趙世杰 齊國(guó)先 田 文 史立業(yè) 陳 玲 盧彥昭
阿托伐他汀預(yù)處理對(duì)缺血再灌注小鼠心肌微血管內(nèi)皮屏障的保護(hù)作用*
趙世杰齊國(guó)先#田文史立業(yè)陳玲盧彥昭
【摘要】目的:探討阿托伐他汀預(yù)處理對(duì)缺血再灌注小鼠心肌微血管血流灌注和滲透性的作用及其機(jī)制。方法:60只昆明小鼠隨機(jī)分為6組:假手術(shù)組(C組)、缺血組(I組)、再灌注組(I/R組)、再灌注前2h阿托伐他汀10mg/kg灌胃組(A102組)、再灌注前2h阿托伐他汀20mg/kg灌胃組(A202組)、再灌注前2h阿托伐他汀20mg/kg灌胃+再灌注前15min L-NAME(N-硝基-L-精氨酸甲酯)15mg/kg尾靜脈注射組(AL202組),每組均10只小鼠。于光鏡下結(jié)扎冠脈制備心肌缺血再灌注模型,以活體冷凍技術(shù)(IVCT)摘取心臟,其中5只小鼠心臟行冷凍置換、石蠟包埋,之后切片行HE染色及Albumin免疫組織化學(xué)染色,并對(duì)間質(zhì)中的Albumin免疫組織化學(xué)染色強(qiáng)度進(jìn)行半定量分析;另5只小鼠采用 Western Blotting檢測(cè)心肌組織內(nèi)皮型一氧化氮合酶(eNOS)、黏附連接蛋白VE-cadherin和緊密連接蛋白Claudin-5的表達(dá)水平。 結(jié)果:與I組及I/R組相比,A202組小鼠心肌細(xì)胞界限較清晰,降落傘型紅細(xì)胞的頂端指向血流方向,Albumin無(wú)明顯向間質(zhì)等部位滲漏。A102組鏡下所見與A202組類似,但微血管內(nèi)紅細(xì)胞數(shù)量、形態(tài)及心肌細(xì)胞界限清晰度等不及A202組,Albumin向間質(zhì)滲漏。AL202組可見心肌細(xì)胞腫脹,微血管內(nèi)紅細(xì)胞減少、缺如,但微循環(huán)血流灌注好于I/R組,Albumin滲漏少于I/R組。與I組和I/R組對(duì)比,A202組和A102組心肌組織中eNOS蛋白表達(dá)均明顯升高(P<0.01),尤以A202組最為明顯(P<0.05)。與A202組相比,AL202組eNOS蛋白表達(dá)降低(P<0.05),且與I/R組差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。與C組相比,I組和I/R組VE-cadherin蛋白和Claudin-5蛋白表達(dá)顯著降低(P<0.05),而A102和A202組VE-cadherin蛋白和Claudin-5蛋白表達(dá)較I組和I/R組明顯升高(P<0.05),尤以A202組升高最為明顯(P<0.05)。AL202組VE-cadherin及Claudin-5蛋白水平較I/R組升高(P<0.05)。結(jié)論:再灌注前短時(shí)間內(nèi)大劑量阿托伐他汀預(yù)處理可改善缺血再灌注心肌微循環(huán)血流灌注及微血管滲透性,其機(jī)制可能不完全依賴于eNOS/NO途徑。
【關(guān)鍵詞】阿托伐他?。蝗毖俟嘧?;微循環(huán);血管滲透性;活體冷凍技術(shù);小鼠
The Protective Effect of Atorvastatin on Microvascular Barrier in Myocardial Ischemia Reperfusion Injury*
ZHAO Shi-jie, QI Guo-xian#, TIAN Wen, SHI Li-ye, CHEN Ling, LU Yan-zhao
Department of Geriatric Cardiology, The First Affiliated Hospital of China Medical University, Shenyang 110001, China;#Corresponding author
【Abstract】Objective: To explore the effect and the probably mechanism of atorvastatin pretreatment before ischemia-reperfusion on epicardial microvascular blood flow and microvascular permeability. Method: 60 mice divided into 6 groups randomly: sham-operated group(C), ischemia group(I), ischemia-reperfusion group(I/R), atorvastatin 10mg/kg gavage 2h before reperfusion group(A102), atorvastatin 20mg/kg gavage 2h before reperfusion group(A202), atorvastatin 20mg/kg gavage 2h before reperfusion+L-NAME 15mg/kg tail vein injection 15min before reperfusion group(AL202). The myocardial ischemia reperfusion model was prepared under light microscopy. IVCT
