董小莉，譚寧，鄧宇珺

(1.海南省人民醫(yī)院心血管內(nèi)科，海南海口570311；2.廣東省醫(yī)學(xué)科學(xué)院廣東省人民醫(yī)院廣東省心血管病研究所心血管內(nèi)科，廣東廣州510080)

·論著·

BNP對在體大鼠I/R損傷心肌細(xì)胞凋亡及氧化應(yīng)激的影響

董小莉1，譚寧2，鄧宇珺2

(1.海南省人民醫(yī)院心血管內(nèi)科，海南?？?70311；2.廣東省醫(yī)學(xué)科學(xué)院廣東省人民醫(yī)院廣東省心血管病研究所心血管內(nèi)科，廣東廣州510080)

目的 探討腦利鈉肽對心肌缺血再灌注損傷心肌細(xì)胞凋亡的保護(hù)作用。方法將24只SD雄性大鼠隨機(jī)分入對照組(S組)、缺血/再灌注組(I/R組)、BNP0.005組及BNP0.01組，每組6只，制作在體心肌缺血再灌注模型，分別使用上述干預(yù)，再灌注結(jié)束后摘取心臟，檢測心肌標(biāo)本超氧化物歧化酶(Superoxidedismutase，SOD)、丙二醛(Malondiadehyde，MDA)及心肌細(xì)胞凋亡指數(shù)(Apoptotic index，AI)。結(jié)果S組、I/R組、BNP 0.005組、BNP 0.01組SOD分別為(195.78±21.45)U/mg、(84.35±9.03)U/mg、(125.66±18.06)U/mg、(161.83±15.49)U/mg；MDA分別為(2.73±0.41)nmol/mg、(7.36±0.51)nmol/mg、(4.46±0.47)nmol/mg、(3.69±0.38)nmol/mg；AI分別為(3.84±2.53)%/ (43.63±3.70)%/(22.13±2.85)%、(14.21±2.77)%。各組間SOD、MDA活力及AI差異均具有統(tǒng)計(jì)學(xué)意義(P＜0.001)；相較于其他組，BNP各組均具有較高的SOD活力和更低的MDA活力及AI水平(P＜0.001)，而相較于BNP 0.005組，BNP 0.01組SOD活力更高(P=0.001)，MDA活力及AI水平更低(P值分別為0.007和0.012)。結(jié)論BNP后處理可能通過減少氧自由基而對缺血再灌注損傷的心肌具有保護(hù)作用，且這種保護(hù)作用可能與濃度相關(guān)。

心肌缺血再灌注損傷；腦利鈉肽；凋亡；氧化應(yīng)激

早期開通閉塞的冠狀動脈是限制和縮小急性心肌梗死(Acute myocardial infarction，AMI)面積、改善左心室功能的關(guān)鍵，但大量的動物實(shí)驗(yàn)和臨床觀察發(fā)現(xiàn)，再灌注在改善心肌供血的同時(shí)可能加重心肌缺血所造成的損傷，即心肌缺血再灌注損傷(Ischemia-reperfusion injury，IRI)[1-2]。如何減輕IRI已成為心臟介入治療的重要課題。心肌缺血預(yù)處理和缺血后處理是近年來研究的重點(diǎn)，尤其是藥物后處理，因?yàn)槠淇刹僮餍愿鼜?qiáng)，可能具有的臨床意義更大而被廣泛研究。近年的研究發(fā)現(xiàn)，腦利鈉肽(Brain Natriuretic Peptide，BNP)在動物離體心臟及細(xì)胞水平實(shí)驗(yàn)研究中具有類似于缺血預(yù)處理和后處理的抗IRI的作用，可以通過激活環(huán)磷鳥嘌呤核苷(Cyclic guanosinc monophosphate，cGMP)，一氧化氮合酶(Nitric oxide synthase，NOS)等途徑產(chǎn)生一系列心肌保護(hù)作用，如減輕再灌注后心肌抑頓，減少心梗面積及細(xì)胞凋亡等[1-5]。而在體實(shí)驗(yàn)相對于離體及細(xì)胞水平實(shí)驗(yàn)可以更為準(zhǔn)確的反映藥物在體內(nèi)的代謝及作用，但目前尚無關(guān)于BNP后處理對IRI時(shí)心肌細(xì)胞凋亡影響的在體動物實(shí)驗(yàn)報(bào)道，因此本研究通過建立SD大鼠在體心肌I/R損傷模型，探討不同濃度BNP后處理對IRI時(shí)心肌細(xì)胞凋亡的保護(hù)作用及其可能機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)動物分組選用健康雄性Sprague-Dawley(SD)大鼠24只，(300±50)g，隨機(jī)分成4組，每組6只：S組、I/R組、BNP 0.005組及BN P 0.01組，各組間重量差異無統(tǒng)計(jì)學(xué)意義(F=0.051，P=0.985)。后三組分別于缺血后10 min開始經(jīng)尾靜脈恒速泵入生理鹽水、BNP 0.005 μg/(kg·min)及BNP 0.01 μg/(kg·min)直至再灌注結(jié)束。

