高建波， 张 颖， 娄建石△

(1天津市药品检验所, 2天津医科大学药理学教研室，天津 300070)

·短篇论著·

无创性延迟肢体缺血预适应对大鼠缺血再灌注损伤心肌持续保护效应*

高建波1,2， 张 颖2， 娄建石2△

(1天津市药品检验所,2天津医科大学药理学教研室，天津 300070)

目的探讨连续不同天数的无创性肢体缺血预适应对大鼠缺血再灌注损伤心肌的延迟保护持续时间的差别。方法雄性Wistar 大鼠随机分为：(1)对照组：包括假手术组、缺血再灌注组、心肌缺血预适应组和股动脉缺血预适应组；(2)无创性延迟肢体缺血预适应组：包括1 d远端肢体缺血预适应间隔1 d组、3 d组和5 d组，3 d远端肢体缺血预适应间隔1 d组、3 d组和5 d组，7 d远端肢体缺血预适应间隔1 d组、3 d组和5 d组。每组分别在间隔末期进行缺血再灌注损伤，监测缺血再灌注期间左心室功能、心律失常情况和S-T段升高幅度，并检测再灌注结束时心型脂肪酸结合蛋白(heart fatty acid binding protein, H-FABP)、糖原磷酸化酶BB (glycogen phosphorylase BB,GPBB)及心肌梗死面积。结果与缺血再灌注组相比，心肌缺血预适应组、股动脉缺血预适应组、3 d和7 d远端肢体缺血预适应间隔1 d组明显改善左心室功能，减少心律失常，降低S-T段抬高幅度，降低H-FABP和GPBB活性，减少心肌梗死面积。结论远端肢体缺血预适应3 d与7 d可获得相似的心肌持续保护效应。

肢体缺血预适应； 心肌缺血再灌注； 心肌保护

无创性远端肢体缺血预适应是在有创性预适应的基础上发展而来的操作简单、损伤小、易重复的预适应方法，具有广阔的临床应用前景。已有研究表明，它可以增强肾、脑、肺、肝、心等多种器官抵抗缺血再灌注(ischemia-reperfusion, I/R)的能力[1-4]，其中，肢体缺血预适应所取得的延迟保护效应即无创性延迟肢体缺血预适应(noninvasive delayed limb ischemic preconditioning, NDLIP)由于持续时间较长而日益成为研究的热点[5-7]。我们前期的研究结果表明大鼠左后肢每天5 min缺血和5 min再灌注，3个循环，连续3 d可以取得明显的保护心肌效应[8-9]。我们设想，增加预适应天数是否可以延长保护效应？因此，我们进行了本研究。

材 料 和 方 法

1动物

雄性Wistar大鼠，250～290 g，购自军事医学科学院动物实验中心，合格证号为SCXK(军)2009-003。

2试剂

心型脂肪酸结合蛋白(heart fatty acid-binding protein, H-FABP)和糖原磷酸化酶BB (glycogen phosphorylase BB, GPBB)活性测试剂盒均购自Abnova公司。

3仪器

BL-420E生物信号系统和HX-300动物呼吸机，均购自成都泰盟科技有限公司。

4方法

雄性Wistar 大鼠随机分为13组(n=8)：(1)假手术(sham)组：左冠状动脉前降支(left anterior descending coronary artery, LAD) 穿线、旷置；(2) I/R组：大鼠行LAD 30 min缺血，继之120 min再灌注；(3) 心肌缺血预适应(myocardial ischemic preconditioning,MIPC)组：大鼠行LAD 5 min缺血，5 min再灌注，3个循环，继之30 min缺血，120 min再灌注；(4) 股动脉缺血预适应(femoral artery ischemic preconditioning, FAIP)组：大鼠行左后肢股动脉 5 min缺血，5 min再灌注，3个循环，继之行LAD 30 min缺血，120 min再灌注；(5)NDLIP组，包括1 d远端肢体缺血预适应间隔1 d组(NDLIP1d+1d)、3 d组(NDLIP1d+3d)和5 d组(NDLIP1d+5d)，3 d远端肢体缺血预适应间隔1 d组(NDLIP3d+1d)、3 d组(NDLIP3d+3d)和5 d组(NDLIP3d+5d)，7 d远端肢体缺血预适应间隔1 d组(NDLIP7d+1d)、3 d组(NDLIP7d+3d)和5 d组(NDLIP7d+5d)，每组分别在间隔末期进行缺血再灌注损伤。