[作者單位]中國(guó)醫(yī)科大學(xué)附屬第一醫(yī)院老年心血管內(nèi)科, 沈陽(yáng)110001;#通訊作者,Tel:024-83282300;E-mail:qigx2002@medmail.com.cn
本文2015-08-20收到,2015-09-29修回
followed by freeze-substitution fixation was used to prepare the heart sample of half of the mice in each group, and then stained with hematoxylin-eosine (HE) and albumin antibody for light microscopy observation. The heart samples of another half of each group were prepared for the expression test of eNOS, VE-cadherin and Claudin-5 by Western Blotting. Results: Compared with I and I/R group, A202 group showed the structure of microenvironment was more clear, the apiculus of parachute-shape erythrocytes directed the orientation of blood flow, and less leakage of albumin in the blood vessel. A102 group showed a similar effect, but less than A202 group. In AL202 group, the slices showed myocardial microvascular slightly narrow and myocardial cell swelling, but the blood flow and leakage of plasma albumin is still less than I/R group. Compared with I and I/R group, the expression level of eNOS in A202 and A102 group were significantly increased (P<0.01), especially in A202 group (P<0.05).Compared with A202 group, the expression level of eNOS in AL202 group reduced significantly (P<0.05), and there was no statistically significant difference with I/R group (P>0.05).Compared with C group, the expression level of VE-cadherin and Claudin-5 significantly reduced (P<0.05). Meanwhile, compared with I and I/R group, the expression level of VE-cadherin and Claudin-5 increased significantly in A102 and A202 group (P<0.05), especially in A202 group (P<0.05). Furthermore, compared with I/R group, the expression level of VE-cadherin and Claudin-5 were also increased in AL202 group (P<0.05). Conclusion: High dose atorvastatin pretreatment 2h before reperfusion can significantly improve the blood flow state and the plasma albumin leakage in microvascular, and the mechanism may related to the protection effect of atorvastatin on microvascular endothelial cell junction proteins such as VE-cadherin and Claudin-5, which probably does not totally depend on the eNOS/NO pathway.