1.2 大鼠在體心肌缺血-再灌流損傷模型建立根據(jù)參考文獻(xiàn)[6-11]并加以改進(jìn)，建立大鼠在體心肌缺血再灌注損傷模型：在體結(jié)扎冠狀動脈前降支近段使心肌缺血35 min后放開活結(jié)再灌注120 min，實(shí)驗(yàn)過程中持續(xù)心電監(jiān)護(hù)，以再灌注15 min內(nèi)ST段、T波回落＞50%作為冠狀動脈再通的指標(biāo)。不符合上述心電圖標(biāo)準(zhǔn)者剔除出實(shí)驗(yàn)。

1.3 檢測指標(biāo)及方法每只均檢測心肌組織SOD及MDA，其中前3只兼用于檢測心肌細(xì)胞AI。檢測方法分別為黃嘌呤氧化酶法、硫代巴比妥法及TUNEL(Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)法。

1.4 統(tǒng)計(jì)學(xué)方法應(yīng)用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。所有實(shí)驗(yàn)數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差(±s)表示，多組間比較使用單因素方差分析，有顯著差異者用LSD檢驗(yàn)進(jìn)行兩兩比較，以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 各組SOD、MDA及AI水平比較經(jīng)單因素方差分析顯示，各組間SOD、MDA活力及AI差異均具有統(tǒng)計(jì)學(xué)意義(P＜0.01)。相較于其他組，BNP各組均具有較高的SOD活力和更低的MDA及AI水平；而相較于BNP 0.005組，BNP 0.01組SOD活力更高，MDA活力及AI水平更低，上述各項(xiàng)指標(biāo)比較差異均具有統(tǒng)計(jì)學(xué)意義(P＜0.01)，見表1。

表1 各組SOD、MDA及AI(±s)

表1 各組SOD、MDA及AI(±s)

注：與BNP 0.005組比較，aP=0.001；與BNP 0.005組比較，bP=0.007；與BNP 0.005組比較abP=0.012，差異有統(tǒng)計(jì)學(xué)意義；其余各組兩兩比較差異均有統(tǒng)計(jì)學(xué)意義(P值均＜0.001)。

2.2 TUENL法檢測心肌細(xì)胞凋亡心肌冰凍切片(3 μm)TUNEL染色，激光掃描共聚焦顯微鏡下拍照(400×)。正常細(xì)胞核顯示藍(lán)色熒光；凋亡細(xì)胞核顯示藍(lán)綠色熒光。S組(圖1A)僅有數(shù)個(gè)凋亡細(xì)胞，I/R組(圖1B)可見大量凋亡細(xì)胞，BNP 0.005組(圖1C)凋亡細(xì)胞較I/R組明顯減少，BNP 0.01組(圖1D)凋亡細(xì)胞減少更為明顯。

