氢吗啡酮后处理对大鼠心肌缺血再灌注损伤的影响及线粒体通透性转换孔在其中的作用：离体实验

Effect of hydromorphone postconditioning on myocardial ischemia-reperfusion injury in isolated rat hearts and the role of mitochondrial permeability transition pore

目的 探讨氢吗啡酮后处理对大鼠心肌缺血再灌注损伤的影响及线粒体通透性转换孔（mPTP）在其中的作用.方法 选择SD雄性大鼠,2～3月龄,体重250～320 g,取Langendorff离体灌流模型制备成功的离体心脏40个,采用随机数字表法分为4组（n=10）：对照组（C组）：K-H液持续灌注120 min;缺血再灌注组（I/R组）：K-H液灌注30 min后全心停灌30 min,再用K-H液灌注60min;氢吗啡酮后处理组（H组）：K-H液灌注30 min后全心停灌30 min,用含0.3 μmol/L氢吗啡酮的K-H液灌注10 min,再用K-H液灌注50 min;氢吗啡酮后处理＋mPTP开放剂氯尼达明组（HL组）：K-H液灌注30 min后全心停灌30 min,用含0.3μmol/L氢吗啡酮和30 μmol/L氯尼达明的K-H液灌注10 min,再用K-H液灌注50 min.于平衡灌注30 min（T0）、再灌注30 min（T2）、60 min（T3）时记录左心室收缩压（LVSP）、左心室舒张末期压（LVEDP）、左心室发展压（LVDP）、左心室内压上升/下降最大速率（±dp/dtmax）、HR和冠状动脉流量（CF）.于T0和T3时收集冠状动脉流出液测定LDH、CK-MB和T型肌钙蛋白（Tn-T）的浓度.T0和再灌注15 min（T1）时取冠状动脉流出液,采用ELISA法测定烟酰胺腺嘌呤二核甘酸（NAD＋）浓度,以反映mPTP开放程度.于T3时采用TTC染色法测定心肌梗死体积.结果 与C组相比,I/R组T2,3时LVDP、HR、±dp/dtmax和CF降低,LVEDP升高,T3时冠状动脉流出液LDH、CK-MB和Tn-T浓度、心肌梗死体积和T1时冠状动脉流出液NAD＋浓度升高（P＜0.05）.与I/R组和HL组相比,H组T23时LVDP、±dp/dtmax、CF和HR升高,LVEDP降低,T3时冠状动脉流出液LDH、CK-MB和Tn-T浓度、心肌梗死体积和T1时冠状动脉流出液NAD＋浓度降低（P＜0.05）.结论 氢吗啡酮后处理可减轻大鼠心肌缺血再灌注损伤,其机制与抑制mPTP开放有关.

Objective To investigate the effect of hydromorphone postconditioning on ischemiareperfusion （I/R） injury in isolated rat hearts and the role of mitochondial permeability transition pore （mPTP）.Methods Male Sprague-Dawley rats, aged 2-3 months, weighing 250-320 g, were used in the study.The rats were heparinized and anesthetized with intraperitoneal 10% chloral hydrate 350 mg/kg.The hearts were excised, and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 36.5-37.5 ℃.Forty isolated rat hearts were randomly divided into 4 groups （n=10 each）using a random number table： control group （group C）, group I/R, hydromorphone postconditioning group （group H）, and hydromorphone postconditioning ＋ mPTP opener lonidamine group （group HL）.Group C was continuously perfused with K-H solution for 120 min.Group I/R was perfused with K-H solution for 30 min, the perfusion was then suspended for 30 min, and group I/R was perfused with K-H solution for another 30 min.Group H was perfused with K-H solution for 30 min, the perfusion was then suspended for 30 min, and group H was perfused with K-H solution containing 0.3 μmol/L hydromorphone for 10 min, and then with K-H solution for 50 min.Group HL was perfused with K-H solution for 30 min, the perfusion was then suspended for 30 min, and group HL was perfused with K-H solution containing 0.3 μmol/L hydromorphone and 30 μmol/L lonidamine for 10 min, and then with K-H solution for 50 min.At 30 min of equilibration （T0）, and 30 and 60 min of reperfusion （T2,3）, left ventricular systolic pressure （LVSP）, left ventricular end-diastolic pressure （LVEDP）, left ventricular developed pressure （LVDP） , ±dp/dtmax, heart rate （HR）, and coronary flow （CF） were measured.The concentrations of lactic dehydrogenase （LDH）, creatine kinase-MB （CK-MB） , and troponin-T （Tn-T） in the coronary effluent were determined at T0 and T3.The coronary effluent was collected at T0 and 15 min of reperfusion （T1）,nicotinamide-adenine dinucleotide （NAD＋） concentrations were measured using enzyme-linked immunosorbent assay to reflect the degree of mPTP opening.The myocardial infarct size was determined at T3 by TTC staining.Results Compared with group C, LVDP, HR, ±dp/dtmax and CF were significantly decreased, and LVEDP was increased at T2,3, and the concentrations of LDH, CK-MB and Tn-T in the coronary effluent, myocardial infarct size at T3, and NAD＋ concentrations in the coronary effluent at T1 were increased in group I/R （P〈0.05）.Compared with I/R and HL groups, LVDP, ±dp/dt CF and HR were significantly increased, and LVEDP was decreased at T2,3, and the concentrations of LDH, CK-MB and Tn-T in the coronary effluent, myocardial infarct size at T3, and NAD＋ concentrations in the coronary effluent at T1 were decreased in group H （P〈0.05）.Conclusion Hydromorphone postconditioning can reduce myocardial I/R injury in isolated rat hearts, and the mechanism is related to inhibition of mPTP opening.