NOD1激活细胞凋亡促进小鼠肝脏缺血再灌注损伤  

Experimental study on liver ischemia reperfusion injury promoted by NOD1 activated pyroptosis

作　　者：希吉日 宋虎[2] 王星星 李世朋 杨爽 蔡金贞[5] 沈中阳[5] Xi Jiri;Song Hu;Wang Xingxing;Li Shipeng;Yang Shuang;Cai Jinzhen;Shen Zhongyang(Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China;First Central Clinical College of Tianjin Medical University, Tianjin 300070, China;Department of General Surgery, Jiaozuo People's Hospital of Xinxiang Medical University, Jiaozuo 454002, China;Department of Medical Molecular Biology, Medical School of Nankai University, Tianjin 300071, China;Oriental Organ Transplant Center of Tianjin First Central Hospital, Tianjin 300192, China)

机构地区：[1]天津中医药大学,300193 [2]天津医科大学一中心临床学院,300070 [3]新乡医学院附属焦作市人民医院普外科,454002 [4]南开大学医学院医学分子生物学教研室,天津300071 [5]天津市第一中心医院器官移植中心,300192

出　　处：《中华器官移植杂志》2019年第1期41-45,共5页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金项目(81670600).

摘　　要：目的探讨核苷酸结合寡聚化结构域1(NOD1)调控肝细胞凋亡对小鼠肝脏缺血再灌注损伤的影响。方法建立缺血再灌注损伤动物模型,选择健康、雄性、清洁级C57 BL小鼠30只,完全随机法分为假手术组和缺血再灌注组(IR2 h、6 h、12 h、24 h),每组6只。血生化检测每组小鼠血清ALT、AST水平,采用HE染色及原位末端标记法(TUNEL法)观察肝脏病理学改变及肝细胞凋亡情况,免疫组化检测各组肝组织NOD1的表达与分布,蛋白质印迹(Western blot)检测NOD1、AIM2、pro-Capase-1、active-Caspase-1的表达情况,在AML12细胞中转染NOD1 siRNA及空载对照siRNA,建立细胞缺氧/复氧模型并收集细胞检测NOD1、AIM2、active-Caspase-1表达。结果IR各组的ALT、AST均比假手术组高,且在IR12 h时达到峰值(P<0.05)。病理检查结果显示再灌注12 h时肝损伤最重,TUNEL结果显示再灌注12 h时凋亡细胞数最多。免疫组化结果显示12 h时NOD1表达最多。Western blot显示再灌注12 h时后NOD1的蛋白表达最高。随着再灌注时间的延长,AIM2、active-Caspase-1的表达逐渐增多,pro-Caspase-1的表达逐渐减少。AML12细胞转染NOD1 siRNA后,NOD1、AIM2及active-Caspase-1表达减少。结论NOD1促进肝脏缺血再灌注损伤,这可能与NOD1通过激活细胞凋亡促进肝脏损伤有关。Objective To investigate the effect of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) on pyroptosis in hepatic ischemia-reperfusion injury. Methods An animal model of ischemia-reperfusion injury was established. Thirty healthy, male, and clean C57 BL mice were randomly divided into sham operation group (sham group) and ischemia-reperfusion group (IR, including 2 h, 6 h, 12 h, 24 h subgroups), 6 per group. Serum ALT and AST levels in each group were measured by blood biochemistry. HE staining and TUNEL were used to observe the pathological changes of liver and hepatocyte apoptosis. Immunohistochemistry was used to detect the expression and distribution of NOD1 in each group. Western blotting was used to detect NOD1, AIM2, pro-Caspase-1 and active-Caspase-1 expression. NOD1 siRNA and empty control siRNA were transfected into AML12 cells, then the hypoxia/reoxygenation model was established and cells were collected to detect the expression of NOD1, AIM2 and active-Caspase-1. Results The ALT and AST levels in IR group were significantly higher than those in sham group, and peaked at IR 12-h subgroup (P<0.05). HE staining showed that hepatic injury was the most severe at 12 h after reperfusion. TUNEL results showed that the number of apoptotic cells was the greated at 12 h after reperfusion. Western blotting showed that NOD1 protein expression was highest at 12 h after reperfusion. With the prolongation of reperfusion time, the expression of AIM2 and active-Caspase-1 gradually increased, and that of pro-Caspase-1 gradually decreased. The expression of NOD1, AIM2 and active-Caspase-1 decreased after transfection of NOD1 siRNA into AML12 cells. Conclusions NOD1 promotes liver ischemia-reperfusion injury, which may be related to NOD1 promoting liver injury by activating pyroptosis.

关 键 词：再灌注损伤 肝脏 细胞凋亡 

分 类 号：R657.3[医药卫生—外科学]