圖1 TUENL法檢心肌細(xì)胞凋亡

3 討論

目前對BNP后處理的相關(guān)動物實(shí)驗(yàn)研究發(fā)現(xiàn)，BNP對缺血再灌注心肌的保護(hù)機(jī)制主要是BNP能與心肌特異性A型利鈉肽受體結(jié)合，通過BNP/GC/ cGMP信號通路，產(chǎn)生應(yīng)激性細(xì)胞保護(hù)作用，增強(qiáng)心肌抗缺血缺氧能力，從而達(dá)到減少IRI所造成的心肌細(xì)胞凋亡、心肌梗死面積及心肌抑頓等保護(hù)作用[12-13]。也有動物實(shí)驗(yàn)報(bào)道BNP對心肌缺血再灌注損傷的保護(hù)作用也通過一氧化氮(NO)的途徑，使線粒體的ATP敏感性鉀通道的開放[14]而獲得。而心肌缺血再灌注時(shí)的心肌組織重新恢復(fù)血流后產(chǎn)生大量活性氧(Active oxygen，ROS)是再灌注損傷最重要的原因之一。

本實(shí)驗(yàn)結(jié)果顯示：BNP后處理各組較I/R組的AI明顯下降，且AI降低幅度與BNP濃度呈正相關(guān)，提示BNP可以減少I/R時(shí)的心肌細(xì)胞凋亡，且該作用與BNP濃度相關(guān)；同時(shí)較之I/R組，BNP后處理各組SOD活性升高，MDA水平降低，提示心肌抗氧化應(yīng)激能力提高，而這種作用在高濃度BNP組中更明顯。由上述結(jié)果可得到以下結(jié)論：BNP后處理減少大鼠在體I/R心肌細(xì)胞凋亡，同時(shí)提高心肌組織SOD活性、降低MDA水平這兩個(gè)間接反映ROS水平下降的指標(biāo)，提示BNP后處理可能通過減輕I/R時(shí)ROS的生成使再灌注時(shí)心肌細(xì)胞凋亡水平下降，進(jìn)而減輕心肌的缺血再灌注損傷，而BNP后處理如何通過ROS途徑減少心肌細(xì)胞凋亡尚需進(jìn)一步研究。

[1]Murry CE,Jennings RB,Reimer KA,et al.Preconditioning with ischemia:a delay of lethal cell injury in ischemic myocardium[J]. Circulation,1986,74(5):1124-1136.

[2]Tsang A,Hausenloy DJ,Mocanu MM,et al.Postconditioning:a form of"modified reperfusion"protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway[J].Circ Res, 2004,95(3):230-232.

[3]Zhao ZQ,Corvera JS,Halkos ME,et al.Inhibition of myocardial injury by ischemic postconditioning during reperfusion:comparison with ischemic preconditioning[J].Am J Physiol Heart Circ Physiol, 2003,285(2):579-588.

[4]Wu B,Jiang H,Lin R,et al.Pretreatment with B-type natriuretic peptide protects the heart from ischemia-reperfusion injury by inhibiting myocardial apoptosis[J].Tohoku J Exp Med,2009,219(2): 107-114.

[5]Ren B,Shen Y,Shao H,et al.Brain natriuretic peptide limits myocardial infarct size dependent of nitric oxide synthase in rats[J]. Clin ChimActa,2007,377(1-2):83-87.

[6]Song DK,Jang Y,Kim JH,et al.Polyphenol(-)-epigallocatechin gallate during ischemia limits infarct size via mitochondrial K (ATP)channel activation in isolated rat hearts[J].J Korean Med Sci,2010,25(3):380-386.

[7]Zhang DW,Liu JG,Feng JT,et al.Effects of effective components compatibility of aqueous extracts of Salviae Miltiorrhizae and Rhizoma Chuanxiong on rat myocardial ischemia/reperfusion injury[J].Zhongguo Wei Zhong Bing Ji Jiu Yi Xue,2010,22(2): 109-112.

[8]謝勇,蔡光先.大鼠心肌缺血再灌注損傷模型的改進(jìn)[J].中華臨床醫(yī)學(xué)研究雜志,2008,14(7):903-904.

[9]Bhindi R,Witting PK,McMahon AC,et al.Rat models of myocardial infarction.Pathogenetic insights and clinical relevance[J]. Thromb Haemost,2006,96(5):602-610.