【Key words】Atorvastatin; Ischemia reperfusion; Microcirculation; Microvascular permeability; In vivo cryotechnique (IVCT); Mice
缺血再灌注損傷(Ischemic Reperfusion Injury,IRI)是困擾臨床的重大難題。心臟再灌注過(guò)程中,冠脈血管內(nèi)皮屏障受損、滲透性增加是損傷的主要始動(dòng)因素,而病理性的微血管滲透性增加主要由內(nèi)皮細(xì)胞間緊密連接和黏附連接處的細(xì)胞間黏附分子來(lái)調(diào)節(jié)[1]。近年來(lái),一系列研究證實(shí)他汀類藥物可能直接保護(hù)內(nèi)皮細(xì)胞間緊密連接及黏附連接組分,從而在內(nèi)皮屏障調(diào)節(jié)中起關(guān)鍵作用[2]。然而,目前大部分使用他汀類藥物防治IRI的研究都是在缺血或再灌注前12h或數(shù)天給予他汀類藥物治療[4],這種給藥方式在急性心肌梗死的再灌注治療中仍有很大的局限性。有研究[4]表明,在心臟移植過(guò)程中,于移植前2h給予供體他汀類藥物口服,可以有效防止心肌再灌注時(shí)微血管內(nèi)皮細(xì)胞的滲透性增加和無(wú)復(fù)流現(xiàn)象。本研究應(yīng)用活體冷凍技術(shù)(In Vivo Cryotechnique,IVCT),觀察再灌注前2h采用阿托伐他汀預(yù)處理對(duì)缺血再灌注小鼠心肌心外膜下微血管血流狀態(tài)及滲透性的影響,探討阿托伐他汀對(duì)微血管內(nèi)皮屏障的保護(hù)作用及機(jī)制。
1材料與方法
60只清潔級(jí)昆明小鼠由中國(guó)醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供,動(dòng)物許可證號(hào):SCXK(遼)2013-0001,雄性,鼠齡5-6w,體重25-30g,普通鼠飼料喂養(yǎng),自由飲水。隨機(jī)分為6組:假手術(shù)組(C組)、缺血組(I組)、再灌注組(I/R組)、再灌注前2h阿托伐他汀10mg/kg灌胃組(A102組)、再灌注前2h阿托伐他汀20mg/kg灌胃組(A202組)、再灌注前2h阿托伐他汀20mg/kg灌胃+再灌注前15min L-NAME(N-硝基-L-精氨酸甲酯)15mg/kg尾靜脈注射組(AL202組)。每組均10只小鼠。
阿托伐他汀片(Pfizer,美國(guó));兔源性抗eNOS抗體(Thermo,美國(guó));兔源性抗Claudin-5抗體(Santa Cruz,美國(guó));山羊源性抗VE-Cadherin抗體(Santa Cruz,美國(guó));超純水系統(tǒng)(NW10LVF,Heal Force,香港);小動(dòng)物呼吸機(jī)(HX-100E,成都泰盟科技有限公司);多導(dǎo)生理記錄儀(PowerLab15T,AD Instrument,澳大利亞);光學(xué)顯微鏡(SMZ-168,Olympus,日本);電子天平(PB303,METTLER TOLEDO,瑞士);電泳儀(BIO-RAD,Model250/2.5,瑞典);冷凍離心機(jī)(Sorvall Super T21,Thermo,美國(guó));超聲細(xì)胞破碎機(jī)(Dr.HIELSCHER,德國(guó))。
戊巴比妥鈉100mg/kg腹腔麻醉,氣管插管,接小動(dòng)物呼吸機(jī),四肢連接生理記錄儀,胸骨左緣第三肋間開胸,逐層分離暴露心臟,在左心耳與肺動(dòng)脈圓
錐間,冠狀動(dòng)脈起始點(diǎn)下方2-3mm處,用6-0帶針線穿線,穿線后墊一乳膠圈,結(jié)扎冠狀動(dòng)脈前降支。生理記錄儀觀察到小鼠心電圖ST段抬高即可確認(rèn)心肌梗死模型制備成功[6]。再灌注時(shí)連同乳膠圈一并剪開結(jié)扎線,解除阻斷血流。
