七氟醚缺血后处理对大鼠离体心脏缺血—再灌注损伤保护的机理研究

发布时间：2018-06-27 13:34

本文选题：七氟醚 + 缺血-再灌注损伤　；参考：《中国协和医科大学》2010年博士论文

【摘要】： 目的： 1、观察七氟醚缺血后处理对健康成年大鼠离体心脏缺血-再灌注损伤的保护作用,研究活性氧(ROS)、细胞外信号调节激酶(ERK1/2)及线粒体通透性转换孔(mPTP)可能的作用； 2、观察七氟醚缺血后处理对慢性心梗大鼠离体心脏缺血-再灌注损伤的保护作用,研究蛋白激酶B (PKB/Akt)、细胞外信号调节激酶(ERK 1/2)及线粒体通透性转换孔(mPTP)在其中的可能作用； 3、比较七氟醚缺血预处理、七氟醚缺血后处理及七氟醚缺血预处理联合七氟醚缺血后处理3种方式对健康成年大鼠离体心脏缺血-再灌注损伤保护效果及相关机制； 4、系统评价和荟萃分析七氟醚、异丙酚用于行冠脉搭桥手术时对患者的心肌保护效果。方法： 1、大鼠心脏离体,以K-H缓冲液灌注,全心缺血30 min后复灌60 min建立缺血-再灌注损伤模型。七氟醚缺血后处理的心脏于缺血后复灌最初15 min以3%七氟醚饱和的K-H缓冲液灌注。分别单独给予或与七氟醚同时给予ROS清除剂NAC (4mM)或ERK 1/2阻断剂PD98059 (20μM),用以评价ROS及ERK 1/2在七氟醚缺血后处理中的作用。比较各组间血流动力学、心肌梗死面积、冠脉流出液中乳酸脱氢酶(LDH)及肌酸肌酶-MB (CK-MB)水平。同时,测定各组缺血30 min复灌60 min后心肌丙二醛(MDA)含量以反映氧化应激损伤程度。Western blotting测定ERK 1／2的磷酸化情况。测定心肌烟酰胺腺嘌呤二核苷酸(NAD+)含量以反映mPTP的开放情况。 2、大鼠在体结扎冠状动脉左前降支(LAD)建立梗死模型,6周后开胸取心,以K-H缓冲液离体灌注,全心缺血30 min后复灌60 min建立缺血-再灌注损伤模型。七氟醚缺血后处理组的心脏在缺血后复灌最初15 min以3%七氟醚饱和的K-H缓冲液灌注。为评价PKB/Akt及ERK 1／2在七氟醚缺血后处理中的作用,分别单独给予或与七氟醚同时给予两激酶的特异性抑制剂LY294002 (15μM)及PD98059 (20μM)。比较各组间血流动力学、心肌梗死面积、冠脉流出液中LDH及CK-MB水平。以Western blotting测定复灌15 min时PKB/Akt及ERK 1/2的磷酸化情况。测定心肌中NAD+含量以反映mPTP的开放情况。 3、健康成年大鼠离体心脏经缓冲液平衡期灌注10 min后,随机分为4组：对照(CTRL)组：继续灌注25 min后,行30 min全心缺血,后复灌120 min,实验过程中未给予其它药物干预；七氟醚缺血预处理(SpreC)组：缺血前,给予3%七氟醚15 min后洗出10 min行预处理；七氟醚缺血后处理(SpostC)组：缺血后复灌最初15 min给予3%七氟醚行后处理；七氟醚缺血预处理联合七氟醚缺血后处理(SpreC+SpostC)组：联合使用SpreC和SpostC组的实验方案。比较组间相同时点及组内不同时点间的血流动力学指标。实验结束后,用TTC染色法比较组间心肌梗死面积。测定平衡期及复灌120 min时冠脉流出液中的LDH和CK-MB作为心肌损伤指标。蛋白印迹法(WB)观察PKB/Akt和ERK 1/2在实验中不同时点的动态变化趋势。通过测定复灌15 min心肌NAD+含量以反映mPTP的开放程度。此外,复灌120 min时左室心肌取材行全基因芯片表达谱分析。 4、计算机检索Pubmed、EMBASE等国外文献数据库及中国生物医学文献数据库、中国期刊网等中文数据库,并配合手工检索所有文献全文,由两位作者分别进行有关资料的提取,主要包括患者一般情况、手术情况及体外循环后心排指数(CI)、术后肌钙蛋白Ⅰ水平(cTnI)、术后机械通气时间、正性肌力药物支持等指标参数、ICU和住院时间、死亡率、心肌梗死、心肌缺血和心房纤颤发生率等终点事件,随后用RevMan 5.0进行荟萃分析。结果： 1、与ISCH组相比,复灌之初给予3%七氟醚可显著改善心功能(增加左室发展压力、左室最大收缩／舒张速率、冠脉流量、心率,并降低左室舒张末期压力)、降低心肌梗死面积及减少LDH及CK-MB释放(P0.05)。