【作者】 殷忠；

【导师】 王海昌；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景WHO《世界卫生统计2008》报告:在未来的二十年,随着中低收入国家人群进入老龄化,非传染性疾病的死亡率将会大幅度增长。从全球范围看,预计到2030年,死于心血管疾病的人数将从2004年的1710万增加到2340万。冠心病( coronary heart disease, CHD )是人类死亡的主要原因之一,每年有约380万男性和340万女性死于冠心病。发生急性心肌梗死(acute myocardial infarction, AMI)后,尽早进行有效的心肌再灌注,无论是使用溶栓治疗、经皮冠状动脉介入治疗( percutaneous coronary intervention, PCI ),还是急诊冠脉搭桥( coronary artery bypass grafting, CABG )治疗都是最有效的减小心肌梗死面积和改善临床预后的策略。然而,缺血心肌恢复血流灌注的过程中,可以诱导损伤,这种现象,称为心肌缺血再灌注损伤,可以减少心肌再灌注产生的有利影响。这种形式的损伤,可以使那些在再灌注前即刻仍然存活的心肌细胞在再灌注后很快死亡。这种形式的心肌损伤本身可以诱导心肌细胞死亡和增加心肌梗死面积,或许可以部分解释为什么尽管给予最适的心肌再灌注治疗,AMI后死亡率仍然接近10%,AMI后心力衰竭的发病率仍然接近25%。A MI动物模型研究表明,最终的梗死面积,其中50%是致命的再灌注损伤引起的;这些实验模型中,许多治疗方法都显示出能够改善致命的再灌注损伤。然而,将这些能够改善再灌注损伤的实验方法运用到临床的时候,结果却令人非常失望。虽然如此,但是在2003年,赵志青教授等报告在体犬心局部缺血45 min后,再灌注开始早期给予三个循环的30 s心肌缺血/30 s心肌再灌注,可明显减小犬心肌梗死面积。并将这种心脏保护作用命名为缺血后处理( ischemic postconditioning IPost )。之后不久,Staat和Laskey分别在临床上进行PCI的AMI患者身上进行IPost,结果发现IPost能够减小心肌梗死面积达36%以及促进心肌再灌注。虽然IPost诱导产生心脏保护作用的确切机制仍然没有完全清楚,但是发现可能是通过减轻氧化应激反应,减少细胞内Ca2 +超载,改善血管内皮功能,减少凋亡心肌细胞的死亡,减少中性粒细胞积累以及延迟中性pH的恢复。此外,发现IPost激活再灌注损伤补救激酶( reperfusion injury salvage kinase, RISK )途径,包括磷脂酰肌醇-3-激酶( phosphatidylinositol-3-kinase, PI3-K )-蛋白激酶B ( Akt )途径和细胞外信号调节激酶( extracellular signal-regulating kinases, ERK )途径。最近,一些研究发现糖原合酶激酶-3β( glycogen synthase kinase-3beta, GSK-3β) Ser9磷酸化能促进心肌对缺血再灌注损伤的耐受。IPost保护心肌缺血再灌注损伤的大多数研究都是在成年鼠类离体心脏上进行的,并发现心脏保护信号及促细胞生存机制可能涉及PI3-K/Akt, ERK1/2以及GSK-3β等。最近有一些研究发现IPost未能保护老年小鼠心脏。但是关于IPost的保护作用能否在老年大鼠或人身上获得及相关机制还是知之甚少。研究目的(1)确定成年大鼠在体心脏急性缺血后再灌注即刻给予缺血后处理能否保护心脏,减轻缺血再灌注损伤。(2)评价缺血后处理能否减轻老年大鼠在体心脏缺血再灌注后的损伤,并比较后处理保护作用在成年大鼠与老年大鼠之间的区别。(3)初步确定老年大鼠缺血后处理的保护作用是否与心肌细胞内PI3-K/Akt及GSK-3β的磷酸化有关,如果有关,则激活PI3-K/Akt和GSK-3β可能是治疗缺血再灌注损伤的新的治疗手段。实验方法1.成年(3-4个月)及老年(16-18个月)雄性SD大鼠,戊巴比妥钠(50 mg/kg)腹腔麻醉后,行左侧开胸术,6-0丝线穿过左冠状动脉前降支(LAD),制备心肌缺血/再灌注大鼠模型。大鼠心脏缺血30min,再灌注3h。2.针形电极置于大鼠四肢皮下,连续记录心电图(electrocardiogram, ECG)。自右侧颈动脉插管至左心室,记录血流动力学参数。左侧颈外静脉插管给药。3.实验共分为10组,成年及老年大鼠各5组(n=8/成年组,n=6/老年组)。①成年大鼠缺血再灌注组( I/Radult)及老年大鼠缺血再灌注组( I/Raged):大鼠造模,缺血前后及再灌注前后不再给予其他干预;②成年大鼠缺血后处理组(IPostadult)及老年大鼠缺血后处理组(IPostaged):大鼠造模,再灌注即刻给予缺血后处理(4×10 s I/ 10 s R),再无其他干预;③成年大鼠缺血后处理联合LY294002组(IPost+LYadult)及老年大鼠缺血后处理联合LY294002组(IPost+LYaged):LY294002是选择性的PI3K抑制剂,大鼠造模,再灌注即刻给予缺血后处理(4×10 s I/ 10 s R),同时由另一术者经大鼠左侧颈外静脉给药LY294002(0.3 mg/kg),再无其他干预;④成年大鼠药物载体组(Vehicleadult)及老年大鼠药物载体组(Vehicleaged):大鼠造模,再灌注即刻给予缺血后处理,同时给予0.02%的DMSO(与IPost+LY组等容积,而不含有LY294002);⑤成年大鼠假手术组( Shamadult )及老年大鼠假手术组(Shamaged):未造模大鼠,颈部切开,气管插管,开胸,前降支下缝线等过程均同造模组,只是不进行冠脉结扎。4.在缺血前及再灌注3 h后取血0.5 mL,离心,取血清-70℃保存,用血清肌酸激酶(CK)和乳酸脱氢酶(LDH)试剂盒检测酶活性。5.再灌注结束时,采用Evans blue与TTC染色测量缺血区及梗死区面积。6.上述10组,各组均额外再做4只大鼠,缺血30 min,在再灌注15 min时,取缺血区组织,采用Western Blotting方法测定缺血心肌组织Akt, GSK-3β蛋白含量及其磷酸化水平。实验结果1.血流动力学参数包括心率( HR )、LVDP以及±dP/dtmax(1)心率:在所有监测的时间点I/Radult,I Postadult及IPost+LYadult组之间; I/Raged,IPostaged及IPost+LYaged组之间没有显著差异。单就I/R组而言,在基线、I 5 min,R 15 min及R 30 min时老年大鼠的心率明显低于成年大鼠组。就IPost及IPost+LY组而言,老年大鼠与成年大鼠心率没有差异。(2)LVDP:所有监测的时间点I/Radult,IP ostadult及IPost + LYadult组之间; I/Raged,IPostaged及IPost + LYaged组之间没有显著差异。单就I/R组而言,基线、R 5min,R 1h及R 2h时老年大鼠的LVDP明显低于成年大鼠组。就IPost组而言,R 5min和R 2h时老年大鼠的LVDP明显低于成年大鼠组。就IPost+LY组而言,R 2h和R 3h时老年大鼠的LVDP明显低于成年大鼠组。(3) +dP/dtmax: R 5min,R 2h及R 3h,IPostadult组大鼠+dP/dtmax明显高于I/Radult及IPost+LYadult。但在所有监测的时间I/Raged,IP ostaged及IPost + LYaged组之间没有显著差异。单就I/R组而言,老年大鼠与成年大鼠之间没有差别。就IPost组而言,整个再灌注期间除了R 1h时,老年大鼠的+dP/dtmax明显低于成年大鼠。