【作者】 卓荦；

【导师】 刘良； Ling Li；

【作者基本信息】 华中科技大学 ， 法医病理学， 2013， 博士

【摘要】 【研究背景】摇头丸，其主要成分为3,4-亚甲基二氧基甲基苯丙胺（MDMA），常见于夜总会、迪吧、歌厅以及各种狂欢聚会场合，使用者多为22岁左右的年轻人。国内外研究及临床数据证实MDMA除了神经毒性之外，还具有肝毒性和心血管毒性。MDMA急性中毒可导致显著的心血管功能异常，表现为心率增加，血压增高以及心肌耗氧量增加引起高血压、心律失常、心肌缺血甚至心衰。已知的研究数据表明，MDMA可通过神经递质途径产生间接的心血管毒性作用，而其氧化代谢产物可破坏细胞抗氧化防御系统诱导氧化应激从而引起心肌细胞损伤。虽然大量的证据表明MDMA能够导致心功能异常，但是其对于心肌细胞的直接毒性作用的机制却尚未探明，MDMA是否还具有其它的心肌毒性作用仍然是未知领域。心肌缝隙连接已经被广泛证实在维持心肌的正常搏动节律中起到至关重要的作用。它能够保持心肌细胞间搏动电传导的致性和稳定性。缝隙连接蛋白的异常，包括蛋白含量，分布以及磷酸化状态的改变，都可导致致死性心律失常的发生。然而，到目前为止，尚未有研究针对MDMA诱发心律失常与心室肌细胞缝隙连接蛋白之间的关联。基于前期的理论和实验基础，我们在本课题中针对MDMA与心肌细胞缝隙连接蛋白的影响进行深入的研究，从而进步揭示MDMA直接诱导心肌毒性的分子毒理机制。【目的】1.观察MDMA急性中毒对大鼠心电功能的影响；2.观察MDMA急性中毒对大鼠心肌细胞缝隙连接蛋白43的影响；3.体外原代培养新生大鼠心室肌细胞并构建体外MDMA急性染毒模型，观察不同浓度MDMA急性染毒对于心室肌细胞CX43蛋白表达含量以及转录水平的影响；4.观察不同浓度MDMA急性染毒对于体外培养新生大鼠心室肌细胞间连接蛋白43含量以及分布的影响；5.观察不同浓度的MDMA急性染毒对体外培养新生大鼠心室肌细胞连接蛋白43磷酸化位点Ser368磷酸化状态的影响；6.观察MDMA急性染毒对于体外培养心室肌细胞内钙振荡模式以及细胞内钙离子浓度的影响。【实验方法】1.将摇头丸片剂研磨、溶解，对获得的溶液进行酸-碱反提萃取法提取MDMA有效成分，并运用气相色谱-质谱联用系统对提取物进行成分以及纯度检验；2.动物实验：将18只SPF级SD大鼠随机分为实验组和对照组。实验组大鼠腹腔注射20mg/kg MDMA后实时记录其心电图以及心率变化。对照组则给予腹腔注射无菌生理盐水后记录其心电图以及心率变化。所有大鼠在24小时后采用颈椎脱臼法处死，迅速剪取心室部分并用生理盐水冲洗干净，多聚甲醛固定、石蜡包埋并制作切片。利用H&E染色观察心肌细胞形态学变化。利用免疫组织化学法对心室肌细胞中连接蛋白43进行定量分析，并观察其分布情况。3.体外实验：选取0天或1天SPF级新生大鼠，消毒后迅速剪取心脏心室部分，利用混合组织消化液分步消化结合差速贴壁法获得纯化的心室肌细胞，置于培养箱中培养。于培养第6天，加入含有10、100、1000μM浓度的MDMA的无血清培养基进行急性染毒1小时，建立MDMA急性染毒体外模型。提取心室肌细胞总膜蛋白，利用蛋白免疫印迹法检测心室肌细胞中总连接蛋白43的表达含量的变化。同时提取细胞总RNA，利用荧光实时定量PCR法检测连接蛋白43mRNA（GJA1）表达含量的变化。4.利用免疫荧光法检测不同浓度（10、100、1000μM）的MDMA染毒的心室肌细胞缝隙连接处连接蛋白43的表达和分布改变。5.向体外培养的心室肌细胞中加入1000μM MDMA急性染毒1小时后，提取实验组和对照组心室肌细胞总蛋白，利用Western Blot法检测连接蛋白43磷酸化位点Ser368的磷酸化状态的改变，并检测心室肌细胞中PKC的含量。利用Fluo-4标记钙离子，在激光共聚焦显微镜下观察MDMA急性染毒组和对照组新生大鼠心室肌细胞内钙振荡模式的变化以及细胞内钙浓度的改变。【数据分析】所有结果采用平均值±标准差。组间比较采用双向方差分析法，两组比较采用非配对t检验法，p值小于0.05为有统计学差异。所有统计学分析采用SPSS13.0软件以及Graph prism医学统计软件。【结果】1.从摇头丸中提取的MDMA成分经质谱检验未见其它活性杂质，MDMA纯度高达95%。2.心电图结果显示急性MDMA腹腔注射后大鼠出现明显的心电图异常，表现为QRS间期延长，波幅增强，并呈现不规则变化。实时心率检测显示，实验组大鼠心率发生显著改变，大鼠心率变化起伏不定，并表现为不规则波动。实验组大鼠平均心率呈下降趋势。3.实验组大鼠心室肌细胞未见明显形态学改变，部分视野下可见心肌纤维萎缩，横纹消失，肌浆呈嗜酸性变性，散在局灶性空泡样变性。4.实验组大鼠中有2只大鼠在注射MDMA后2小时内发生自发性死亡。免疫组织化学结果显示，实验组大鼠心肌细胞连接蛋白43表达含量显著下降。自发性死亡大鼠心肌细胞可见连接蛋白43分布异常，沿心肌细胞两侧长轴方向分布。5.体外培养的新生大鼠心室肌细胞可见成片搏动。MDMA急性染毒组心肌细胞总连接蛋白43含量以及连接蛋白43mRNA表达均显著下降。免疫荧光检测见100、1000μM高浓度MDMA染毒可引起缝隙连接中连接蛋白43的含量下降及分布异常。MDMA还引起心肌细胞N-钙粘蛋白的表达显著下降。6.高浓度MDMA可导致连接蛋白43磷酸化状态改变，表现为非磷酸化状态比例上升，同时可引起磷酸化位点Ser368的磷酸化水平增加，但却对心肌细胞内PKC含量未造成影响。高浓度MDMA同时还引起心肌细胞内钙振荡模式的改变和钙离子浓度的上升。【结论】1.动物实验证实MDMA可诱发大鼠心率改变以及心电异常，甚至猝死，这些毒性作用与其引起心肌细胞连接蛋白43表达含量下降有关。2.体外实验表明MDMA可直接作用于心室肌细胞引起连接蛋白43基因转录水平以及蛋白表达显著下降，同时其引起心肌N-钙粘蛋白水平的下调与MDMA导致连接蛋白43的分布异常有关。3. MDMA可诱发心室肌细胞内钙振荡模式的改变以及细胞内钙离子浓度的增加介导相应蛋白激酶的活性引起连接蛋白43磷酸化磷酸化位点Ser368磷酸化水平升高。4.本研究首次证实了MDMA可破坏心肌细胞内钙稳态，影响心肌细胞连接蛋白43的表达、分布以及磷酸化状态，诱发心电功能障碍，从而导致心律失常的发生。这也是MDMA直接心肌毒性的潜在作用机制之。更多还原

【Abstract】 BACKGROUND3,4-methylenedioxymethamphetamine （MDMA）, also known as ‘Ecstasy’, ispopular among the young adults around25years old in night clubs and rave parties.The research and clinical data have determined that MDMA can induce not onlyneurotoxicity but also hepatoxicity and cardiovascular toxicity. Acute MDMAadministration can cause markedly cardiovascular dysfunction, including heartbeatrate, blood pressure and myocardial oxygen consumption increase, which result inhypertension, cardiac arrhythmia, myocardial infarction and even heart failure or heartarrest. Previous studies show MDMA may induce cardiotoxicity through indirect ordirect ways. Alteration of monoaminergic systems via the neurotoxic actions ofMDMA has the potential to cause cardiovascular changes. The oxidative