α-硫辛酸对大鼠肝缺血再灌注损伤的影响及乙醛脱氢酶2在其中的作用  

Effect of alpha-lipoic acid on hepatic ischemia/reperfusion injury and role of ALDH2

作　　者：刘宽智[1] 沈璞[1] 张涛[1] 黄文起[1] Liu Kuanzhi;Shen Pu;Zhang Tao;Huang Wenqi(Department of Anesthesiology,the First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]中山大学附属第一医院麻醉科,广州510080

出　　处：《中华麻醉学杂志》2022年第2期155-160,共6页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81770619);广东省自然科学基金(2018A0303130332)。

摘　　要：目的:评价α-硫辛酸对大鼠肝缺血再灌注损伤的影响及乙醛脱氢酶2(ALDH2)在其中的作用。方法:在体实验:雄性SD大鼠24只,体重250~300 g,8~10周龄,采用随机数字表法分为4组(n=6):假手术组(Sham组)、肝缺血再灌注组(IR组)、肝缺血再灌注+α-硫辛酸组(IR+ALA组)和肝缺血再灌注+α-硫辛酸+黄豆苷组(IR+ALA+D组)。采用阻断肝左叶及中叶肝蒂60 min后再灌注的方法建立大鼠肝缺血再灌注损伤模型。缺血前45 min时,IR+ALA+D组腹腔注射ALDH2拮抗剂黄豆苷50 mg/kg;缺血前30 min时,IR+ALA组和IR+ALA+D组腹腔注射α-硫辛酸100 mg/kg。于再灌注6 h时,采集下腔静脉血标本,检测血清AST和ALT活性;然后处死大鼠,取肝组织,行HE染色光镜下观察病理学结果,并行肝损伤评分,测定ALDH2活性和ROS水平,采用免疫组化法测定4-羟基壬烯醛(4-HNE)和MDA表达。离体实验:体外培养的大鼠肝细胞BRL-3A,采用随机数字表法分为4组(n=15):对照组(C组)、缺氧复氧组(HR组)、缺氧复氧+α-硫辛酸组(HR+ALA组)和缺氧复氧+α-硫辛酸+黄豆苷组(HR+ALA+D组)。HR组、HR+ALA组和HR+ALA+D组于37℃、95%N 2+5%CO_(2)条件下培养6 h,然后于37℃、95%空气+5%CO_(2)条件下培养24 h;于缺氧前60 min时,HR+ALA组加入α-硫辛酸100μmol/L,HR+ALA+D组加入α-硫辛酸100μmol/L和黄豆苷60μmol/L。于复氧24 h时,采用CCK-8法检测细胞活力,分光光度法检测ALDH2活性,DCFH-DA荧光探针法检测ROS水平,JC-1法检测线粒体膜电位。结果:在体实验:与Sham组比较,IR组血清AST和ALT活性、肝损伤评分、肝组织ROS水平、4-HNE和MDA表达水平升高(P<0.05),ALDH2活性差异无统计学意义(P>0.05);与IR组比较,IR+ALA组血清AST和ALT活性、肝损伤评分、肝组织ROS水平、4-HNE和MDA表达水平降低,ALDH2活性升高(P<0.05);与IR+ALA组,HR+ALA+D组血清AST和ALT活性、肝损伤评分、肝组织ROS水平、4-HNE和MDA表达水平升高,ALDH2活性降低(P<0.05)。离体实验:与C组比Objective To evaluate the effects of the alpha-lipoic acid on hepatic ischemia/reperfusion(I/R)injury and the role of(ALDH2).Methods This experiment was performed in two parts in vivo and in vitro experiments.In vivo experiment Twenty-four male Sprague-Dawley rats,aged 8-10 weeks,weighing 250-300 g,were divided into 4 groups(n=6 each)by the random number table method:sham operation group(Sham group),hepatic I/R group(IR group),and hepatic I/R plusα-lipoic acid group(IR+ALA group)and hepatic I/R+α-lipoic acid+daidzin group(IR+ALA+D group).Hepatic I/R was induced by occlusion of the left and middle hepatic lobes for 60 min,followed by 6 h of reperfusion in anesthetized rats.In IR+ALA+D group,ALDH2 inhibitor daidzin 50 mg/kg was intraperitoneally injected at 45 min before ischemia.Alpha-lipoic acid 100 mg/kg was intraperitoneally injected at 30 min before ischemia in IR+ALA group and IR+ALA+D group.Blood samples from the inferior vena cava were collected at the end of reperfusion to determine serum AST and ALT activities.Then the rats were sacrificed,and livers were removed for microscopic examination of pathological changes of the lung tissues which were scored and for determination of ALDH2 activity,level of reactive oxygen species(ROS)and expression of 4-hydroxy-trans-2-nonenal(4-HNE)and malondialdehyde(MDA)(by immuno-histochemistry).In vitro experiment Rat BRL-3A hepatocytes cultured in vitro were divided into 4 groups(n=15 each)by the random number table method:control group(C group),hypoxia-reoxygenation group(HR group),hypoxia-reoxygenation+α-lipoic acid group(HR+ALA group)and hypoxia-reoxygenation+α-lipoic acid+daidzin group(HR+ALA+D group).BRL-3A hepatocytes were exposed to 95%N2-5%CO_(2) in an incubator at 37℃for 6 h followed by reoxygenation with 95%O2-5%CO_(2) for 24 h.At 60 min before hypoxia,alpha-lipoic acid 100μmol/L was addded in HR+ALA group,and alpha-lipoic acid 100μmol/L and daidzin 60μmol/L were added in HR+ALA+D group.At 24 h of reoxygenation,cell viability was measured by CCK-8 method

关 键 词：硫辛酸 再灌注损伤 肝 醛脱氢酶 

分 类 号：R575[医药卫生—消化系统]