阿托伐他汀预处理对缺血再灌注小鼠心肌微血管内皮屏障的保护作用

The Protective Effect of Atorvastatin on Microvascular Barrier in Myocardial Ischemia Reperfusion Injury

目的:探讨阿托伐他汀预处理对缺血再灌注小鼠心肌微血管血流灌注和渗透性的作用及其机制。方法:60只昆明小鼠随机分为6组:假手术组(C组)、缺血组(I组)、再灌注组(I/R组)、再灌注前2h阿托伐他汀10mg/kg灌胃组(A102组)、再灌注前2h阿托伐他汀20mg/kg灌胃组(A202组)、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尾静脉注射组(AL202组),每组均10只小鼠。于光镜下结扎冠脉制备心肌缺血再灌注模型,以活体冷冻技术(IVCT)摘取心脏,其中5只小鼠心脏行冷冻置换、石蜡包埋,之后切片行HE染色及Albumin免疫组织化学染色,并对间质中的Albumin免疫组织化学染色强度进行半定量分析;另5只小鼠采用Western Blotting检测心肌组织内皮型一氧化氮合酶(eNOS)、黏附连接蛋白VE-cadherin和紧密连接蛋白Claudin-5的表达水平。结果:与I组及I/R组相比,A202组小鼠心肌细胞界限较清晰,降落伞型红细胞的顶端指向血流方向,Albumin无明显向间质等部位渗漏。A102组镜下所见与A202组类似,但微血管内红细胞数量、形态及心肌细胞界限清晰度等不及A202组,Albumin向间质渗漏。AL202组可见心肌细胞肿胀,微血管内红细胞减少、缺如,但微循环血流灌注好于I/R组,Albumin渗漏少于I/R组。与I组和I/R组对比,A202组和A102组心肌组织中eNOS蛋白表达均明显升高(P<0.01),尤以A202组最为明显(P<0.05)。与A202组相比,AL202组eNOS蛋白表达降低(P<0.05),且与I/R组差异无统计学意义(P>0.05)。与C组相比,I组和I/R组VE-cadherin蛋白和Claudin-5蛋白表达显著降低(P<0.05),而A102和A202组VE-cadherin蛋白和Claudin-5蛋白表达较I组和I/R组明显升高(P<0.05),尤以A202组升高最为明显(P<0.05)。AL202组VEcadherin及Claudin-5蛋白水平较I/R组升高(P<0.05)。结论:再灌注前短时间内大剂量阿托伐他汀预处理可改善缺血再灌注心肌微循环血流灌注及微血管渗透性,其机制可能不完全依赖于eNOS/NO途径。

Objective：To explore the effect and the probably mechanism of atorvastatin pretreatment before is-chemia-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）,atorvasta-tin 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 1 5mg/kg tail vein injection 1 5min before reperfusion group（AL202）.The myocardial ischemia reperfusion model was prepared under light microscopy.IVCT 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 VEcadherin and Claudin-5,which probably does not totally depend on the eNOS/NO pathway.