就IPost+LY组而言,只是在R 30min时老年大鼠的+dP/dtmax明显低于成年大鼠组。(4) -dP/dtmax: R 2h及R 3h,IPostadult组大鼠-dP/dtmax明显高于I/Radult及IPost+LYadult。但在所有监测的时间I/Raged,IPostaged及IPost+LYaged组之间没有显著差异。在I/R组和IPost+LY组,只有I 5min时,老年大鼠的-dP/dtmax明显高于成年大鼠。但在IPost组,R 5min和R 15min时,老年大鼠的-dP/dtmax都明显低于成年大鼠组。2.冠脉LAD阻塞后缺血危险区(area at risk , AAR以%LV表示)在除Sham组外的其余各组之间是可比的(I/Radult,52.8±5.2%;IPostadult, 52.0±5.0%;IPost+LYadult ,52.2±5.8%;I/Raged, 51.3±7.2%;IPostaged, 53.8±3.5%;IPost+LYaged,51.1±5.8%;所有P =NS)。正如所期望的,与I/R组相比,IPost明显减小梗死面积(Infarct size,IS;以%AAR表示) (IPostadult vs. I/Radult:11.9±1.6% vs. 30.2±2.8%,P<0.05;IPostaged vs. I/Raged:13.7±2.5% vs. 27.5±3.5%,P<0.05)。再灌注同时给予PI3K抑制剂LY294002,结果取消IPost的减小IS的作用(IPost+LYadult vs. IPostadult: 30.1±2.9% vs. 11.9±1.6%,P<0.05;IPost + LYaged vs. IPostaged:33.4±7.0% vs. 13.7±2.5%,P <0.05)。而在I/Radult组与I/Raged组、IP ostadult组与IPostaged组、IP ost+LYadult组与IPost+LYaged组IS之间均无统计学差异(所有P =NS)。3.在术前,老年组血清CK的水平均明显低于成年组,LDH水平老年与成年组之间均无差异。术前,CK与LDH水平在老年组内部及成年组内部各组间也无差异。与术前相比,再灌注3 h后各组CK及LDH水平显著升高(所有P<0.05)。再灌注3 h后IPost组CK与LDH水平升高幅度均显著低于I/R组(所有P<0.05)。给予PI3K抑制剂LY294002后,CK与LDH水平升高幅度均比IPost组高(所有P<0.05),但与I/R组之间无统计学差异。4.任两组间总Akt(t-Akt)与总GSK-3β(t-GSK-3β)水平无差异。但是不论是成年组大鼠还是老年组,与I/R组相比,IPost均显著增加p-Akt水平(p-Akt/t-Akt: IPostadult vs. I/Radult: 0.64±0.04 vs. 0.40±0.09,P<0.05; IPostaged vs. I/Raged:0.63±0.03 vs. 0.39±0.01, P<0.05)。给予LY294002后可阻断Akt磷酸化(p-Akt/t-Akt:IPost + LYadult vs. IPostadult: 0.34±0.07 vs. 0.64±0.04,P<0.05;IPost + LYaged vs. IPostaged:0.35±0.09 vs 0.63±0.03, P<0.05)。同样,IPost也明显增加GSK-3β磷酸化水平,而LY294002则阻断GSK-3β的磷酸化。结论1.再灌注即刻给予缺血后处理能够减小成年SD大鼠心肌梗死面积,降低CK及LDH升高水平,并能够部分改善心脏功能。2.缺血后处理可减小老年SD大鼠急性缺血/再灌注时心肌梗死面积,降低CK及LDH升高水平,但未能改善心脏功能。3.缺血后处理对成年及老年大鼠心脏保护作用可能与Akt及GSK-3β磷酸化有关。更多还原

【Abstract】 BackgroundAs populations age in middle- and low-income countries over the next 25 years, the proportion of deaths due to noncommunicable diseases will rise significantly. Globally, deaths from cancer will increase from 7.4 million in 2004 to 11.8 million in 2030, and deaths from cardiovascular diseases will rise from 17.1 million to 23.4 million in the same period ( world health statistics 2008 ).Coronary heart disease is the leading cause of death worldwide, and 3.8 million men and 3.4 million women die of the disease each year. After an acute myocardial infarction, early and successful myocardial reperfusion with the use of thrombolytic therapy or primary percutaneous coronary intervention ( PCI ) or emergency coronary artery bypass grafting ( CABG ) is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome. The process of restoring blood flow to the ischemic myocardium, however, can induce injury. This phenomenon, termed myocardial reperfusion injury, can paradoxically reduce the beneficial effects of myocardial reperfusion. The injury culminates in the death of cardiac myocytes that were viable immediately before myocardial reperfusion. This form of myocardial injury, which by itself can induce cardiomyocyte death and increase infarct size ,may in part explain why, despite optimal myocardial reperfusion, the rate of death after an acute myocardial infarction approaches 10%, and the incidence of cardiac failure after an acute myocardial infarction is almost 25%.Studies in animal models of acute myocardial infarction suggest that lethal reperfusion injury accounts for up to 50% of the final size of a