無創(chuàng)性延遲肢體缺血預(yù)適應(yīng)對(duì)大鼠缺血再灌注損傷心肌持續(xù)保護(hù)效應(yīng)*

高建波， 張 穎， 婁建石△

(1天津市藥品檢驗(yàn)所, 2天津醫(yī)科大學(xué)藥理學(xué)教研室，天津 300070)

·短篇論著·

無創(chuàng)性延遲肢體缺血預(yù)適應(yīng)對(duì)大鼠缺血再灌注損傷心肌持續(xù)保護(hù)效應(yīng)*

高建波1,2， 張 穎2， 婁建石2△

(1天津市藥品檢驗(yàn)所,2天津醫(yī)科大學(xué)藥理學(xué)教研室，天津 300070)

目的探討連續(xù)不同天數(shù)的無創(chuàng)性肢體缺血預(yù)適應(yīng)對(duì)大鼠缺血再灌注損傷心肌的延遲保護(hù)持續(xù)時(shí)間的差別。方法雄性Wistar 大鼠隨機(jī)分為：(1)對(duì)照組：包括假手術(shù)組、缺血再灌注組、心肌缺血預(yù)適應(yīng)組和股動(dòng)脈缺血預(yù)適應(yīng)組；(2)無創(chuàng)性延遲肢體缺血預(yù)適應(yīng)組：包括1 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)間隔1 d組、3 d組和5 d組，3 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)間隔1 d組、3 d組和5 d組，7 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)間隔1 d組、3 d組和5 d組。每組分別在間隔末期進(jìn)行缺血再灌注損傷，監(jiān)測(cè)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(cè)再灌注結(jié)束時(shí)心型脂肪酸結(jié)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(jié)果與缺血再灌注組相比，心肌缺血預(yù)適應(yīng)組、股動(dòng)脈缺血預(yù)適應(yīng)組、3 d和7 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)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(jié)論遠(yuǎn)端肢體缺血預(yù)適應(yīng)3 d與7 d可獲得相似的心肌持續(xù)保護(hù)效應(yīng)。

肢體缺血預(yù)適應(yīng)； 心肌缺血再灌注； 心肌保護(hù)

無創(chuàng)性遠(yuǎn)端肢體缺血預(yù)適應(yīng)是在有創(chuàng)性預(yù)適應(yīng)的基礎(chǔ)上發(fā)展而來的操作簡(jiǎn)單、損傷小、易重復(fù)的預(yù)適應(yīng)方法，具有廣闊的臨床應(yīng)用前景。已有研究表明，它可以增強(qiáng)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(yù)適應(yīng)所取得的延遲保護(hù)效應(yīng)即無創(chuàng)性延遲肢體缺血預(yù)適應(yīng)(noninvasive delayed limb ischemic preconditioning, NDLIP)由于持續(xù)時(shí)間較長(zhǎng)而日益成為研究的熱點(diǎn)[5-7]。我們前期的研究結(jié)果表明大鼠左后肢每天5 min缺血和5 min再灌注，3個(gè)循環(huán)，連續(xù)3 d可以取得明顯的保護(hù)心肌效應(yīng)[8-9]。我們?cè)O(shè)想，增加預(yù)適應(yīng)天數(shù)是否可以延長(zhǎng)保護(hù)效應(yīng)？因此，我們進(jìn)行了本研究。

材 料 和 方 法

1動(dòng)物

雄性Wistar大鼠，250～290 g，購自軍事醫(yī)學(xué)科學(xué)院動(dòng)物實(shí)驗(yàn)中心，合格證號(hào)為SCXK(軍)2009-003。

2試劑

心型脂肪酸結(jié)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(cè)試劑盒均購自Abnova公司。

3儀器

BL-420E生物信號(hào)系統(tǒng)和HX-300動(dòng)物呼吸機(jī)，均購自成都泰盟科技有限公司。

4方法

雄性Wistar 大鼠隨機(jī)分為13組(n=8)：(1)假手術(shù)(sham)組：左冠狀動(dòng)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(yù)適應(yīng)(myocardial ischemic preconditioning,MIPC)組：大鼠行LAD 5 min缺血，5 min再灌注，3個(gè)循環(huán)，繼之30 min缺血，120 min再灌注；(4) 股動(dòng)脈缺血預(yù)適應(yīng)(femoral artery ischemic preconditioning, FAIP)組：大鼠行左后肢股動(dòng)脈 5 min缺血，5 min再灌注，3個(gè)循環(huán)，繼之行LAD 30 min缺血，120 min再灌注；(5)NDLIP組，包括1 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)間隔1 d組(NDLIP1d+1d)、3 d組(NDLIP1d+3d)和5 d組(NDLIP1d+5d)，3 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)間隔1 d組(NDLIP3d+1d)、3 d組(NDLIP3d+3d)和5 d組(NDLIP3d+5d)，7 d遠(yuǎn)端肢體缺血預(yù)適應(yīng)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(jìn)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(dòng)物無創(chuàng)血壓測(cè)試儀自制套管套住大鼠左后肢根部，通過監(jiān)測(cè)足背動(dòng)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(xù)3個(gè)循環(huán)，每天1次。

