無創性延遲肢體缺血預適應對大鼠缺血再灌注損傷心肌持續保護效應*

高建波， 張 穎， 婁建石△

(1天津市藥品檢驗所, 2天津醫科大學藥理學教研室，天津 300070)

·短篇論著·

無創性延遲肢體缺血預適應對大鼠缺血再灌注損傷心肌持續保護效應*

高建波1,2， 張 穎2， 婁建石2△

(1天津市藥品檢驗所,2天津醫科大學藥理學教研室，天津 300070)

目的探討連續不同天數的無創性肢體缺血預適應對大鼠缺血再灌注損傷心肌的延遲保護持續時間的差別。方法雄性Wistar 大鼠隨機分為：(1)對照組：包括假手術組、缺血再灌注組、心肌缺血預適應組和股動脈缺血預適應組；(2)無創性延遲肢體缺血預適應組：包括1 d遠端肢體缺血預適應間隔1 d組、3 d組和5 d組，3 d遠端肢體缺血預適應間隔1 d組、3 d組和5 d組，7 d遠端肢體缺血預適應間隔1 d組、3 d組和5 d組。每組分別在間隔末期進行缺血再灌注損傷，監測缺血再灌注期間左心室功能、心律失常情況和S-T段升高幅度，并檢測再灌注結束時心型脂肪酸結合蛋白(heart fatty acid binding protein, H-FABP)、糖原磷酸化酶BB (glycogen phosphorylase BB,GPBB)及心肌梗死面積。結果與缺血再灌注組相比，心肌缺血預適應組、股動脈缺血預適應組、3 d和7 d遠端肢體缺血預適應間隔1 d組明顯改善左心室功能，減少心律失常，降低S-T段抬高幅度，降低H-FABP和GPBB活性，減少心肌梗死面積。結論遠端肢體缺血預適應3 d與7 d可獲得相似的心肌持續保護效應。

肢體缺血預適應； 心肌缺血再灌注； 心肌保護

無創性遠端肢體缺血預適應是在有創性預適應的基礎上發展而來的操作簡單、損傷小、易重復的預適應方法，具有廣闊的臨床應用前景。已有研究表明，它可以增強腎、腦、肺、肝、心等多種器官抵抗缺血再灌注(ischemia-reperfusion, I/R)的能力[1-4]，其中，肢體缺血預適應所取得的延遲保護效應即無創性延遲肢體缺血預適應(noninvasive delayed limb ischemic preconditioning, NDLIP)由于持續時間較長而日益成為研究的熱點[5-7]。我們前期的研究結果表明大鼠左后肢每天5 min缺血和5 min再灌注，3個循環，連續3 d可以取得明顯的保護心肌效應[8-9]。我們設想，增加預適應天數是否可以延長保護效應？因此，我們進行了本研究。

材 料 和 方 法

1動物

雄性Wistar大鼠，250～290 g，購自軍事醫學科學院動物實驗中心，合格證號為SCXK(軍)2009-003。

2試劑

心型脂肪酸結合蛋白(heart fatty acid-binding protein, H-FABP)和糖原磷酸化酶BB (glycogen phosphorylase BB, GPBB)活性測試劑盒均購自Abnova公司。

3儀器

BL-420E生物信號系統和HX-300動物呼吸機，均購自成都泰盟科技有限公司。

4方法

雄性Wistar 大鼠隨機分為13組(n=8)：(1)假手術(sham)組：左冠狀動脈前降支(left anterior descending coronary artery, LAD) 穿線、曠置；(2) I/R組：大鼠行LAD 30 min缺血，繼之120 min再灌注；(3) 心肌缺血預適應(myocardial ischemic preconditioning,MIPC)組：大鼠行LAD 5 min缺血，5 min再灌注，3個循環，繼之30 min缺血，120 min再灌注；(4) 股動脈缺血預適應(femoral artery ischemic preconditioning, FAIP)組：大鼠行左后肢股動脈 5 min缺血，5 min再灌注，3個循環，繼之行LAD 30 min缺血，120 min再灌注；(5)NDLIP組，包括1 d遠端肢體缺血預適應間隔1 d組(NDLIP1d+1d)、3 d組(NDLIP1d+3d)和5 d組(NDLIP1d+5d)，3 d遠端肢體缺血預適應間隔1 d組(NDLIP3d+1d)、3 d組(NDLIP3d+3d)和5 d組(NDLIP3d+5d)，7 d遠端肢體缺血預適應間隔1 d組(NDLIP7d+1d)、3 d組(NDLIP7d+3d)和5 d組(NDLIP7d+5d)，每組分別在間隔末期進行缺血再灌注損傷。