七氟醚的心肌保护作用同样表现在降低缺血-再灌注损伤后心肌的MDA含量(P0.05)。然而,给予NAC或PD98059不仅可消除上述保护作用,而且可以抑制七氟醚增强ERK 1/2磷酸化及抑制mPTP开放的保护作用(P0.05)。 2、与缺血-再灌注损伤组相比,复灌之初给予3%七氟醚可显著改善心功能(增加左室发展压力、左室最大收缩／舒张速率、冠脉流量、心率、并降低左室舒张末期压力)、降低心肌梗死面积及减少LDH及CK-MB释放(P0.05)。然而,给予LY或PD分别抑制PKB/Akt或ERK 1/2磷酸化后可消除上述保护作用(P0.05),而且导致心肌NAD+含量降低(P0.05)。 3、与CTRL组相比,七氟醚处理(SpreC、SpostC及SpreC+SpostC)的3组心脏缺血-再灌注损伤后心功能改善(表现为LVDP、+dp/dt、-dp/dt、CF和HR的升高及LVEDP的降低)、梗死面积降低、LDH和CK-MB释放减少(P0.05)。就上述指标在3个七氟醚处理组间行组内比较可见：SpreC+SpostC的保护效果最佳(P0.05),而SpreC和SpostC间比较差异无统计学意义(P0.05)。WB分析显示,CTRL组心脏缺血-再灌注后PKB/Akt和ERK-1/2的磷酸化程度较平衡期增强(P0.05)；SpreC和SpreC+SpostC组心脏均出现双时相的PKB/Akt和ERK-1/2的磷酸化增强(分别出现在预处理和后处理后)；与此不同的是SpostC组仅出现单时相的PKB/Akt和ERK-1/2磷酸化增强(后处理后)。组间比较：SpreC+SpostC组中先后施行的SpreC和SpostC对PKB/Akt及ERK-1/2的磷酸化增强作用在复灌15 min和120 min出现累加。CTRL组复灌15 min时心肌NAD+含量最低(P0.05),SpreC+SpostC组最高(P0.05),提示SpreC和SpostC抑制mPTP开放的作用也可累加。以Agilent大鼠全基因表谱芯片对3种七氟醚缺血处理方式后的心肌和未经处理的缺血-再灌注损伤心肌进行基因差异表达分析,仅有少数基因同时受SpreC、SpostC和SpreC+SpostC同向调节。 4、荟萃分析共纳入13项前瞻性随机对照研究(RCT),包括696例患者,其中七氟醚组402人,异丙酚组294人。结果显示：两组患者术后机械通气时间、正性肌力药物支持、死亡率、心梗和房颤发生率等方面的差异均无统计学意义(P0.05)。与异丙酚组相比,七氟醚组患者体外循环后CI增高(P=0.003),术后cTnI降低(P0.00001),术后心肌缺血发生率降低(P=0.02),ICU和住院时间缩短(P=0.24和P=0.21)。结论： 1、3%七氟醚缺血后处理通过ROS-ERK 1/2-mPTP信号通路可为健康大鼠离体心脏的缺血-再灌注损伤提供保护。 2、3%七氟醚缺血后处理通过激活PI3K-PKB/Akt及MEK 1/2-ERK 1/2途径,并抑制mPTP开放,从而为慢性梗死大鼠离体心脏的缺血-再灌注损伤提供保护。 3、3%的SpreC和SpostC对离体心脏的心肌缺血-再灌注损伤的保护效果相当,主要表现在改善心功能、降低梗死面积、减少心肌损伤、促进保护性激酶活化及抑制mPTP开放。联合应用SpreC和SpostC则能够提供额外的心肌保护效果。三种七氟醚缺血处理后心肌基因表达谱的差异悬殊。 4、现有证据表明,对行冠脉搭桥手术的患者而言,七氟醚较异丙酚显示了更好的心肌保护效果。
[Abstract]:Objective:
1, to observe the protective effect of sevoflurane ischemic postconditioning on ischemic reperfusion injury in healthy adult rats, and to study the possible role of reactive oxygen species (ROS), extracellular signal regulated kinase (ERK1/2) and mitochondrial permeability transition pore (mPTP).
