張?jiān)剖?滕天明 康毅 崔健 夏釗 張文娟

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·基礎(chǔ)研究·

藥物后處理對離體大鼠心肌缺血再灌注損傷影響的對比研究

張?jiān)剖?滕天明 康毅 崔健 夏釗 張文娟

急性心肌梗死(acute myocardial infarction)是嚴(yán)重影響人類健康和生活質(zhì)量的常見病、多發(fā)病。及早恢復(fù)冠狀動脈血流是治療的關(guān)鍵。但心肌在恢復(fù)血供后，可出現(xiàn)一系列再灌注損傷。因此，探討如何減輕缺血再灌注損傷是目前研究的熱點(diǎn)。預(yù)處理雖然可顯著減輕再灌注損傷，但因心肌缺血難以預(yù)測，從而限制了預(yù)處理的應(yīng)用。2005年，Staat等[1]首次將缺血后處理應(yīng)用到臨床，指出缺血后處理組與對照組相比肌酸激酶的釋放曲線下面積明顯減少，即心肌梗死面積減少36%。本實(shí)驗(yàn)室已經(jīng)驗(yàn)證缺血后處理可有效減輕缺血再灌注心肌組織的壞死、改善心臟泵功能以及降低再灌注心律失常(reperfusion arrhythmia,RA)的發(fā)生率[2]。

1 材料與方法

1.1 材料與試劑

1.2.1 離體大鼠心肌缺血/再灌注模型制作 Wistar大鼠(雄性)，水合氯醛(40 mg/kg)、肝素5000 U/kg腹腔注射，麻醉完全后開胸迅速取出心臟置于4℃ K-H液中，快速擠出心腔內(nèi)殘留血液，然后將其迅速套扎于離體心臟灌流系統(tǒng)，保持灌注壓力為90 cmH2O(1 cmH2O=0.098 kPa)， 37℃ K-H液持續(xù)灌流。迅速連接BL-420S系統(tǒng)，用容積導(dǎo)體法記錄心電圖。在靠近肺動脈圓錐處用3/0號醫(yī)用縫線的3/8彎針勾繞前降支，待心臟穩(wěn)定片刻后將壓力球囊從左心耳插入左心室內(nèi)，向球囊內(nèi)注入80～100 μl水調(diào)節(jié)壓力前負(fù)荷為5～10 mmHg(1 mmHg=0.133 kPa)。記錄心電圖、左心室壓力變化等指標(biāo)。待心臟穩(wěn)定灌流30 min后，實(shí)驗(yàn)組結(jié)扎前降支，球囊內(nèi)加壓至6 kPa。心電圖可見ST 段抬高，造成心肌梗死模型，結(jié)扎30 min后迅速松開球囊，打開結(jié)扎線。對照組穿線但不進(jìn)行結(jié)扎。待灌流結(jié)束后，部分進(jìn)行1%Evans blue-1% TTC雙染色，測定心肌梗死面積。

N組，正常對照組；I-R組，缺血再灌注組；LAD，前降支圖1 實(shí)驗(yàn)流程示意圖

1.2.3 血流動力學(xué)參數(shù)統(tǒng)計(jì) 根據(jù)左心室壓力曲線，分析記錄穩(wěn)定灌流30 min時(shí)(結(jié)扎前基礎(chǔ)狀態(tài)值)、缺血15 min、缺血30 min、再灌注30 min及再灌注45 min左心室發(fā)展壓(left ventricular developed pressure,LVDP)，LVDP=左心室壓力曲線最高值-左心室壓力曲線最低值。記錄再灌注30 min及45 min時(shí)左心室內(nèi)壓力最大上升/下降速率(±dp/dtmax)，各取10個(gè)連續(xù)心動周期的平均值為基準(zhǔn)。

1.2.4 心電指標(biāo)統(tǒng)計(jì) 觀察各組離體大鼠心臟再灌注期間室性心律失常(VA)出現(xiàn)的時(shí)間和持續(xù)時(shí)間，記錄室性早搏的個(gè)數(shù)，統(tǒng)計(jì)室性心動過速(室速)及心室顫動(室顫)發(fā)生率和總持續(xù)時(shí)間。以在室速或室顫后出現(xiàn)3個(gè)及以上的正常竇性心律作為室速或室顫終止的指標(biāo)。心律失常的評分依據(jù)CURTIS評分方案[7]，記錄各組大鼠RA評分(表1，圖2)。

