牛磺酸在大鼠急性心肌缺血模型中心肌保护作用的机制研究

【作者】 张悦；

【导师】 葛均波；

【作者基本信息】 复旦大学 ， 内科学， 2013， 博士

【摘要】 背景急性心肌缺血是目前最为常见的致死性心血管疾病。尽管目前的再灌注治疗能够部分挽救心肌缺血、顿抑心肌,但已损伤心肌的修复困难加上缺血再灌注引起的新的心肌损伤使得即使接受再灌注治疗的患者仍然会有一部分出现心力衰竭和左心室重构。因此在给予再灌注治疗的同时给予心肌保护药物可能会起到锦上添花的作用。牛磺酸(Taurine, Tau)在机体内是含量最丰富的游离氨基酸,能够通过抑制细胞凋亡、稳定细胞膜、抗脂质过氧化损伤、清除氧自由基、调节细胞渗透压、维持细胞内钙稳态等可能的机制保护心肌细胞。牛磺酸通过细胞膜上的牛磺酸转运体(taurine transporter,TAUT)进行转运,TAUT对于维持细胞内牛磺酸的高浓度起着至关重要的作用。冠脉搭桥手术以及高血压可以引起心肌细胞内牛磺酸含量的减少,其具体机制尚不清楚。那么在急性心肌缺血的的病理状态下,心肌细胞内的牛磺酸水平会不会发生改变,补充外源性的牛磺酸能否提升细胞内的牛磺酸水平从而达到对心肌细胞的保护作用,本研究旨在对上述问题进行探索,并且进一步深入探究其可能的机制,为牛磺酸在临床中的应用打下理论基础。方法构建大鼠急性心肌缺血模型,并给与外源性补充牛磺酸治疗,观察缺血后不同时间点心肌组织中牛磺酸含量的改变以及补充牛磺酸能否增加细胞内的牛磺酸含量,并探究具体的机制。利用H9C2细胞株进行体外实验,观察不同缺糖和缺氧时间点心肌细胞内牛磺酸含量的改变,以及不同浓度牛磺酸治疗对于缺糖缺氧损伤的心肌细胞内牛磺酸含量和细胞凋亡的影响,并进一步探究牛磺酸浓度改变的可能机制。结果急性心肌缺血引起心肌组织结构的破坏,导致心肌细胞凋亡的增加,外源性补充牛磺酸能够减少缺血引起的心肌组织的结构破坏和心肌细胞的凋亡,改善缺血引起的心脏功能减弱。心肌细胞在缺糖和缺氧30mmin后即出现细胞凋亡的增加和增殖的减少,补充低剂量牛磺酸能减少缺氧引起的心肌细胞凋亡并逆转缺糖和缺氧导致的心肌细胞增殖减少。低剂量牛磺酸预处理减弱了缺糖引起的细胞氧化应激,抑制了缺糖诱导的心肌细胞线粒体膜电位的破坏,减弱了缺糖诱导的ER/SR介导的凋亡通路,并减少了缺糖诱导的UPR相关蛋白表达的增加。急性心肌缺血大鼠心肌组织内的牛磺酸浓度较对照组明显减少,大鼠血浆中的牛磺酸浓度明显增加,外源性补充牛磺酸能够增加心肌组织中的牛磺酸浓度。急性心肌梗塞患者血浆中的牛磺酸浓度也较对照组明显升高。体外实验显示低剂量牛磺酸逆转缺糖和缺氧引起的心肌细胞内牛磺酸含量的降低。急性心肌缺血大鼠建模2h后心肌细胞内CDO表达较对照组有所减少,建模0.5h和2h后CSD表达以及建模0.5h后CDO表达均没有变化,缺糖损伤的心肌细胞在缺糖60mmin后CDO和CSD表达都有所减少,而缺氧损伤的心肌细胞在不同缺氧时间点CDO和CSD的表达都没有变化。急性心肌缺血大鼠心肌细胞TAUT的表达明显下调,外源性补充低剂量牛磺酸能够明显上调TAUT的表达。体外实验显示缺糖和缺氧损伤下调心肌细胞TAUT的表达,而低剂量牛磺酸预处理能够上调TAUT的表达。利用TAUT过表达以及TAUT敲低的心肌细胞证实了TAUT表达的高低对于细胞内牛磺酸浓度的影响以及对于细胞增殖能力的影响。低剂量牛磺酸上调TAUT表达的机制可能与其逆转了缺血缺氧引起的TAUT上游的调节基因TonEBP表达的下降有关。结论：急性心肌缺血以及缺糖缺氧损伤时心肌细胞内牛磺酸含量在缺血以及缺糖缺氧损伤早期(30min)即明显减少,这可能与急性心肌缺血和缺糖缺氧损伤下调心肌细胞内TonEBP表达从而下调心肌细胞TAUT表达有关,TAUT表达的下调减少了牛磺酸向心肌细胞内的转运。外源性补充低剂量牛磺酸对于急性心肌缺血的大鼠心肌细胞和缺糖缺氧损伤下的心肌细胞有保护作用,明显减少心肌细胞的凋亡。外源性补充低剂量牛磺酸明显增加了心肌细胞内牛磺酸的含量,这可能是通过上调心肌细胞内TonEBP的表达从而上调TAUT的表达,使得牛磺酸向心肌细胞内的转运明显增加。应用价值：本研究阐明了急性心肌缺血的病理状态下,心肌细胞内牛磺酸含量的变化,提出了心肌细胞内牛磺酸浓度可能成为急性心肌梗塞的一个新的诊断指标。并验证了外源性补充低剂量牛磺酸对于心肌细胞的保护作用,且解释了外源性补充低剂量牛磺酸增加心肌细胞内牛磺酸含量的可能机制,为牛磺酸早日在临床上使用以及其使用剂量提供了理论依据。创新点：首次发现不同浓度的牛磺酸对于心肌细胞TAUT表达有着不同的调节功能。首次提出心肌细胞内牛磺酸浓度有望成为急性心肌梗塞的早期诊断指标。更多还原