NDLIP操作：將大鼠用戊巴比妥鈉溶液腹腔注射(30 mg/kg)麻醉，以改良的動物無創血壓測試儀自制套管套住大鼠左后肢根部，通過監測足背動脈血壓、脈搏確定操作是否成功，缺血5 min，再灌5 min，每次連續3個循環，每天1次。

手術操作：按Li等[8]和Shahid等[10]描述的方法進行，腹腔注射烏拉坦(1 g/kg)麻醉大鼠，剝離右側頸總動脈，將導管經由該動脈插入左心室，導管另一端連接生物信號記錄儀。氣管插管，于開胸后連接動物呼吸機，每分55～60循環，潮氣量7～8 mL/kg。開胸后，3-0號線在左前降支下穿過，絲線兩端共同穿過末端為球形的聚乙烯小管，形成一活結。拉緊活結造成LAD 供血區心肌缺血，放松活結使心肌恢復血流灌注。大鼠行LAD 30 min缺血，繼之120 min再灌注。監測左室末期舒張壓(left ventricular end-diastolic pressure, LVEDP)、左室壓收縮壓 (left ventricular systolic pressure, LVSP)、左室壓最大上升速率 (the maximal rate of rise of left ventricular pressure, dp/dtmax)、ST段、室性早搏(ventricular premature contraction, VPC) 及室性心動過速(ventricular tachycardia, VT) 的出現和持續時間，按Lambeth Conventions 標準進行判定[11]。再灌注末取靜脈血測定H-FABP和GPBB活性，TTC染色檢測心肌梗死區面積(infarct size, IS) 和危險區面積(area at risk, AAR)的比值。

5統計學處理

數據用均值±標準差(mean±SD)表示，采用t檢驗或單因素方差進行均數比較，使用SPSS 11.5 軟件，以P<0.05為差異有統計學意義。

結 果

1對照組

與I/R組相比，MIPC和FAIP組dp/dtmax顯著升高，LVEDP明顯降低(P<0.01),見表1；S-T段升高幅度及IS/AAR 顯著降低(P<0.01)，見圖1、表2；VPC和VT的發生時間推遲，持續時間縮短(P<0.01)，見圖2～5；H-FABP 和GPBB活性降低(P<0.05)，見圖6、7。

表1缺血再灌注期間左心室功能的變化

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.

圖1缺血期ST段變化

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.

圖2缺血期房早的出現時間

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.

圖3缺血期室早的出現時間

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.

圖4缺血期房早的持續時間

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.

圖5缺血期室早的持續時間

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.

圖6缺血再灌注末期心型脂肪酸結合蛋白的活性

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.

圖7缺血再灌注末期糖原磷酸化酶同功酶BB的活性

2無創性延遲肢體缺血預適應組

與I/R組相比，1 d肢體缺血預適應不能提供有效的心肌保護，3 d和7 d遠端肢體缺血預適應間隔1 d組可以取得與MIPC和FAIP相似的心肌保護效應(P<0.05)，且組間相比無明顯差別；間隔3 d后心肌保護效應消失，見表1、2和圖1～7。

討 論

肢體缺血預適應僅1 d尚不能提供有效的心肌延遲保護，表明機體產生足夠的內源性延遲保護物質需要一定的時間，不是短時完成的；而連續3 d肢體缺血預適應后，間隔1 d可提供與MIPC和FAIP相似程度的心肌保護效應，間隔3 d后，其保護作用消失，表明肢體缺血預適應3 d后方可產生足量的物質進而提供內源性心肌保護，且其保護效應最多維持3 d；增加預適應天數至7 d，與預適應3 d比較，并沒有延長保護時間，其心肌保護作用同樣于間隔3 d后基本消失，表明預適應3 d內源性保護物質的生成與消耗既可達到平衡，再延長肢體預適應時間并不能加強其產生并延長心肌保護的維持時間。連續預

表2缺血期梗死面積/缺血面積比值的變化

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.