表1 心律失常CURTIS評分方案

圖2 再灌注心律失常示意圖 A.正常心電圖;B.頻發(fā)室性早搏;C.室性心動過速;D.心室顫動

1.2.5 心肌Evans-blue和TTC雙染色 灌流結(jié)束后將進(jìn)行再灌注組大鼠的前降支重新結(jié)扎，將1% Evans-blue 2 ml經(jīng)主動脈根部逆行注入心臟，洗去浮色后，凍存于-20℃冰箱1 h后取出，沿心臟長軸方向由心尖部向心底部均勻切割成2 mm薄片共7片，置于1% TTC溶液，水浴(37℃)加熱10 min，取出后以10%甲醛溶液浸泡2 h固定染色，區(qū)分正常區(qū)域(深藍(lán)色)、缺血未梗死區(qū)域(紅色，TTC染色陽性)及梗死區(qū)域(灰白色，TTC染色陰性)。用高清數(shù)碼相機(jī)抓拍圖像后，利用Image Proplus 7.0圖像分析軟件計(jì)算心肌梗死區(qū)(infarcted region，IR)及缺血區(qū)域(area at risk，AAR)，以IR/AAR(%)表示心肌梗死面積百分比。

1.3 統(tǒng)計(jì)學(xué)分析

所有數(shù)據(jù)均采用SPSS 22.0統(tǒng)計(jì)軟件進(jìn)行處理。正態(tài)分布的計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示，LVDP、±dp/dtmax采用重復(fù)測量方差分析，心肌梗死面積比較采用單因素方差分析，組間比較采用LSD法檢驗(yàn)。非正態(tài)分布的計(jì)量資料用中位數(shù)和四分位數(shù)[M(Q1，Q3)]表示，組間比較采用Kruskal-Wallis-H秩和檢驗(yàn)，不校正α水準(zhǔn)[8]。計(jì)數(shù)資料比較采用卡方檢驗(yàn)或者Fisher確切概率法。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 各組大鼠左心室壓力變化比較(表2)

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2.2 各組大鼠左心室心功能變化比較(表3)

2.3 各組大鼠再灌注心律失常比較(表4)

2.4 各組大鼠心肌梗死面積比較

3 討論

缺血的組織、器官經(jīng)恢復(fù)血液灌注后不但不能使其功能和結(jié)構(gòu)恢復(fù)，反而加重其功能障礙和結(jié)構(gòu)損傷的現(xiàn)象稱為心肌缺血再灌注損傷(myocardial ischemia-reprefusioninjury，MIRI)。研究表明，MIRI的發(fā)生可能與活性氧的作用、鈣超載、白細(xì)胞的炎癥反應(yīng)作用、心肌細(xì)胞高能磷酸化合物生成障礙以及細(xì)胞內(nèi)酸中毒等相關(guān)[9]。藥物后處理對預(yù)防MIRI的發(fā)生及減輕MIRI帶來的損傷，具有臨床價(jià)值。

表2 各組大鼠不同狀態(tài)下LVDP的比較

表3 各組大鼠不同狀態(tài)下左心室±dp/dtmax比較

表4 各組大鼠再灌注室性心律失常發(fā)生率、時(shí)程及RA評分(n=11)