[10]王硯青,江時(shí)森,劉平,等.大鼠心肌缺血再灌注模型心電圖變化分析[J].醫(yī)學(xué)研究生學(xué)報(bào),2006,8(8):700-702.

[11]Fang WY,Zhao HY.Study on experimental model of coronary artery thrombogenesis and acute myocardial infarction:changes in epicardial ECG,platelet function and biochemistry,and pathology [J].J Tongji Med Univ,1989,9(2):117-120.

[12]Gorbe A,Giricz Z,Szunyog A,et al.Role of cGMP-PKG signaling in the protection of neonatal rat cardiac myocytes subjected to simulated ischemia/reoxygenation[J].Basic Res Cardiol,2010,5(5): 643-650.

[13]Giricz Z G?rbe A,Pipis J,et al.Hyperlipidaemia induced by a high-cholesterol diet leads to the deterioration ofguanosine-3', 5'-cyclic monophosphate/protein kinase G-dependent cardioprotection in rats[J].Br J Pharmacol,2009,158(6):1495-1502.

[14]Sun Y,Zhang Y,Yan M,et al.B-type natriuretic peptide-induced cardioprotection against reperfusion is associated with attenuation of mitochondrial permeability transition[J].Biol Pharm Bull,2009,32 (9):1545-1551.

Effect of BNP on myocardium apoptosis and oxidative stress induced by ischemia/reperfusion injury in rat.

DONG Xiao-li1,TAN Ning2,DENG Yu-jun2.1.Department of Cardiology,People's Hospital of Hainan Province,Haikou 570311,Hainan,CHINA;2.Guangdong Academy of Medical Sciences,People's Hospital of Guangdong Province, Guangdong Provincial Academy of Medicine,Guangzhou 510080,Guangdong,CHINA

ObjectiveTo evaluate the effects of brain natriuretic peptide(BNP)postconditioning on myocardial apoptosis during myocardial ischemia/reperfusion injury in a rat model.MethodsTwenty four male Sprague Dawley rats were randomized into four groups:sham operation group(S group,n=6,the breast tissue was dissected without ligation of left anterior descending branch);ischemic/reperfusion group(I/R group,n=6,ischemia/reperfusion surgery by ligation of left anterior descending branch,followed by saline injected to caudal vein through syringe pump for 10 min);BNP 0.005 group(n=6,0.005 μg/(kg·min)BNP injected to caudal vein by syringe pump for 10 min after ischemia surgery);BNP 0.01 group(n=6,0.005 μg/(kg·min)BNP injected to caudal vein by syringe pump for 10 min after ischemia surgery).At the end of reperfusion,heart was harvested,and myocardium superoxide dismutase(SOD),Malondiadehyde(MDA)and Apoptotic index(AI)were detected.ResultsIn S group,I/R group,BNP 0.005 group and BNP 0.01 group,SOD levels were(195.78±21.45)U/mg,(84.35±9.03)U/mg,(125.66±18.06)U/mg,(161.83±15.49)U/mg,respectively;MDA levels were(2.73±0.41)nmol/mg,(7.36±0.51)nmol/mg,(4.46±0.47)nmol/mg,(3.69±0.38)nmol/mg, respectively;AI were(3.84±2.53)%,(43.63±3.70)%,(22.13±2.85)%,(14.21±2.77)%,respectively.The differences between the groups were all statistically significant(P＜0.001).BNP 0.005 group and BNP 0.01 group had significantly higher SOD activity,lower MDA activity and lower AI level than other groups(P＜0.001).Compared with BNP 0.005 group,BNP 0.01 group had significantly higher SOD activity(P=0.001),lower MDA activity(P=0.007)and lower AI level(P=0.012).ConclusionBNP postconditioning could reduce the oxygen free radicals level to protect the ischemia-reperfusion myocardial injury.This effect is positively correlated with BNP levels.

Myocardial ischemia-reperfusion injury;Brain natriuretic peptide(BNP);Apoptosis;Oxidative stress

R-332

A

1003—6350(2014)21—3124—03

10.3969/j.issn.1003-6350.2014.21.1227

2014-02-15)