神经生长因子β预先给药对大鼠离体心脏缺血再灌注损伤的影响：与预处理法比较

Effects of pretreatment with nerve growth factor-beta on ischemia-reperfusion injury in isolated rat hearts：a comparison with preconditioning

目的 评价神经生长因子β（NGF-β）预先给药对大鼠离体心脏缺血再灌注损伤的影响.方法 雄性SD大鼠,8～ 10周龄,体重200～300 g,制备Langendorff离体心脏灌注模型.取模型制备成功的心脏24个,采用随机数字表法,将其分为3组（n=8）：缺血再灌注组（I/R组）、NGF-β预先给药组（NGFPt组）、NGF-β预处理组（NGFPc组）.各组先用K-H液平衡灌注10 min；I/R组K-H液继续灌注30 min,NGFPt组用含0.1 μg/ml NGF-β的K-H液继续灌注20 min,NGFPc组用含0.1 μg/ml NGF-β的K-H液继续灌注20 min,再用K-H液冲洗10 min；各组均停灌30 min,K-H液复灌120 min.于平衡灌注末、停灌前即刻、再灌注5、30、60和120 min（T1-6）时,记录心率（HR）、左室舒张末压（LVEDP）、左室发展压（LVDP）、左室内压最大上升速率（＋dp/dtmax）；于T1和T3-6时收集冠脉流出液2 ml,测定肌酸激酶（CK-MB）、乳酸脱氢酶（LDH）的活性；T6时取心肌组织测定心肌细胞凋亡情况,观察心肌病理学改变.结果 与T1时比较,各组T3-6时＋ dp/dtmax降低,LVEDP、冠脉流出液CK-MB、LDH的活性升高,I/R组T3-6时LVDP、HR降低,NGFPt组T3,4时和NGFPc组T3-6时LVDP降低,NGFPt组和NGFPc组T2-6时HR升高（P＜0.05）.与I/R组比较,NGFPt组和NGFPc组LVDP、＋dp/dtmax和HR升高,LVEDP、冠脉流出液CK-MB、LDH活性和心肌细胞凋亡指数降低（P＜0.05）；与NGFPc组比较,NGFPt组LVDP、HR和＋ dp/dtmax升高,冠脉流出液LDH活性降低,心肌细胞凋亡指数降低（P＜0.05）,LVEDP和冠脉流出液CK-MB活性差异无统计学意义（P＞0.05）.I/R组心肌病理学损伤明显,而NGFPt组和NGFPC组明显减轻.结论 NGF-β（0.1 μg/ml）预先给药可减轻大鼠离体心脏缺血再灌注损伤,其效果优于预处理法.

Objective To evaluate the effect of pretreatment with nerve growth factor-beta （NGF-β） on ischemia-reperfusion （I/R） injury in isolated rat hearts by comparing it with NGF-β preconditioning.Methods Pathogen-free male Sprague-Dawley rats,weighing 200-300 g,aged 8-10 weeks,were anesthetized with intraperitoneal 10% chloral hydrate 300 mg/kg.Their hearts were excised and perfused in a Langendorff apparatus with KH solution aerated with 95% O2 and 5% CO2 at 37℃.Twenty-four isolated rat hearts were randomly divided into 3 groups （n =8 each） using a random number table：I/R group,NGF-β pretreatment group （group NGFPt） and NGF-β preconditioning group （group NGFPc）.The hearts were perfused with K-H solution for 10 min （equilibration） in each group.In group I/R,the hearts were continuously perfused with K-H solution for 30 min.The hearts were continuously perfused with K-H solution containing NGF 0.1 μg/ml for 20 min before ischemia in group NGF-Pt.The hearts were continuously perfused with K-H solution containing NGF 0.1 μg/ml for 20 min followed by 10 min washout before ischemia in group NGFPc.The perfusion was suspended for 30 min followed by 120 min of reperfusion with K-H solution in each group.HR,left ventricular end-diastolic pressure （LVEDP）,left ventricular developed pressure （LVDP） and ＋ dp/dtmax were measured at the end of 10 min equilibration （baseline,T1）,immediately before suspension of perfusion （T2）,and at 5,30,60 and 120 min of reperfusion （T3-6）.The activities of creatine kinase isoenzyme-MB （CK-MB） and lactate dehydrogenase （LDH） in coronary effluent were measured at T1 and T3-6.Myocardial specimens were obtained at T6 for detection of myocardial apoptosis （by TUNEL） and for microscopic examination.Apoptotic index （AI） was calculated.Results Compared with the baseline value at T1,＋ dp/dtmax was significantly decreased,and LVEDP and activities of CK-MB and LDH were increased at T3-6 in each group,LVDP and HR were decreased at T3-6 in group I/R,LVDP was decreased at T3,4 in group NGFPt and at T3-6 in group NGFPc,and HR was increased at T2-6 in NGFPt and NGF Pc groups （P 〈 0.05）.Compared with group I/R,LVDP,＋ dp/dtmax and HR were significantly increased and LVEDP and activities of CK-MB and LDH and AI were decreased in NGFPt and NGFPc groups （P 〈 0.05）.Compared with group NGFPc,LVDP,＋ dp/dtmax and HR were significantly increased,while the LDH activity and AI were decreased （P 〈 0.05） and no significant changes were found in LVEDP and CK-MB activity in group NGFPt （P 〉 0.05）.The pathologic changes of myocardium were significantly reduced in NGFPt and NGFPc groups as compared with I/R group.Conclusion Pretreatment with 0.1 μg/ml NGF-β attenuates I/R injury in isloated rat hearts,and the efficacy is superior to that of NGF-β preconditioning. Keywords：Nerve growth factor; Ischemic preconditioning; Myocardial reperfusion injury; Pretreatment