2, to observe the protective effect of sevoflurane ischemic postconditioning on myocardial ischemia reperfusion injury in rats with chronic myocardial infarction, and to study the possible role of protein kinase B (PKB/Akt), extracellular signal regulated kinase (ERK 1/2) and mitochondrial permeability transition pore (mPTP).
3, compared with sevoflurane ischemic preconditioning, sevoflurane ischemic postconditioning and sevoflurane ischemic preconditioning combined with sevoflurane ischemic postconditioning, the protective effect and mechanism of 3 ways of ischemic reperfusion injury in healthy adult rat heart were compared.
4, systematic review and meta analysis of sevoflurane and propofol for myocardial protection in patients undergoing coronary artery bypass grafting.
Method:
1, rat heart was perfused in vitro, perfusion of K-H buffer, and reperfusion injury model of reperfusion 60 min after 30 min of heart ischemia. After ischemia, the heart of sevoflurane ischemic reperfusion was initially 15 min with 3% sevoflurane saturated K-H buffer. They were given individually or in conjunction with sevoflurane, NAC (4mM) or ERK 1/2 blocking with sevoflurane. PD98059 (20 mu M) was used to evaluate the role of ROS and ERK 1/2 in the post-treatment of sevoflurane ischemia. The hemodynamics, myocardial infarction area, lactate dehydrogenase (LDH) and creatine muscle enzyme -MB (CK-MB) in the coronary effluent were compared between each group. Meanwhile, the content of MDA (MDA) in the 30 min reperfusion 60 min in each group was measured to reflect the oxidative stress. The degree of phosphorylation of ERK 1 / 2 was measured by.Western blotting, and the content of nicotinamide adenine dinucleotide (NAD+) was measured to reflect the opening of mPTP.
2, the rat model was established by ligation of the left anterior descending branch of the coronary artery (LAD). After 6 weeks, the heart was taken out of the chest and perfused in vitro with K-H buffer. The ischemia reperfusion injury model was established after 30 min of the whole heart ischemia. The heart of the sevoflurane ischemic post treatment group was perfused with the K-H buffer saturated with 3% sevoflurane at the beginning of ischemia reperfusion after ischemia. The role of PKB/Akt and ERK 1 / 2 in the post-treatment of sevoflurane ischemia was given separately or with the specific inhibitor LY294002 (15 M) and PD98059 (20 micron M) given to the two kinase of sevoflurane respectively. The hemodynamics, myocardial infarction area, and the level of LDH and CK-MB in the coronary outflow fluid were compared between each group. The Western blotting was used for the determination of 15 min P. The phosphorylation of KB/Akt and ERK 1/2 was measured. The NAD+ content in myocardium was measured to reflect the opening of mPTP.
3, after perfusion of 10 min in the balance period of the isolated heart of healthy adult rats, they were randomly divided into 4 groups: control (CTRL) group: after continuous perfusion of 25 min, 30 min whole heart ischemia, and then reperfusion 120 min, no other drug intervention was given, sevoflurane ischemic preconditioning (SpreC) group: before ischemia, 3% sevoflurane 15 min were given after 15 min to wash 10 min Preconditioning, sevoflurane ischemic postconditioning (SpostC) group: 3% sevoflurane was given at the first 15 min after ischemia, and sevoflurane ischemic preconditioning combined with sevoflurane ischemic postconditioning (SpreC+SpostC) group: the combined use of SpreC and SpostC groups. Index. After the experiment, TTC staining was used to compare the area of myocardial infarction. LDH and CK-MB in the flow of coronary flow were measured at the balance stage and 120 min as the index of myocardial injury. The dynamic change trend of the difference of PKB/Akt and ERK 1/2 in the experiment was observed by the Western blot (WB). The content of NAD+ in the 15 min myocardium was measured to reflect the mP. In addition, the gene expression profiles of left ventricular myocardium were analyzed at 120 min after reperfusion for TP.
4, the computer retrieves foreign literature databases such as Pubmed, EMBASE, Chinese biomedical literature database, Chinese Journal Network and other Chinese databases, and with manual retrieval of the full text of all the documents. The related data are extracted by two authors respectively, including the general situation of the patients, the operation situation and the cardiac exclusion index after cardiopulmonary bypass (CI), after the operation. Cardiac troponin I level (cTnI), postoperative mechanical ventilation time, positive inotropic drug support and other index parameters, ICU and time of hospitalization, mortality, myocardial infarction, myocardial ischemia and atrial fibrillation, were followed by a meta-analysis of RevMan 5.