C組:以6-0帶針線由冠脈下穿過(guò),不結(jié)扎;I組:結(jié)扎冠脈30min;I/R組:結(jié)扎冠脈30min,再灌注1h;A202組及A102組:再灌注前2h予阿托伐他汀20mg/kg或10mg/kg灌胃,結(jié)扎冠脈30min,再灌注1h;AL202組:再灌注前2h予阿托伐他汀20mg/kg灌胃,結(jié)扎冠脈15min(即再灌注前15min)時(shí)予L-NAME 15mg/kg尾靜脈注射,至結(jié)扎冠脈30min后再灌注1h。均以IVCT技術(shù)摘取心臟,其中5只小鼠心臟行冷凍置換、石蠟包埋,之后切片,行蘇木素-伊紅(HE)染色及Albumin免疫組織化學(xué)染色;另5只小鼠心肌組織采用 Western Blotting檢測(cè)內(nèi)皮型一氧化氮合酶(eNOS)、黏附連接蛋白(VE-cadherin)和緊密連接蛋白-5(Claudin-5)的表達(dá)水平。
將預(yù)先在液態(tài)氮(-196℃)中預(yù)冷的異戊烷-丙烷制成的冷凍劑(-193℃)直接傾倒在小鼠跳動(dòng)的心臟組織表面,然后將小鼠迅速移到液態(tài)氮中。在液態(tài)氮中分離心臟組織后將心臟移到由干冰丙酮制冷的含2%多聚甲醛的丙酮溶液中(-80℃)進(jìn)行冷凍至少48h,之后分別在-30℃、-10℃、4℃和室溫下放置2h。純丙酮脫水3次,每次1h。二甲苯透明,低溫石蠟(50-52℃)溫箱(60℃)過(guò)夜,混合蠟(56-58℃石蠟與低溫石蠟按3∶1混合)孵育6h后包埋。
將上述包埋的心臟組織切片(厚3μm),常規(guī)去石蠟后行HE染色,于顯微鏡下觀察心肌微血管(約5μm)內(nèi)紅細(xì)胞灌注情況。
采用免疫組化染色法。將上述石蠟切片常規(guī)去石蠟后用0.3%過(guò)氧化氫溶液去除內(nèi)源性過(guò)氧化酶活性,之后用5%魚精蛋白封閉,一抗(1∶100)4℃過(guò)夜,二抗(1∶1 000)室溫孵育1h,ABC-DAB顯色5min,0.04%鋨酸處理1min,以降低背景,增加顯色對(duì)比效果。顯微鏡下觀察,拍照。每只小鼠隨機(jī)抽取5張顯微照片(每組共25張),觀察記錄心肌間質(zhì)內(nèi)Albumin染色強(qiáng)度,分為5級(jí):陰性(-),弱陽(yáng)性(±),陽(yáng)性(+),中強(qiáng)度陽(yáng)性(2+),強(qiáng)陽(yáng)性(3+)。結(jié)果分別來(lái)自3名觀察者。Albumin陽(yáng)性呈棕黃色。
采用Western Blotting。取-80℃凍存的各組心肌組織,參考文獻(xiàn)[7]方法配制細(xì)胞裂解液,提取胞漿總蛋白,按BCA蛋白含量測(cè)定試劑盒說(shuō)明書進(jìn)行蛋白定量,SDS-PAGE后蛋白轉(zhuǎn)印于PVDF膜,一抗孵育:分別加入兔源性抗eNOS抗體(1∶500)、抗Claudin-5抗體(1∶200)及山羊源性抗VE-Cadherin抗體(1∶100);4℃封閉過(guò)夜,洗膜后二抗孵育:加入羊抗兔二抗,驢抗山羊二抗(1∶500),室溫孵育1h。洗膜后于暗室中行ECL顯色。用凝膠圖像處理系統(tǒng)(Gel-Pro-Analyzer軟件)分析目標(biāo)條帶和β-actin條帶的光密度值,用兩者比值表示其相對(duì)表達(dá)量。
2結(jié)果
顯微鏡下,C組可見大量具有方向性和流動(dòng)狀態(tài)的降落傘樣紅細(xì)胞保存在心肌細(xì)胞間的各級(jí)血管內(nèi),心肌細(xì)胞界限清晰。I組和I/R組血管內(nèi)紅細(xì)胞形態(tài)不一,或破碎,部分血管內(nèi)紅細(xì)胞明顯減少或缺如,心肌細(xì)胞腫脹,界限模糊。與I/R組相比,A202組心肌細(xì)胞界限較清晰,降落傘樣紅細(xì)胞的頂端指向血流方向。A102組鏡下所見與A202組類似,但微血管內(nèi)紅細(xì)胞數(shù)量、形態(tài)及心肌細(xì)胞界限等不及A202組。AL202組可見心肌細(xì)胞腫脹,微血管內(nèi)紅細(xì)胞減少、缺如,存在少量破碎紅細(xì)胞,但微血管血流灌注情況仍優(yōu)于I/R組。見圖1。