【Abstract】 BackgroundInducing arrhythmias and even heart failure, acute myocardial ischemia (AMI) is one of the most serious cardiovascular events and the most common cause of mortality worldwide. Although percutaneous coronary intervention (PCI) has been one of the usual therapeutic interventions for coronary heart disease, ischemia reperfusion injury will still lead to the myocardial damage. So supplementation of myocardial preservation drug along with percutaneous coronary intervention will have the effect of icing on the cake. Taurine, a β-aminosulfonic acid, is the most abundant free amino acid in excitable mammalian tissues, with intracellular concentrations of20-70mmol/kg in the heart. Accumulating evidence indicates that taurine may play a cytoprotective role in the heart. Indeed, the oral supplementation of taurine has been shown to be effective in animal models and human patients with congestive heart failure and cardiomyopathy. The main mechanisms behind the cytoprotective effects of taurine include the maintenance of calcium (Ca2+), homeostasis, the regulation of osmotic balance and antioxidant and anti-apoptotic activity. Maintenance of the large intracellular taurine pool mainly depends upon the activity of taurine transporter (TAUT). Coronary artery bypass grafting and the spontaneously hypertension have been demonstrated to decrease the the taurine content in the myocardium with unknow mechanism. Whether the taurine content in the myocardium will be changed in acute myocardial ischemia, and whether the supplementation of exogenous taurine will be protective in myocardium, will be explored in this study. And the specific mechanism will also be detected.MethodsThe cultured cardiocytes which were exposed to glucose deprivation(GD) injury and hypoxia and the acute myocardial ischemic rats with or without taurine treatment were investigated. The morphology of cardiac tissues, the apoptosis of cardiocytes and the heart function were detected. Taurine contents were measured by HPLC. The mRNA and protein expressions of TAUT and TonEBP were measured by RT-PCR and western-blot respectively.ResultsTaurine treatment could reverse the apoptosis induced by GD injury, hypoxia and acute myocardial ischemia and effectively protect the myocardium. Our data showed that taurine contents and TAUT expression levels were significantly decreased when cultured cardiocytes and cardiac tissues subjected to GD injury, hypoxic or ischemic stress. Moreover, taurine treatment (lOOmg/kg/d) clearly up-regulated the TAUT expressions and elevated the taurine contents in ischemic myocardial tissues. In vitro, Low dose (40mM or80mM) but not high dose (120mM) of taurine significantly induced the TAUT expressions and elevated the intracellular taurine contents in GD injury and hypoxic cardiocytes. TAUT expressions were in accordance with the TonEBP expressions.ConclusionsTaurine provided the protective effects on ischemic myocardium. Low^dose but not high dose of taurine up-regulated the TAUT expressions through up-regulated the TonEBP expressions and the increased the intra-cardiocyte taurine contents in GD injury, hypoxia and acute myocardial ischemia.The potential application of this workThe downregulation of TAUT expression and decreased concentrations of taurine occurred30min after modeling, which indicates that the concentration of taurine may be an early diagnosis marker for AMI. The mechanism of myocardial protective effect of taurine was explained to provide the theoretical and experimental evidence for the clinical application of taurine in acute myocardial infarction patients.Originalities of this workIt is the first time to reveal that different concentrations of taurine have the different function of regulation of TAUT expressions. It is the first time to reveal that concentration of taurine in myocardium may be an early diagnosis marker for acute myocardial infarction patients.更多还原