適應不同天數后間隔3 d心肌保護作用均消失，表明通過無創性肢體缺血預適應獲得的延遲保護的持續時間不超過3 d。

H-FABP和GPBB是近年來嘗試用于檢測心肌損傷程度的生化指標[12-15]。本研究表明血清H-FABP和GPBB的活性有相同的變化趨勢，與心肌梗死呈正相關，與預適應的心肌保護強度呈負相關，這說明H-FABP和GPBB有望作為臨床檢測心肌梗死及判斷預適應提供心肌保護效應強度的指標。

[1] Wever KE, Warlé MC, Wagener FA, et al. Remote ischaemic preconditioning by brief hind limb ischaemia protects against renal ischaemia-reperfusion injury: the role of adenosine[J]. Nephrol Dial Transplant, 2011, 26(10):3108-3117.

[2] Yuan HJ, Zhu XH, Luo Q, et al. Noninvasive delayed limb ischemic preconditioning in rats increases antioxidant activities in cerebral tissue during severe ischemia-reperfusion injury[J]. J Surg Res, 2012, 174(1):176-183.

[3] 王良榮，鄭瀏璞，蔣柳明，等. 無創性缺血預處理對肢體缺血再灌注性肺換氣功能損傷及一氧化氮/內皮素-1失衡的影響[J]. 中國病理生理雜志，2010， 26(2):322-326.

[4] Cao Y, Zhang SZ, Zhao SQ, et al. The mitochondrial Ca2+-activated K+channel contributes to cardioprotection by limb remote ischemic preconditioning in rat[J]. Life Sci, 2011, 88(23-24):1026-1030.

[5] Thielmann M, Wendt D, Tsagakis K, et al. Remote ischemic preconditioning: the surgeon’s perspective[J]. J Cardiovasc Med (Hagerstown), 2013,14(3):187-192.

[6] Czigány Z, Turóczi Z, Bulhardt O, et al.Remote ischemic conditioning: short-term effects on rat liver ischemic-reperfusion injury[J]. Orv Hetil, 2012,153(40):1579-1587.

[7] Li C, Li YS, Xu M, et al. Limb remote ischemic preconditioning for intestinal and pulmonary protection during elective open infrarenal abdominal aortic aneurysm repair: a randomized controlled trial[J]. Anesthesiology,2013,118(4):842-852.

[8] Li SJ, Wu YN, Kang Y, et al. Noninvasive limb ischemic preconditioning protects against myocardial I/R injury in rats[J]. J Surg Res, 2010, 164(1):162-168.

[9] Wu YN, Yu H, Zhu XH, et al. Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial KATPchannel-dependent mechanism[J]. Physiol Res, 2011, 60(2):271-279.

[10] Shahid M, Tauseef M, Sharma KK, et al. Brief femoral artery ischaemia provides protection against myocardial ischaemia-reperfusion injury in rats: the possible mechanisms[J]. Exp Physiol, 2008, 93(8):954-968.

[11] Walker MJ, Curtts MJ, Hearse DJ, et al. The Lambeth Conventions: guidelines for the study of arrhythmias in ischemia infarction, and reperfusion[J]. Cardiovasc Res,1988, 22(7): 447-455.

[12] Williams K, George K, Hulton A, et al. A unique case series of novel biomarkers of cardiac damage in cyclists completing the 4800 km Race Across America (RAAM) [J]. Curr Med Chem, 2011, 18(23):3446-3451.

[13] Lippi G, Schena F, Montagnana M, et al. Influence of acute physical exercise on emerging muscular biomarkers[J]. Clin Chem Lab Med, 2008, 46(9):1313-1318.

[14] Horacek JM, Vasatova M, Tichy M, et al. The use of cardiac biomarkers in detection of cardiotoxicity associated with conventional and high-dose chemotherapy for acute leukemia[J]. Exp Oncol, 2010, 32(2):97-99.

[15] 劉海波，郭小芳，張麗梅，等. 心肌脂肪酸結合蛋白濃度對急性冠脈綜合征臨床轉歸的預測價值[J]. 中國病理生理雜志, 2011，27(2): 238-242.

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

國家自然科學基金資助項目(No.81072631); 天津市自然科學基金資助項目(No.09JCZDJC21100)

△通訊作者 Tel: 022-23513782; E-mail: jianshilou@126.com

10.3969/j.issn.1000- 4718.2013.09.027