Result:
1, compared with the ISCH group, 3% sevoflurane at the beginning of reinjection could significantly improve cardiac function (increased left ventricular development pressure, maximum left ventricular systolic / diastolic velocity, coronary flow rate, heart rate, and lower left ventricular end diastolic pressure), lower myocardial infarction area and reduction of LDH and CK-MB release (P0.05). The myocardial protection of sevoflurane also showed a decrease in deficiency. MDA content (P0.05) of myocardium after blood reperfusion injury. However, giving NAC or PD98059 not only eliminates the above protective effect, but also inhibits the protective effect of sevoflurane enhanced ERK 1/2 phosphorylation and inhibition of mPTP opening (P0.05).
2, compared with the ischemia-reperfusion injury group, 3% sevoflurane at the early stage of reperfusion could significantly improve cardiac function (increased left ventricular development pressure, left ventricular maximum contraction / diastolic velocity, coronary flow rate, heart rate, and lower left ventricular end diastolic pressure), reduced infarct size and reduced LDH and CK-MB release (P0.05). However, LY or PD was given to inhibit PKB/, respectively. After Akt or ERK 1/2 phosphorylation, the protective effect (P0.05) was eliminated, and the NAD+ content of myocardium decreased (P0.05).
3, compared with group CTRL, 3 groups of sevoflurane treated (SpreC, SpostC, and SpreC+SpostC) improved cardiac function after ischemia reperfusion injury (LVDP, +dp/dt, -dp/dt, CF and HR increase and LVEDP decrease), infarct size, LDH and CK-MB release decreased. The above indicators were compared in the group of 3 sevoflurane treatment groups: The protective effect of reC+SpostC was the best (P0.05), while the difference between SpreC and SpostC was not statistically significant (P0.05).WB analysis showed that the degree of phosphorylation of PKB/Akt and ERK-1/2 after ischemia and reperfusion in the CTRL group was stronger than that in the equilibrium phase (P0.05), and both SpreC and SpreC+SpostC groups had a dual phase and phosphorylation enhancement (respectively). The difference was that the PKB/Akt and ERK-1/2 phosphorylation enhanced (after treatment) in the SpostC group only. Comparison between groups: the enhancement of the phosphorylation of PKB/Akt and ERK-1/2 by SpreC and SpostC in the SpreC+SpostC group was 15 min and 120 min in the complex irrigation of 15 min. The NAD+ content of myocardium was the lowest (P0.05) and the highest (P0.05) in group SpreC+SpostC (P0.05), suggesting that the effect of SpreC and SpostC on the inhibition of mPTP opening could also be accumulative. Gene differential expression analysis of myocardial and untreated ischemia reperfusion injury after 3 sevoflurane ischemic treatment was carried out with a small number of genes. At the same time, SpreC, SpostC and SpreC+SpostC are regulated in the same direction.
4, the meta-analysis was included in 13 prospective randomized controlled studies (RCT), including 696 patients, of which 402 were sevoflurane and 294 in the propofol group. The results showed that there were no significant differences in mechanical ventilation time, positive inotropic drug support, mortality, myocardial infarction and atrial fibrillation in the two groups (P0.05). Compared with the patients in the sevoflurane group, the CI increased (P=0.003) after cardiopulmonary bypass, and the postoperative cTnI decreased (P0.00001), the incidence of myocardial ischemia was reduced (P=0.02), and the time of ICU and hospitalization were shortened (P=0.24 and P=0.21).
Conclusion:
1,3% sevoflurane ischemic postconditioning can protect the isolated rat heart from ischemia-reperfusion injury through ROS-ERK 1/2-mPTP signaling pathway.
2,3% sevoflurane is treated by ischemic postconditioning by activating PI3K-PKB/Akt and MEK 1/2-ERK 1/2 pathway and inhibiting the opening of mPTP, thus providing protection for ischemic reperfusion injury in the isolated heart of chronic infarcted rats.
The protective effect of 3,3%'s SpreC and SpostC on myocardial ischemia and reperfusion injury in isolated heart is similar, mainly in improving cardiac function, reducing infarct size, reducing myocardial damage, promoting protective kinase activation and inhibiting the opening of mPTP. Combined use of SpreC and SpostC can provide additional myocardial protection effect. Three kinds of sevoflurane ischemic place There is a great difference in the gene expression profiles of the posterior myocardium.
4, available evidence shows that sevoflurane is better than propofol for coronary protection in patients undergoing coronary artery bypass grafting.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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