自噬在饱和氢盐水减轻肝移植术大鼠肝细胞凋亡中的作用  

Role of autophagy in reduction of apoptosis in liver cells by hydrogen-rich saline in rats undergoing liver transplantation

作　　者：施东婧 杨超[1] 喻文立[2] 吴莉[1] 盛明薇[2] 翁亦齐[2] 王永旺[2] 王树森[3] 杜洪印[2] 

机构地区：[1]天津医科大学一中心临床学院,300192 [2]天津市第一中心医院麻醉科,300192 [3]卫生部危重病急救医学重点实验室,天津市300384

出　　处：《中华麻醉学杂志》2016年第5期546-550,共5页Chinese Journal of Anesthesiology

基　　金：天津市卫生行业重点攻关项目（12KG101,13KG105）

摘　　要：目的探讨自噬在饱和氢盐水减轻肝移植术大鼠肝细胞凋亡中的作用。方法SPF级成年健康雄性SD大鼠56只，体重200～250g，采用随机数字表法分为4组：假手术组（s组，n=8）、肝移植组（OLT组，n=16）、饱和氢气盐水组（HS组，n=16）和自噬抑制剂3-甲基腺嘌呤组（M组，n=16）。OLT组、HS组和M组受体和供体各8只。M组术前1h腹腔注射3-甲基腺嘌呤2．5mg／kg。采用改良的Kamada二袖套法建立肝移植模型。于肝下下腔静脉开放即刻HS组和M组经肝下下腔静脉注射4℃饱和氢盐水6ml／kg，OLT组注射4oC生理盐水6ml／kg。OLT组、HS组和M组于门静脉开放后6h，S组于术毕后6h时，经肝下下腔静脉采集血样，检测血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）浓度。取血结束后，取肝脏组织，观察病理学结果，并行病理学评分；检测MDA含量和SOD活性；采用TUNEL法检测细胞凋亡水平，计算凋亡指数；电镜下观察超微结果；采用West．ernblot法检测肝组织细胞质总p53、磷酸化p53（p-p53）、微管相关蛋白轻链3Ⅱ（LC31I）、Beelin．1、caspase-3和细胞色素C（Cytc）的表达水平。以p-p53／总p53比值反映p53的磷酸化水平。结果与s组比较，OLT组、HS组和M组血清ALT和AST浓度、肝组织病理学评分、MDA含量和凋亡指数升高，SOD活性降低，caspase-3和CytC表达上调，OLT组和HS组肝组织LC3I／和Beclin-1表达上调，HS和M组肝组织p-p53／总p53比值升高（P〈0．05）；与OLT组比较，HS组血清ALT和AST浓度、肝组织病理学评分、MDA含量和凋亡指数降低，SOD活性升高，caspase．3、CytC、LC31I和Beclin-1表达下调，p-p53／总p53比值升高（P〈0．05），肝细胞超微结构改变减轻，自噬小体减少；与HS组比较，M组血清ALT和AST浓度、肝组织病理学评分、MDA含量和凋亡指数降低，SOD活性升高，caspase-3、CytC、LC311和Beclin-1表达下调（P〈0．05�Objective To investigate the role of autophagy in reduction of apoptosis in liver cells by hydrogen-rich saline in the rats undergoing liver transplantation. Methods Fifty-six healthy adult male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 4 groups using a random number table： sham operation group （ group S, n = 8） ; orthotopic liver transplantation （OLT） group （ n = 16） ; hydrogen-rich saline group （ group HS, n = 16） ; autophagy inhibitor 3-methyladenine group （ group M, n= 16）. The number of donors was 8, and recipients 8 in OLT, HS and M groups. 3-methyladenine 2.5 mg/kg was injected intraperitoneally at 1 h before surgery in group M. The model of OLT was established ac- cording to the modified Kamada＇s two-cuff technique. Immediately after opening of infra-hepatic vena cava, 4 ~C hydrogen-rich saline 6 ml/kg was injected through the infra-hepatic vena cava in HS and M groups, and 4 ~C normal saline 6 ml/kg was given in group OLT. At 6 h after portal vein opening in OLT, HS and M groups, or at 6 h after the end of surgery in group S, blood samples were collected from the infra-hepatic vena cava for determination of serum alanine transaminase （ALT） and aspartate transaminase （AST） con- centrations. After blood sampling, livers were removed for microscopic examination of the pathological changes which were scored and ultrastructure, and for determination of the malondialdehyde （MDA） con- tent, superoxide dismutase （SOD） activity, apoptotic cells, and expression of total p53 in cytoplasm, phosphorylated p53 （p-p53）, microtubule-associated proteins light chain 3 II （LC3 I1 ） and Beclin-1, caspase-3 and cytochrome c （Cyt c） in liver tissues （by Western blot）. The ratio of p-p53 to total p53 was used to reflect the phosphorylation of p53. Results Compared with group S, the serum ALT and AST concentrations, pathological score, MDA content and apoptosis index were significantly increased, the SOD activity was decreased, and the expression

关 键 词：氢 肝移植 细胞凋亡 自噬 

分 类 号：R614[医药卫生—麻醉学]