presence of Ca ~ ( 2 + ) ( 1.27 mmol / L ) and Ca ~ ( 2 + ) without Ca ~ ( 2 + ) , the intracellular Ca ~ ( 2 + ) increased more rapidly ( 5 min ) than Ca ~ ( 2 + ) .
The acute cardiac function caused by TE can be significantly antagonized in the isolated heart and the whole animal level , and the CICR signal pathway plays an important role in the cardiac toxicity of TE .

The expression of 尾 1 - AR in the cells increased significantly with the concentration of cAMP , suggesting that the cardiac toxicity of TE might be related to the excessive activation of 尾 - adrenergic - cAMP - 1 - RyR2 - Ca ~ ( 2 + ) signal pathway .
The concentration of cAMP in the myocardial cells induced by TE could be significantly antagonized by the intervention of 尾 - adrenergic receptor blocker ( Pnerol , Atenolol , Esmolol ) .
Under the condition of no Ca ~ ( 2 + ) in extracellular Ca ~ ( 2 + ) , the survival rate of neonatal rat cardiomyocytes after TE was significantly increased . The results showed that 尾 - adrenergic receptor - cAMP - protein - RyR2 - Ca ~ ( 2 + ) signaling pathway might also be one of the important mechanisms of TE - induced cardiac dysfunction .

Conclusion :

1 . It is demonstrated that TE has a direct toxic effect on the heart at isolated heart levels by measuring various cardiac function indices , monitoring changes in ECG , and changes in cardiac injury - related enzymes after TE .

2 . The CICR signal pathway plays an important role in the acute cardiac dysfunction caused by TE in whole animal , isolated heart , myocardial cell and molecular level .

3 . At the level of cell and molecular level , it is shown that the 尾 - adrenergic receptor - cAMP - protein - RyR2 - Ca ~ ( 2 + ) signal pathway is related to the abnormal cardiac function caused by TE .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R595.8

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