注：RA，再灌注心律失常；N組，正常對照組； I-R組，缺血再灌注組； a，與I-R組比較，P<0.05

N組，正常對照組；I-R組，缺血再灌注組圖3 各組心臟染色切片

硝酸甘油(nitroglycerin)是硝酸酯類藥物的基礎(chǔ)藥物，臨床上主要用于治療心絞痛、急性心肌梗死、重度高血壓病和冠狀動脈痙攣等疾病[10]。硝酸甘油經(jīng)過線粒體ALDH2等途徑在機(jī)體內(nèi)生物轉(zhuǎn)化生成小分子物質(zhì)即一氧化氮(NO)[11]，NO可以通過G-蛋白偶聯(lián)受體系統(tǒng)的級聯(lián)反應(yīng)舒張血管，發(fā)揮對心血管系統(tǒng)的保護(hù)作用。低濃度硝酸甘油(10-8mol/L)對心肌具有明顯的保護(hù)作用，表現(xiàn)為促進(jìn)心室力學(xué)指標(biāo)恢復(fù)、心肌梗死面積減小及乳酸脫氫酶釋放減少[3]。RA發(fā)生的基本條件是冠狀動脈阻斷15～45 min后進(jìn)行再灌注，缺血時(shí)間過長及過短，其發(fā)生率均降低。本實(shí)驗(yàn)采用結(jié)扎30 min后進(jìn)行再灌注。與I-R組相比，硝酸甘油組在再灌注后壓力降低減緩，心肌梗死面積明顯減少，室顫與室速的發(fā)生率與I-R組比較則差異無統(tǒng)計(jì)學(xué)意義(P>0.05)，但可以縮短室顫與室速的發(fā)生時(shí)間(P<0.05)，總體可以降低RA評分(P<0.05)。

尼可地爾(nicorandil)是一種硝酸酯樣作用的鉀通道開放劑，既可以發(fā)揮開放細(xì)胞膜與線粒體鉀離子通道的作用，也可以發(fā)揮類硝酸酯樣擴(kuò)張冠狀動脈血管的作用。尼可地爾通過激活胞漿內(nèi)鳥苷酸環(huán)化酶(GA)致使環(huán)磷酸鳥苷酸(cGMP)細(xì)胞水平增多和胞漿鈣減少，從而使血管平滑肌松弛，發(fā)揮擴(kuò)張血管的活性作用[16]。尼可地爾可以增加鉀離子從細(xì)胞的流出，使靜息膜電位負(fù)值更大(超極化)，由于膜的超級化致使鈣離子內(nèi)流減少，減少了缺血再灌注部位心肌早期和晚期后除極，因此有抗RA的作用。有實(shí)驗(yàn)研究發(fā)現(xiàn)，尼可地爾在抗心律失常方面效果優(yōu)異，心臟泵功能得到了改善，心肌梗死面積減少[17]。與I-R組比較，尼可地爾組室速、室顫的發(fā)生率均降低(P<0.05)。

腺苷(adenosine)是一種內(nèi)源性嘌呤核苷，同時(shí)也是一種細(xì)胞膜ATP-K+通道開放劑，在機(jī)體內(nèi)參與重要的病理生理過程。根據(jù)腺苷與腺苷受體結(jié)合能力的差異以及對腺苷環(huán)化酶的活性調(diào)控作用不同，腺苷受體分為4個(gè)亞型[18]：A1、A2A、A2B、A3，腺苷可以通過激活腺苷受體參與心肌再灌注損傷的保護(hù)作用。此外，腺苷還可以減輕鈣超載，減輕中性粒細(xì)胞的激活，使超氧陰離子等自由基減少[19]。本實(shí)驗(yàn)中，與I-R組相比，腺苷組在再灌注后壓力降低減緩，心肌梗死面積減少，同時(shí)在減少RA方面效果更佳。

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Comparative study of the effect of different drugs on post-conditioning of myocardial ischemia reperfusion injury on isolated rat heart models

ZHANG Yun-sheng,TENG Tian-ming,KANG Yi,CUI Jian,XIA Zhao,ZHANG Wen-juan.