NDLIP操作：将大鼠用戊巴比妥钠溶液腹腔注射(30 mg/kg)麻醉，以改良的动物无创血压测试仪自制套管套住大鼠左后肢根部，通过监测足背动脉血压、脉搏确定操作是否成功，缺血5 min，再灌5 min，每次连续3个循环，每天1次。

手术操作：按Li等[8]和Shahid等[10]描述的方法进行，腹腔注射乌拉坦(1 g/kg)麻醉大鼠，剥离右侧颈总动脉，将导管经由该动脉插入左心室，导管另一端连接生物信号记录仪。气管插管，于开胸后连接动物呼吸机，每分55～60循环，潮气量7～8 mL/kg。开胸后，3-0号线在左前降支下穿过，丝线两端共同穿过末端为球形的聚乙烯小管，形成一活结。拉紧活结造成LAD 供血区心肌缺血，放松活结使心肌恢复血流灌注。大鼠行LAD 30 min缺血，继之120 min再灌注。监测左室末期舒张压(left ventricular end-diastolic pressure, LVEDP)、左室压收缩压 (left ventricular systolic pressure, LVSP)、左室压最大上升速率 (the maximal rate of rise of left ventricular pressure, dp/dtmax)、ST段、室性早搏(ventricular premature contraction, VPC) 及室性心动过速(ventricular tachycardia, VT) 的出现和持续时间，按Lambeth Conventions 标准进行判定[11]。再灌注末取静脉血测定H-FABP和GPBB活性，TTC染色检测心肌梗死区面积(infarct size, IS) 和危险区面积(area at risk, AAR)的比值。

5统计学处理

数据用均值±标准差(mean±SD)表示，采用t检验或单因素方差进行均数比较，使用SPSS 11.5 软件，以P<0.05为差异有统计学意义。

结 果

1对照组

与I/R组相比，MIPC和FAIP组dp/dtmax显著升高，LVEDP明显降低(P<0.01),见表1；S-T段升高幅度及IS/AAR 显著降低(P<0.01)，见图1、表2；VPC和VT的发生时间推迟，持续时间缩短(P<0.01)，见图2～5；H-FABP 和GPBB活性降低(P<0.05)，见图6、7。

表1缺血再灌注期间左心室功能的变化

Table 1. Left ventricular function changes during ischemia and reperfusion (Mean±SD.n=8)

GroupLVSP(mmHg)LVEDP(mmHg)dp/dtmax(mmHg/s)Sham107.3±10.5**10.4±2.1**4069.8±670.1**I/R85.4±13.223.3±6.22415.9±363.5MIPC100.3±12.014.3±3.4**3279.1±552.1**FAIP100.5±13.114.2±3.6**3327.6±748.5**NDLIP1d+1d87.9±14.620.2±5.02733.5±452.7NDLIP3d+1d82.8±18.820.4±7.72705.8±918.1NDLIP7d+1d83.1±17.321.2±6.32512.6±888.1NDLIP1d+3d100.4±11.7*15.8±4.6*3047.3±678.2*NDLIP3d+3d89.1±13.218.7±5.02787.8±939.8NDLIP7d+3d88.4±13.920.4±4.92449.6±539.0NDLIP1d+5d97.6±5.8*15.5±5.1*3151.4±697.8*NDLIP3d+5d90.4±11.319.1±5.32830.4±614.1NDLIP7d+5d83.6±18.023.4±6.22646.1±730.0

*P<0.05,**P<0.01vsI/R group.