手術(shù)操作：按Li等[8]和Shahid等[10]描述的方法進(jìn)行，腹腔注射烏拉坦(1 g/kg)麻醉大鼠，剝離右側(cè)頸總動(dòng)脈，將導(dǎo)管經(jīng)由該動(dòng)脈插入左心室，導(dǎo)管另一端連接生物信號(hào)記錄儀。氣管插管，于開胸后連接動(dòng)物呼吸機(jī)，每分55～60循環(huán)，潮氣量7～8 mL/kg。開胸后，3-0號(hào)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(jié)。拉緊活結(jié)造成LAD 供血區(qū)心肌缺血，放松活結(jié)使心肌恢復(fù)血流灌注。大鼠行LAD 30 min缺血，繼之120 min再灌注。監(jiān)測(cè)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(dòng)過速(ventricular tachycardia, VT) 的出現(xiàn)和持續(xù)時(shí)間，按Lambeth Conventions 標(biāo)準(zhǔn)進(jìn)行判定[11]。再灌注末取靜脈血測(cè)定H-FABP和GPBB活性，TTC染色檢測(cè)心肌梗死區(qū)面積(infarct size, IS) 和危險(xiǎn)區(qū)面積(area at risk, AAR)的比值。

5統(tǒng)計(jì)學(xué)處理

數(shù)據(jù)用均值±標(biāo)準(zhǔn)差(mean±SD)表示，采用t檢驗(yàn)或單因素方差進(jìn)行均數(shù)比較，使用SPSS 11.5 軟件，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1對(duì)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(fā)生時(shí)間推遲，持續(xù)時(shí)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(xiàn)時(shí)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(xiàn)時(shí)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(xù)時(shí)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(xù)時(shí)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(jié)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(chuàng)性延遲肢體缺血預(yù)適應(yīng)組

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

討 論

肢體缺血預(yù)適應(yīng)僅1 d尚不能提供有效的心肌延遲保護(hù)，表明機(jī)體產(chǎn)生足夠的內(nèi)源性延遲保護(hù)物質(zhì)需要一定的時(shí)間，不是短時(shí)完成的；而連續(xù)3 d肢體缺血預(yù)適應(yīng)后，間隔1 d可提供與MIPC和FAIP相似程度的心肌保護(hù)效應(yīng)，間隔3 d后，其保護(hù)作用消失，表明肢體缺血預(yù)適應(yīng)3 d后方可產(chǎn)生足量的物質(zhì)進(jìn)而提供內(nèi)源性心肌保護(hù)，且其保護(hù)效應(yīng)最多維持3 d；增加預(yù)適應(yīng)天數(shù)至7 d，與預(yù)適應(yīng)3 d比較，并沒有延長(zhǎng)保護(hù)時(shí)間，其心肌保護(hù)作用同樣于間隔3 d后基本消失，表明預(yù)適應(yīng)3 d內(nèi)源性保護(hù)物質(zhì)的生成與消耗既可達(dá)到平衡，再延長(zhǎng)肢體預(yù)適應(yīng)時(shí)間并不能加強(qiáng)其產(chǎn)生并延長(zhǎng)心肌保護(hù)的維持時(shí)間。連續(xù)預(yù)

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(yīng)不同天數(shù)后間隔3 d心肌保護(hù)作用均消失，表明通過無創(chuàng)性肢體缺血預(yù)適應(yīng)獲得的延遲保護(hù)的持續(xù)時(shí)間不超過3 d。

H-FABP和GPBB是近年來嘗試用于檢測(cè)心肌損傷程度的生化指標(biāo)[12-15]。本研究表明血清H-FABP和GPBB的活性有相同的變化趨勢(shì)，與心肌梗死呈正相關(guān)，與預(yù)適應(yīng)的心肌保護(hù)強(qiáng)度呈負(fù)相關(guān)，這說明H-FABP和GPBB有望作為臨床檢測(cè)心肌梗死及判斷預(yù)適應(yīng)提供心肌保護(hù)效應(yīng)強(qiáng)度的指標(biāo)。

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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

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

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

10.3969/j.issn.1000- 4718.2013.09.027