免疫組織化學(xué)染色結(jié)果顯示,C組Albumin主要分布于微血管內(nèi),間質(zhì)、閏盤等處僅有少量分布。I組和I/R組微血管與心肌細(xì)胞界限模糊,結(jié)構(gòu)破壞,Albumin向心肌間質(zhì)滲出。與I/R組相比,A202組微血管、心肌細(xì)胞等各結(jié)構(gòu)間界限清晰,Albumin主要分布于微血管內(nèi),向心肌間質(zhì)等滲漏不明顯。A102組與A202組類似,但微血管、心肌細(xì)胞等各結(jié)構(gòu)間界限清晰程度稍差,Albumin向心肌間質(zhì)的滲漏稍多。AL202組心肌細(xì)胞腫脹,各結(jié)構(gòu)間界限模糊,Albumin向心肌間質(zhì)內(nèi)滲漏明顯增多,但仍少于I/R組。見圖2。各組Albumin免疫組織化學(xué)染色強(qiáng)度的統(tǒng)計(jì)學(xué)比較結(jié)果見表1。
表1 各組心肌間質(zhì)Albumin染色強(qiáng)度比較
注:與C組比較,1)P<0.05;與I組和I/R組比較,2)P<0.05
2.3.1eNOS蛋白表達(dá):I組和I/R組與C組比較,eNOS表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05),A202組和A102組與I組和I/R組比較,eNOS表達(dá)均明顯升高(P<0.01),A202組升高更明顯(P<0.05)。AL202組與A202組比較,eNOS表達(dá)降低(P<0.05),且與I/R組差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。見圖3。
2.3.2VE-cadherin蛋白表達(dá):與C組相比,I組和I/R組VE-cadherin蛋白表達(dá)顯著降低(P<0.05)。A102和A202組VE-cadherin蛋白表達(dá)較I組和I/R組明顯升高(P<0.05),A202組升高更明顯(P<0.05)。AL202組VE-cadherin蛋白水平較I/R組升高(P<0.05)。見圖4。
注:與I組和I/R組比較,*P<0.01;與A102組比較,
2.3.3Claudin-5蛋白表達(dá):與C組相比,I組和I/R組Claudin-5蛋白表達(dá)顯著降低(P<0.05)。A102和A202組Claudin-5蛋白表達(dá)較I組和I/R組明顯升高(P<0.05),A202組升高更明顯(P<0.05)。AL202組Claudin-5表達(dá)較I/R組升高(P<0.05)。見圖5。
[本文圖1、圖2見封2]
3討論
IRI是指組織、器官供血中斷一定時(shí)間后再恢復(fù)血供時(shí),組織器官功能障礙和結(jié)構(gòu)損傷反而進(jìn)一步加重的現(xiàn)象,是臨床亟待解決的關(guān)鍵問(wèn)題之一。再灌注過(guò)程中,冠脈血管內(nèi)皮屏障受損、滲透性增加是構(gòu)成IRI病理生理過(guò)程的基礎(chǔ),并可導(dǎo)致微血管受壓、血管痙攣、血栓形成等一系列病理過(guò)程[8]。本文結(jié)果顯示,I組和I/R組小鼠心肌血管內(nèi)紅細(xì)胞形態(tài)紊亂、破碎,部分區(qū)域紅細(xì)胞明顯減少或缺如,血管和心肌界限明顯模糊,結(jié)構(gòu)破壞,Albumin向心肌間質(zhì)明顯滲出。而A102組和A202組血管內(nèi)紅細(xì)胞數(shù)量較I/R明顯增加,形態(tài)較I/R組明顯改善,微血管和心肌細(xì)胞界限清晰,Albumin向間質(zhì)滲漏不明顯,尤以A202組作用更顯著,表明再灌注前2h給予小鼠阿托伐他汀20mg/kg灌胃可明顯改善缺血再灌注心肌微循環(huán)的血流狀態(tài)及微血管滲透性,且效果優(yōu)于10mg/kg,進(jìn)一步證實(shí)了再灌注前短時(shí)間大劑量他汀類藥物強(qiáng)化治療的額外獲益。