Figure 1. ST-segment changes during ischemia.I/R: ischemia-reperfusion;MIPC: myocardial ischemic preconditioning;FAIP: femoral artery ischemic preconditioning;NDLIP: noninvasive delayed limb ischemic preconditioning.Mean±SD.n=8.**P<0.01vsI/R group.

图1缺血期ST段变化

Figure 2. Onset of ventricular premature contraction (VPC) during ischemia.I/R: ischemia-reperfusion; MIPC: myocardial ischemic preconditioning;FAIP: the femoral artery ischemic preconditioning; NDLIP: noninvasive delayed limb ischemic preconditioning.Mean±SD.n=8.**P<0.01vsI/R group.

图2缺血期房早的出现时间

Figure 3. Onset of ventricular tachycardia (VT) during ischemia.I/R: ischemia-reperfusion; MIPC: myocardial ischemic preconditioning; FAIP: the femoral artery ischemic preconditioning; NDLIP: noninvasive delayed limb ischemic preconditioning.Mean±SD.n=8.**P<0.01vsI/R group.

图3缺血期室早的出现时间

Figure 4. Duration of ventricular premature contraction (VPC) during ischemia. I/R: ischemia-reperfusion;MIPC: myocardial ischemic preconditioning;FAIP: the femoral artery ischemic preconditioning;NDLIP: noninvasive delayed limb ischemic preconditioning.Mean±SD.n=8.**P<0.01vsI/R group.

图4缺血期房早的持续时间

Figure 5. Duration of ventricular tachycardia (VT) during ischemia.I/R: ischemia-reperfusion;MIPC: myocardial ischemic preconditioning;FAIP: the femoral artery ischemic preconditioning;NDLIP: noninvasive delayed limb ischemic preconditioning.Mean±SD.n=8.**P<0.01vsI/R group.

图5缺血期室早的持续时间

Figure 6. Activity of heart fatty acid-binding protein (H-FABP) after ischemia and reperfusion. I/R: ischemia-reperfusion; MIPC: myocardial ischemic preconditioning;FAIP: the femoral artery ischemic preconditioning;NDLIP: noninvasive delayed limb ischemic preconditioning. Mean±SD.n=8.*P<0.05vsI/R group.

图6缺血再灌注末期心型脂肪酸结合蛋白的活性

Figure 7. Activity of glycogen phosphorylase BB (GPBB) after ischemia and reperfusion. I/R: ischemia-reperfusion;MIPC: myocardial ischemic preconditioning;FAIP: the femoral artery ischemic preconditioning;NDLIP: noninvasive delayed limb ischemic preconditioning.Mean±SD.n=8.*P<0.05vsI/R group.

图7缺血再灌注末期糖原磷酸化酶同功酶BB的活性

2无创性延迟肢体缺血预适应组

与I/R组相比，1 d肢体缺血预适应不能提供有效的心肌保护，3 d和7 d远端肢体缺血预适应间隔1 d组可以取得与MIPC和FAIP相似的心肌保护效应(P<0.05)，且组间相比无明显差别；间隔3 d后心肌保护效应消失，见表1、2和图1～7。

讨 论

肢体缺血预适应仅1 d尚不能提供有效的心肌延迟保护，表明机体产生足够的内源性延迟保护物质需要一定的时间，不是短时完成的；而连续3 d肢体缺血预适应后，间隔1 d可提供与MIPC和FAIP相似程度的心肌保护效应，间隔3 d后，其保护作用消失，表明肢体缺血预适应3 d后方可产生足量的物质进而提供内源性心肌保护，且其保护效应最多维持3 d；增加预适应天数至7 d，与预适应3 d比较，并没有延长保护时间，其心肌保护作用同样于间隔3 d后基本消失，表明预适应3 d内源性保护物质的生成与消耗既可达到平衡，再延长肢体预适应时间并不能加强其产生并延长心肌保护的维持时间。连续预

