心肌缺血／再灌注后多巴酚丁胺最佳应用时间探讨

Discussion of the best time to use dobutamine after myocardium ischemia/reperfusion

目的 制作大鼠离体心脏缺血/再灌注损伤（ischemia/reperfusion injury,I/RI）模型,比较再灌注后不同时间点给予多巴酚丁胺对心功能和心肌损伤的影响,以探索心肌缺血/再灌注（ischemia/reperfusion,I/R）后给予多巴酚丁胺的最佳时间.方法 雄性SD大鼠36只,按完全随机法分为4组（每组9只）,各组在Langendorff灌注装置上建立离体心脏I/RI模型.平衡灌注15 min,缺血30 min,再灌注60 min,于再灌注期采取不同处理措施.单纯I/R组（I/R组）再灌注期全程以克-亨氏（Kreb＇sHenseleit,K-H）液灌注;多巴酚丁胺一组（D1组）再灌注5 min时给予多巴酚丁胺灌注30 min,其余时间以K-H液灌注;多巴酚丁胺二组（D2组）再灌注15 min时给予多巴酚丁胺灌注30 min,其余时间以K-H液灌注;多巴酚丁胺三组（D3组）再灌注25 min时给予多巴酚丁胺灌注30 min,其余时间以K-H液灌注.其中,多巴酚丁胺输注剂量均为10 μg· kg-1·min-1.记录各组平衡灌注末（T0）,再灌注10 min（T1）、20min（T2）、30min（T3）、60 min（T4）时的血流动力学指标：HR、左室舒张末压（left ventricular end diastolic pressure,LVEDP）、左室发展压（left ventricular developed pressure,LVDP）、左室内压上升/下降最大速率（the maximum rate of left ventricular pressure change,±dp/dtmax）及冠状动脉流量（coronary flow,CF）.留取T0～T4各时点冠状动脉流出液,使用乳酸脱氢酶（lactate dehydrogenase,LDH）和肌酸磷酸激酶（creatine kinase,CK）试剂盒测定冠状动脉流出液中LDH和CK的活性.2,3,5-氯化三苯基四氮唑（2,3,5-triphenyl tetrazolium chloride,TTC）染色法测定心肌梗死面积（myocardial infarct size,MIS）.Western blot法检测肌浆网钙泵（sarcoendoplasmic reticulum Ca2＋-ATPase,SERCA2a）和兰尼碱受体（ryanodine receptors,RyR2）蛋白表达量. 结果 D1组在给予多巴酚丁胺后,HR、LVEDP、LDH、CK、MIS均比I/R组高,差异有统计学意义（P＜0.05）;D2组、D3组在给予多巴酚丁胺后,HR、CF、LVDP、±dp/dtmax均高于I/R组,差异有统计学意义（P＜0.05）,而LVEDP、LDH、CK、MIS比较,差异无统计学意义（JP＞0.05）.多巴酚丁胺各组SERCA2a蛋白表达量和I/R组比较,差异无统计学意义（P＞0.05）,而RyR2蛋白表达量则高于I/R组（P＜0.05）.D2组与D3组相比在给予多巴酚丁胺后上述指标比较,差异均无统计学意义（P＞0.05）. 结论 大鼠心肌FR 15 min时应用多巴酚丁胺要优于其他时间点.在这一时间点用药可以及时而有效地提高大鼠HR,增加CF,改善心肌收缩功能,且不会加重心肌损伤.

Objective Using isolated rat heart ischemia/reperfusion injury （FRI） model to compare the effects of dobutamine giving at different time points after reperfusion on cardiac function and myocardial injury and to explore the best time to use dobutamine after myocardium ischemia/reperfusion（I/R）.Methods Thirty-six male SD rats,establishing isolated heart I/RI model in Langendorff perfusion apparatus with 15 min equilibrium,30 min global ischemia and followed by 60 min reperfusion,were randomly divided into four groups（n=9）.Different treatment was taken in reperfusion period.Group I/R：reperfused with Kreb＇s-Henseleit（K-H） solution,dobutamine group one （group D1）：received dobutamine for 30 min after 5 min of reperfusion,other time perfused with K-H solution.Dobutamine group two （group D2）：given dobutamine for 30 min after 15 min of reperfusion,other time perfused with K-H solution.Dobutamine group three （group D3）：administrated dobutamine for 30 min after 25 min of reperfusion,other time perfused with K-H solution.Dobutamine infusion dose was 10 μg ·kg-1 ·min-1.Heart function indexes of each group at the end of balance reperfusion（T0） and 10 min （T1）,20 min（T2）,30 min（T3）,60 min（T4） of reperfusion：HR,left ventricular end diastolic pressure（LVEDP）,left ventricular developed pressure（LVDP）,the maximum rate of left ventricular pressure change（±dp/dtmax） and coronary flow（CF） were recorded.The coronary flow at T0-T4 time points was used to measure the activity of lactate dehydrogenase（LDH） and creatine kinase （CK） according to the test methods attached to LDH and CK test kit.Myocardial infarct size （MIS） were measured with 2,3,5-triphenyl tetrazolium chloride （TTC） staining method.Sarcoendoplasmic reticulum Ca2＋-ATPase （SERCA2a） and ryanodine receptors（RyR2） expression were determined by Western bloting.Results After giving dobutamine,HR,LVEDP,LDH and CK activity and MIS of group D1 were higher than those in group I/R （P〈0.05）.Meanwhile after administrating dobutamin,HR,CF,LVDP,±dp/dtmax of group D2 and D3 were higher than that of group I/R,but there was no significant difference in LVEDP,LDH and CK activity and MiS in contrast with group I/R（P〉0.05）.SERCA2a expression between the above groups was no significant difference.While RyR2 expression was higher in groups with dobutamin compared to group I/R.There was no significant difference in above data between group D2 and D3 after using dobutamine.Conclusions Giving dobutamine after 15 min of myocardial I/R in rats is superior to the other time points.Using dobutamine at this time point can timely and effectively improve HR,increase CF,improve myocardial contractile function,and does not aggravate myocardial injury.