myocardial infarct, and in these models a number of strategies have been shown to ameliorate lethal reperfusion injury. Yet, the translation of these beneficial effects into the clinical setting has been disappointing. But in 2003, Prof Zhao Zhiqing et al. showed that after a 45-minute episode of sustained myocardial ischemia, the interruption of myocardial reperfusion with three 30-second cycles of myocardial ischemia and reperfusion could reduce the myocardial infarct size significantly. They named this form of cardioprotection ischemic postconditioning. Shortly after that,Staat and Laskey respectively used ischemic postconditioning in patients with acute myocardial infarction who are undergoing PCI with a protocol that has reduced myocardial infarct size by 36% and improved myocardial reperfusion.Although the precise mechanism of ischemic postconditioning–induced protection is not fully understood, but the procedure has been shown to target the important mediators of lethal reperfusion injury by reducing oxidative stress, decreasing intracellular Ca2 + overload, improving endothelial function, attenuating apoptotic cardiomyocyte death, reducing neutrophil accumulation, and delaying the restoration of neutral pH. Furthermore, ischemic postconditioning activates the reperfusion injury salvage kinase (RISK) pathway, which include the phosphatidylinositol-3-kinase-Akt pathway ( PI3K-Akt ) and the extracellular signal-regulating kinases ( ERK ) pathway. Recently, several studies have suggested that phosphorylation of glycogen synthase kinase-3beta (GSK-3β) at Ser9 enhances myocardial tolerance during ischemia- reperfusion injury.In the majority of studies, ischemic postconditioning (IPost) has been demonstrated to protect adult rodent hearts from myocardium ischemia- reperfusion (I/R) injury in vitro, and has implicated the involvement of several candidate signaling pathways such as PI3-K/Akt, ERK1/2, and GSK-3βin cardioprotective signaling and pro-cell survival mechanisms. Recently, some researches have found that the cardioprotection of IPost is lost in aged mice. However, little is known with regards to these signaling mechanisms during advanced age.Aim(1) to determine whether ischemic postconditioning applied at the beginning of reperfusion could protect adult hearts in vivo from ischemia reperfusion injury .(2) to evaluate the hypothesis that ischemic postconditioning could protect ischemia/reperfusion aged hearts in vivo. And to compare the cardioprotection effects of ischemic postconditioning between aged and adult heats.(3) to investigate whether IPost’s cardioprotection is dependent on the activation of PI3-K/Akt and GSK-3β, and if activation of PI3-K/Akt or GSK-3βmay be a novel therapeutic strategy for reducing myocardial I/R injury. Methods1. Male Sprague Dawley (SD) rats (adult, 3-4 months aged, 16-18 months) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and experienced left thoracotomy. a 6-0 silk suture was placed under the left anterior descending coronary artery (LAD), and ends of the suture were threaded through a segment of polyethylene tube to form a snare for the reversible LAD occlusion. Rats were subiected to 30 min myocardial ischemia and 3 h reperfusion.2. Needle electrodes were placed subcutaneously on the limbs, and an electrocardiogram was