metabolismof MDMA can also cause the oxidative stress in myocardial cells. Although plenty ofevidences have demonstrated that MDMA induce cardiac function disorder, the directeffect on the cardiomyocytes is still unclear. Whether MDMA has any othercardiotoxicity mechanisms is still unknown.Myocardial gap junctions are widely known to play a significant role in thevelocity and the safety of impulse propagation in cardiac tissue, maintaining thenormal cardiac rhythm. Changes in gap junction proteins, including proteinexpression, distribution and phosphorylation status, may lead to fetal cardiacarrhythmia. However, there is no previous study focuses on the relationship betweenMDMA-induced arrhythmia and myocardial gap junctions alterations. Therefore,based on previous research findings, we firstly investigated the effect of MDMA onmyocardial gap junction proteins in this study. The results will further disclose thedirect mechanism of MDMA-induced cardiotoxicity. OBJECTIVE1. To observe the effect on rat’s electro-cardiac function after a single dose MDMAacute administration;2. To investigate the change of myocardial gap junction connexin43proteinexpressions induced by acute MDMA administration3. To investigate the alteration of total connexin43expression and Cx43mRNA levelin the primary cultured neonatal rats’ ventricular myocytes exposed to differentconcentration of MDMA;4. To observe the changes of the amount and the distribution of junctional connxin43in cultured ventricular myocytes treated with MDMA;5. To investigate the changes of phosphorylation status in connexin43Ser368ofcultured cells after exposure to different concentrations of MDMA.6. To test the changes of intracellular Ca²⁺oscillation patterns and calciumconcentration during MDMA exposure to the cultured ventricular myocytes.METHODS1. Ecstasy tablets were grinded into powder and then added pure water to solve itcompletely. Extraction of MDMA was performed by acid-base reactions. Thecomponents and purity of product was analyzed by GC-MS methods.2. In vivo study: a total of18male SD rats （SPF grade） were divided into two groupsrandomly. The rats in experimental group were administrated by20mg/kg MDMA i.p..Subsequently, ECG was detected and recorded by ECG detector. Rats treated withsaline were used as control group. All the rats were sacrificed24hours after druginjections. The heart tissues were taken and washed by saline, and then fixed in4%paraformaldehyde for48hours prior to paraffin embedded. Sections of heart tissueswere HE stained. Immunohistochemical staining was performed to investigate theexpression and distribution of connexin43proteins.3. in vitro study: newborn rats were sterilized and anesthetized. Cardiac ventricular was excised and shredded into tissue fragments. Step-wise digestion and90minutesdifferential adhesion were performed in order to enrich the culture with ventricularmyocytes. The cells were incubation in5%CO2at37. At the6thday, cultured cellswere added into10,100,1000μM MDMA with serum-free culture medium andincubated at37for1hour in order to establish MDMA intoxication models.Total membrane proteins of ventricular myocytes were extracted by using BioVisionprotein kit. Western blot was applied to test the change of total connexin43expressions. In addition, total RNA was extracted and the mRNA level of connexin43was quantitatively analyzed by application of Real time qPCR associated with2-（Δ（ΔCt））method.4. The junctional connexin43in cultured cells