表2缺血期梗死面积/缺血面积比值的变化

Table 2. Infarct size (IS)/ area at risk (AAR) changes during ischemia(Mean±SD.n=8)

GroupsIS/AARSham0.099±0.035**I/R0.343±0.066MIPC0.195±0.020**FAIP0.196±0.046**NDLIP1d+1d0.327±0.061NDLIP3d+1d0.340±0.048NDLIP7d+1d0.343±0.066NDLIP1d+3d0.212±0.054**NDLIP3d+3d0.290±0.053NDLIP7d+3d0.311±0.058NDLIP1d+5d0.202±0.041**NDLIP3d+5d0.298±0.091NDLIP7d+5d0.299±0.093

**P<0.01vsI/R group.

适应不同天数后间隔3 d心肌保护作用均消失，表明通过无创性肢体缺血预适应获得的延迟保护的持续时间不超过3 d。

H-FABP和GPBB是近年来尝试用于检测心肌损伤程度的生化指标[12-15]。本研究表明血清H-FABP和GPBB的活性有相同的变化趋势，与心肌梗死呈正相关，与预适应的心肌保护强度呈负相关，这说明H-FABP和GPBB有望作为临床检测心肌梗死及判断预适应提供心肌保护效应强度的指标。

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Durativeprotectionofnoninvasivedelayedlimbischemicpreconditioningagainstmyocardialischemia-reperfusioninjuryinrats

GAO Jian-bo1,2, ZHANG Ying2, LOU Jian-shi2

(1TianjinInstituteforDrugControl,2DepartmentofPharmacology,TianjinMedicalUniversity,Tianjin300070,China.E-mail:jianshilou@126.com)

AIM: To test the difference of the duration of delayed protection among different continuous days of noninvasive delayed limb ischemic preconditioning (NDLIP) for cardioprotection against ischemia-reperfusion (I/R) injury in rats.METHODSMale Wistar rats were randomized into the following groups: (1) control groups, including sham operation, I/R, myocardial ischemic preconditioning (MIPC) and femoral artery ischemic preconditioning (FAIP) groups; (2) NDLIP groups, including 1-day NDLIP plus 1-day interval (NDLIP1d+1d), 1-day NDLIP plus 3-day interval (NDLIP1d+3d), 1-day NDLIP plus 5-day interval (NDLIP1d+5d), 3-day NDLIP plus 1-day interval (NDLIP3d+1d), 3-day NDLIP plus 3-day interval (NDLIP3d+3d), 3-day NDLIP plus 5-day interval (NDLIP3d+5d), 7-day NDLIP plus 1-day interval (NDLIP7d+1d), 7-day NDLIP plus 3-day interval (NDLIP7d+3d) and 7-day NDLIP plus 5-day interval (NDLIP7d+5d) groups. Myocardial I/R injury was performed at the end of the intervals. The left ventricular function, ventricular arrhythmia incidence and ST-segment were measured during I/R. Myocardial infarct size, heart fatty acid-bin-ding protein (H-FABP) and glycogen phosphorylase BB (GPBB) were determined at the end of the experiment.RESULTSCompared with I/R group, MIPC, FAIP, NDLIP3d+1d and NDLIP7d+1d groups showed attenuated ventricular arrhythmia, improved left ventricular function, lowered ST-segment elevation, reduced myocardial infarct size, and decreased H-FABP and GPBB activity.CONCLUSIONNDLIP for 3 and 7 days can provide the similar duration of cardioprotection in rats.

Limb ischemic preconditioning; Myocardial ischemia-reperfusion; Cardioprotection

R96

A

1000- 4718(2013)09- 1691- 05

2013- 04- 02

2013- 07- 18

国家自然科学基金资助项目(No.81072631); 天津市自然科学基金资助项目(No.09JCZDJC21100)

△通讯作者 Tel: 022-23513782; E-mail: jianshilou@126.com

10.3969/j.issn.1000- 4718.2013.09.027