continuously recorded.the right carotid artery was cannulated with a 24-gauge angiocath connected to a fluid-filled pressure transducer for monitoring Hemodynamic properties. The left external jugular vein was cannulated for drug administration.3. The rats were assigned to one of the following groups based upon the intervention performed (n = 8 per adult group, n = 6 per aged group): (1) I/Radult and I/Raged: received no other intervention either before or after LAD occlusion (2) IPostadult and IPostaged: received ischemic postconditioning induced by four cycles of 10 seconds of ischemia and 10 seconds of reperfusion at the beginning of reperfusion (3) IPost+LYadult and IPost+LYaged: received ischemic postconditioning in the presence of the selective PI3K inhibitor, LY294002 (0.3 mg/kg) administered via the left external jugular vein immediately at the beginning of reperfusion (4) Vehicleadult and Vehicleaged: received ischemic postconditioning in the presence of an equal volume of vehicle, 0.02% dimethyl sulfoxide (DMSO) without LY294002 at the beginning of reperfusion (5) Shamadult and Shamaged:p lacement of the silk suture under the LAD and no other intervention. 4. Blood samples (0.5 mL) were collected at baseline and the end of reperfusion and centrifuged at 2500×g and 4°C for 10 min. The samples were then stored at -70°C until further study. The activity of CK and LDH was analyzed at 25°C using commercially available kits.5. At the end of reperfusion,ischemic and infarct areas were measured by Evans blue and triphenyltetrazolium chloride ( TTC ) staining respectively.6. In separate experiments (n=4 rats per experimental group), phosphorylation of Akt and GSK-3βwere analyzed by Western blotting after 15 min of reperfusion.Results1. Hemodynamic data includes heart rate ( HR ), LVDP and±dP/dtmax (1) HR: At all time points, there were no significant differences among I/Radult, IPostadult, and IPost+LYadult groups, and no significant differences among the I/Raged, IPostaged, and IPost+LYaged groups. At baseline, I 5 min, R 15 min, R 30 min, the HR of I/Raged group were lower than that of I/Radult group. At all time points, there were no significant differences between IPostadult,and IPostaged , between IPost+LYadult and IPost+LYaged group.(2) LVDP: At all time points, there were no significant differences among I/Radult, IPostadult, IPost+LYadult groups, and no significant differences among the I/Raged, IPostaged, IPost+LYaged groups. At points of R 5 min, R1 h, and R 2 h, the LVDP of I/Raged group were lower than that of I/Radult group. At points of R 5 min, R 2 h, the LVDP of IPostaged group were lower than that of IPostadult group. At points of R 2 h and R 3 h, the LVDP of IPost+LYaged group were lower than that of IPost+LYadult group. (3) +dP/dtmax:At R 5 min, R 2 h, and R 3 h, +dP/dtmax of IPostadult were significantly higher than that of I/Radult or IPost+LYadult. But at all time points, there were no significant differences among the I/Raged, IPostaged, IPost+LYaged groups. In the I/Rgroup, there was no significant difference between aged and adult rats. But in the IPost groups, +dP/dtmax of adult rats were significantly higher than that of aged rats during reperfusion except R 1 h. In the IPost+LY groups only at R 30 min +dP/dtmax of adult rats were significantly higher than that of aged rats.