Cardiology of Tianjin Medical University General Hospital,Tianjin 300052, China

ZHANG Wen-juan，Email：zwjzyy2013@163.com

Objective To observe and compare the effects of nitroglycerin, diltiazem, nicorandil and adenosin on isolated rat myocardial ischemia reperfusion injury modles, and to investigate the protective effect and related mechanisms of respective agents. Methods Sixty-six Wistar rats were randomly divided into six groups: normal group (N Group), ischemia-reperfusion group (I-R group), nitroglycerin+ischemia-reperfusion group, diltiazem+ischemia-reperfusion group, adenosine+ischemia-reperfusion group, nicorandil+ischemia-reperfusion group. The normal group (N group) was given continued perfusion of normal liquid for 150 min; the I-R group was given stable perfusion for 30 min followed by ligating the LAD for 30 min and subsequent reperfusion for 90 min. For the drug post-conditioning groups, the models were given reperfusion with nitroglycerin (10-8mol/L), diltiazem (5 μmo/L)， nicorandil (200 μmo/L) and adenosine (100 μmo/L) for 15 min respectively, and then perfused with K-H liquid for 75 min. Observed and recorded reperfusion arrhythmia (RA), Left ventricular developed pressure (LVDP)，±dp/dtmax by Taimeng (Chengdu) BL-420s system and calculated myocardial infarct size.Results (1) LVDP was lowest in the I-R group compared with the other groups after 30 min and 45 min of reperfusion (P<0.05). In the drug post-conditioning groups after 30 min reperfusion, LVDP in the diltiazem group [(92.68±5.09) mmHgvs. (84.26±3.02) mmHgvs. (83.35±2.88) mmHg] and in the nicorandil group [(88.95±1.75) mmHgvs. (84.26±3.02) mmHgvs. (83.35±2.88) mmHg] was higher than the nitroglycerine and the adenosine treated group. LVDP after 45 min reperfusion in the diltiazem group [(90.39±4.29) mmHgvs. (82.09±4.24) mmHgvs. (80.98±3.89) mmHg] and in the nicorandil group [(86.13±2.38) mmHgvs. (82.09±4.24) mmHgvs. (80.98±3.89) mmHg] was higher than the nitroglycerin and adenosine groups respectively. The diltiazem group has higher LVDP than the nicorandil group after reperfusion (P<0.05). The difference between the nitroglycerin group and the adenosine group was not statistically significant (P>0.05). (2)±dp/dtmax was lowest in the I-R group than the other groups after 30 min and 45 min reperfusion (P<0.05). ±dp/dtmax in the diltiazem group and the nicorandil group were significantly higher than those in the nitroglycerin group and the adenosine group at 30 min and 45 min after reperfusion. ±dp/dtmax of the diltiazem group was higher than the nicorandil group, and the difference was significant (P<0.05). There was no difference between the nitroglycerin group and the adenosine group (P>0.05). (3) Comparison of the RA score showed the score of the IR group [5(3,6), 57.36] was significantly higher than all the drug post-conditioning groups as nicorandil group [1(1,3), 22.05], diltiazem group [3(1,4), 34.77], nitroglycerin group [4(1,4), 45.41] and adenosine group [2(1,3), 23.14] (P=0.000,P=0.000,P=0.004,P=0.000). The nicorandil group had the lowest score. There was no significant difference between the nicorandil group and the adenosine group (P=0.771). (4) Myocardial infarct size was (27.04±2.45)% in the nitroglycerin group, (17.01±1.13)% in the diltiazem group, (47.97±1.22)% in the adenosine group and (34.95±1.25) in the nicorandil group. IR group (55.51±1.43)% had the largest infarct size among all the groups statistical difference (allP<0.01). The infarct size in the diltiazem group was the smallest.Conclusions In the treatment of myocardial ischemia and reperfusion, all of the four drugs(nitroglycerin, diltiazem, nicorandil and adenosine) can reduce myocardial ischemia-reperfusion injury, and play a protective role in ischemic myocardium. Diltiazem showed promising improvement in cardiac pump function and reducing the area of myocardial infarction. In the post-conditioning for RA, nicorandil and adenosine showed better performance.

Drug post-conditioning； Ischemia/reperfusion; Nitroglycerin; Diltiazem; Nicorandi; Adenosine

10.3969/j.issn.1004-8812.2016.10.008

天津市高等學(xué)校科技發(fā)展基金計(jì)劃項(xiàng)目(20110151)

300052 天津，天津醫(yī)科大學(xué)總醫(yī)院心血管內(nèi)科(張?jiān)剖?、滕天明、崔健、夏釗、張文?；天津醫(yī)科大學(xué)藥理學(xué)實(shí)驗(yàn)室(康毅)

張文娟，Email：zwjzyy2013@163.com

R542.2

2016-06-18)