注:與C組比較,*P<0.05;與I/R組比較,#P<0.05;
注:與C組比較,*P<0.05;與I/R組比較,#P<0.05;
構(gòu)成血管內(nèi)皮屏障的連接結(jié)構(gòu)中,黏附連接主要由黏附連接蛋白Cadherin及Catenin組成,其中Cadherin的作用更為關(guān)鍵[9]。緊密連接則主要由緊密連接蛋白Occludins及Claudins等組成,既往研究報(bào)道Occludin缺失并不顯著影響緊密連接的形態(tài)及內(nèi)皮屏障功能[10],因而Claudin-5在調(diào)節(jié)緊密連接組成及血腦屏障功能中起重要作用[11],且可能是雌激素保護(hù)心血管內(nèi)皮屏障新的作用靶點(diǎn)[12]。而本研究結(jié)果顯示,阿托伐他汀預(yù)處理可明顯改善缺血再灌注引起的心肌微血管滲透性增加,改善內(nèi)皮細(xì)胞間黏附連接蛋白VE-cadherin和緊密連接蛋白Claudin-5的表達(dá),且該作用不可能被L-NAME所完全消除,進(jìn)一步證實(shí)阿托伐他汀對(duì)缺血再灌注過(guò)程中細(xì)胞間連接蛋白的保護(hù)作用,而且該保護(hù)作用并非既往研究認(rèn)為的完全通過(guò)eNOS/NO途徑實(shí)現(xiàn)[13],其具體機(jī)制可能與他汀類藥物對(duì)微血管中RhoA(一種小GTP酶)/Rho激酶通路活性的抑制作用相關(guān)[14]。
傳統(tǒng)組織制備方法因血液組分丟失及可溶性血清蛋白移位等問(wèn)題易致微環(huán)境內(nèi)的人工假象,故檢測(cè)心臟IRI微血管滲透性多需通過(guò)尾靜脈注射伊文斯藍(lán)等染色劑,觀察其在心臟大體標(biāo)本中的滲出來(lái)完成。本研究應(yīng)用IVCT技術(shù)實(shí)現(xiàn)了活體狀態(tài)下于微循環(huán)水平直接評(píng)估血流狀態(tài)及微血管滲透性的變化,具備良好的可信性。
綜上,本研究證實(shí)再灌注前短時(shí)間內(nèi)大劑量阿托伐他汀強(qiáng)化治療可明顯改善缺血再灌注心肌微循環(huán)血流狀態(tài),降低微血管滲透性,并可增加內(nèi)皮細(xì)胞間連接蛋白VE-cadherin、Claudin-5等的表達(dá),其機(jī)制不完全依賴于eNOS/NO途徑。
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趙世杰(1982—),男,滿族,博士,主治醫(yī)師,從事冠心病的臨床防治研究
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作者簡(jiǎn)介:本文第一
*[基金項(xiàng)目]國(guó)家自然科學(xué)青年基金項(xiàng)目(81200083);教育部重點(diǎn)實(shí)驗(yàn)室課題(KF201312)
[中圖分類號(hào)]R541.4
[文獻(xiàn)標(biāo)識(shí)碼]A
[文章編號(hào)]1005-1740(2015)04-0001-06
doi:10.3969/j.issn.1005-1740.2015.04.001
微循環(huán)學(xué)雜志,2015,25(4):1-6
? 2015 CHINESE JOURNAL OF MICROCIRCULATION