(4) -dP/dtmax: At R2 h, R 3 h, -dP/dtmax of IPostadult were significantly higher than that of I/Radult or IPost+LYadult. But at all time points, there were no significant differences among the I/Raged, IPostaged, IPost+LYaged groups. In the I/R and IPost+LY group, only at I 5 min -dP/dtmax of adult rats were significantly lower than that of aged rats. But in the IPost groups, -dP/dtmax of adult rats were significantly higher than that of aged rats at R 5 min and R 15 min.2. The area at risk ( in percentage of the left ventricle ) by LAD occlusion was comparable among six groups ( I/Radult, 52.8±5.2% IPostadult, 52.0±5.0% Ipost + LYadult, 52.2±5.8% I/Raged, 51.3±7.2% IPostaged, 53.8±3.5% IPost+LYaged, 51.1±5.8% ). As expected, the postconditioned hearts developed significantly smaller infarct sizes ( expressed as percentage of the area at risk, IS/AAR ) than I/R hearts (11.9±1.6% in IPostadult vs. 30.2±2.8% in I/Radult, P<0.05 13.7±2.5% in IPostaged vs. 27.5±3.5% in I/Raged, P<0.05 ). The PI3K inhibitor, LY294002, abolished IPost’s cardioprotection in aged and adult rats (30.1±2.9% in IPost + LYadult vs. 11.9±1.6% in IPostadult, P<0.05; 33.4±7.0% in IPost + LYaged vs. 13.7±2.5% in IPostaged, P<0.05 ). There were no significant differences between I/Radult and I/Raged, IPostadult and IPostaged, IPost + LYadult and IPost+LYaged. ( all P =NS ). 3. At baseline, the levels of CK in aged groups were lower than that in adult groups, and no differences within aged groups and within adult groups. The levels LDH were no differences between the six groups. Compared with the baseline, CK and LDH release was significantly increased after 3 hours of reperfusion in all I/R, IPost, and IPost+LY groups ( all P < 0.05 vs. baseline ). At 3 h of reperfusion, CK and LDH release in IPost rats was significantly lower than that in I/R groups ( all P< 0.05 vs. I/R groups ). LY294002 prevented the decrease of CK and LDH release in IPost group ( all P<0.05 vs. IPost, not significantly different from I/R groups ).4. There were no differences in total Akt ( t-Akt ) and total GSK-3β( t-GSK-3β) levels between every two groups. But in both adult and aged rat hearts, IPost significantly increased levels of Akt phosphorylation to levels greater than I/R hearts ( p-Akt/t-Akt: 0.64±0.04 in IPostadult vs. 0.40±0.09 in I/Radult, P<0.05 0.63±0.03 in IPostaged vs. 0.39±0.01 in I/Raged, P<0.05 ). The PI3K inhibitor, LY294002, blocked Akt phosphorylation ( p-Akt/t-Akt: 0.34±0.07 in IPost + LYadult vs. 0.64±0.04 in IPostadult, P<0.05 0.35±0.09 in IPost + LYaged vs. 0.63±0.03 in IPostaged ,P<0.05 ). Similarly, IPost significantly increased levels of GSK-3βphosphorylation to levels greater than I/R hearts, and LY294002 attenuated the levels of phosphorylated-GSK-3βin both adult and aged hearts.Conclusion1. Ischemic postconditioning at the onset of reperfusion reduces myocardial infarct size, attenuates the increased levels of CK and LDH, and improve functional preservation after reperfusion in adult SD rats in vivo.2. An infarct sparing effect for IPost in in vivo heart model of aged rats, which unlike the adult rat model, was not associated with functional preservation.3. IPost’s cardioprotection in both adult and aged SD rat hearts may be associated with Akt and GSK-3βphosphorylation.更多还原