exposed to MDMA （10,100,1000μM）were investigated by immunofluorescent staining in order to see the change of Cx43level and distribution.5.1000μM MDMAwith serum-free culture medium was added onto the cultured cellsand incubated for1hour. Western blot was applied in order to test the alteration ofphosphorylation status in specific phosphorylated site Ser368of connexin43, as wellas the amount of PKCalpha. Besides, the intracellular calcium was label by Fluo-4and observed under confocal laser scanning microscope. The change of Ca²⁺oscillation patterns and the intracellular Ca²⁺concentration induced by MDMA weretest.STATISTICAL ANALYSISResults are shown as mean±SEM, multiple comparisons between groups wereanalyzed by the two-way ANOVA. For two group comparisons, unpaired Student’st-test was used. A value of p<0.05was considered significant. All the statisticalanalysis were performed by using Graph prism software.RESULTS1. MDMA which was extracted from Ecstasy tablets were determined that no other pharmacological active components existed. The purity of extraction was95%.2. Abnormal ECG findings were observed in experimental rats after a single doseMDMA acute administration, which was characterized by prolonged QRS duration,enhanced amplitudes associated with irregular changes. The heart rates of rats treatedwith MDMA showed significant changes with time. The average heart rate ofexperimental rats was significant decreased compared to control group.3. H&E staining showed unremarkable morphological findings. Atrophy, eosinophilicdegeneration, myocytolysis and vacuolization were seen in the rats after MDMAadministration.4. Two rats of experimental group died suddenly nearly2hours after MDMAinjection. IHC results showed the expression of connexin43was significantlydecreased in MDMA-treated rats compared to controls. In addition, it was observed indead rats that distributions of Cx43were strewn in longitudinally orientated arraysalong the lateral interfaces instead of confining to the sites of intercalated disks.5. In vitro study, significant decrease in total connexin43expression was found inMDMA-treated cultured cells associated with the obvious down-regulation of Cx43mRNA level. Immunofluorescent detection showed high concentration of MDMA ledto significant decrease of junctional connexin43and MDMA caused parallel reductionof N-cadherin level in cultured ventricular myocytes.6. high concentration of MDMA （1000μM） also increased the non-phosphorylatedconnexin43, and induced the increase of phosphorylated Cx43at site Ser368. Therewas no significant difference in PKCalpha between MDMA-treated and controlgroups. However, intracellular Ca²⁺oscillation patterns alterations and increase ofintracellular Ca²⁺concentrations were found induced by high concentration ofMDMA.CONCLUSIONS1. A single dose of MDMA acute administration can cause reduction of myocardialconnexin43and induce changes in heart rate and ECG patterns of rats. 2. MDMA directly affects the cardiomyocytes and results in decrease of totalconnexin43and abnormal connexin43distribution which may be related to thereduction of N-cadherin induced by MDMA.3. MDMA induces the changes in intracellular Ca²⁺oscillation patterns and increaseof Ca²⁺concentration, which may activate related protein kinases and up-regulate thephosphorylated Cx43Ser368.4. Our findings provide first evidence of MDMA-mediated changes in cardiac gapjunctions that may underlie MDMA-induced cardiac arrhythmia.更多还原