黑色素瘤缺乏因子2在缺氧复氧损伤介导的肝细胞焦亡中的作用  

Role of absent in melanoma 2 in hepatocyte pyroptosis mediated by hypoxia-reoxygenation injury

作　　者：李春桃 唐苏婷 喻淋淋 徐云柯 郭勇 李波 LI Chun-tao;TANG Su-ting;YU Lin-lin;XU Yun-ke;GUO Yong;LI Bo(Department of Hepatobiliary Surgery,Traditional Chinese Medicine Hospital Affiliated to Southwest Medical University,Luzhou 646000,China;Department of Hepatobiliary Surgery,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属中医医院肝胆外科,四川省泸州市646000 [2]西南医科大学附属医院肝胆外科,四川省泸州市646000

出　　处：《广西医学》2020年第20期2674-2679,2702,共7页Guangxi Medical Journal

基　　金：西南医科大学附属中医医院自然科学青年苗圃项目(2018XYLH-051)。

摘　　要：目的探讨黑色素瘤缺乏因子2(AIM2)在缺氧复氧损伤介导的肝细胞焦亡中的作用。方法(1)采用缺氧复氧法干预L02细胞以构建肝脏缺血再灌注损伤体外模型。检测未建模L02细胞(空白对照组)以及缺氧后复氧1 h、3 h、6 h、12 h、24 h的L02细胞的活性、细胞焦亡率以及焦亡相关蛋白[半胱氨酸天冬氨酸蛋白酶(Caspase)-1、白细胞介素(IL)-1β、IL-18]、AIM2蛋白的表达水平。(2)将L02细胞分为空白对照组、缺氧复氧模型组、缺氧复氧+si-AIM2组、缺氧复氧+si-NC组。缺氧复氧+si-AIM2组、缺氧复氧+si-NC组细胞分别转染si-AIM2、si-NC后进行缺氧6h/复氧培养12 h的干预;缺氧复氧模型组细胞仅进行缺氧培养6 h、复氧培养12 h,空白对照组细胞在常氧条件下等时长培养。检测各组细胞活性、焦亡率及焦亡相关蛋白表达水平。结果(1)与空白对照组相比,复氧6 h后L02细胞活性开始降低,复氧3 h后L02细胞焦亡率开始上升(P<0.05);复氧3 h、6 h、12 h、24 h后L02细胞活性依次降低而焦亡率依次升高(均P<0.05)。与空白对照组相比,复氧3 h后L02细胞AIM2蛋白的表达水平开始增加,复氧6 h后L02细胞Caspase-1、IL-1β、IL-18表达水平开始升高(均P<0.05);复氧24 h后L02细胞以上蛋白的表达水平高于2个或2个以上不同复氧时间组(均P<0.05)。(2)与缺氧复氧模型组和缺氧复氧+si-NC组比较,缺氧复氧+si-AIM2组细胞活性升高,且焦亡率、Caspase-1和IL-1β的表达水平均降低(P<0.05),而缺氧复氧模型组和缺氧复氧+si-NC组之间差异并无统计学意义(P>0.05);缺氧复氧+si-AIM2组IL-18的表达水平亦低于缺氧复氧+si-NC组(P<0.05)。结论AIM2参与缺氧后复氧诱导的L02细胞焦亡;干扰AIM2基因的表达可能通过抑制Caspase-1、IL-1β、IL-18的表达水平来改善缺氧后复氧引起的L02细胞焦亡。Objective To elucidate the role of absent in melanoma 2(AIM2)in hepatocyte pyroptosis mediated by hypoxia-reoxygenation injury.Methods(1)An in vitro model of hepatic ischemia reperfusion injury(HIRI)was developed by intervention on L02 cells using the hypoxia-reoxygenation method.The measurement of cell viability,pyroptosis rate as well as expression levels of pyroptosis-related proteins(cysteinyl aspartate specific proteinase 1[Caspase-1],interleukin[IL]-1β,IL-18)and AIM2 protein was conducted in unmodeled L02 cells(blank control group)and L02 cells after one,three,six,12 and 24 hours of post-hypoxia reoxygenation.(2)L02 cells were divided into blank control group,hypoxia-reoxygenation model group,hypoxia-reoxygenation+si-AIM2 group,and hypoxia-reoxygenation+si-NC group.Cells in the hypoxia-reoxygenation+si-AIM2 and hypoxia-reoxygenation+si-NC groups were transfected with si-AIM2 and si-NC respectively to perform intervention with 6-hour hypoxia culture/12-hour reoxygenation culture;cells in the hypoxia-reoxygenation group only received 6-hour hypoxia culture and 12-hour reoxygenation culture,while cells in the blank control group received isochronous culture under normoxic condition.Cell viability,pyroptosis rate,and expression levels of pyroptosis-related proteins were detected in each group.Results(1)Compared with the blank control group,in L02 cells viability began to decline after six hours of reoxygenation,pyroptosis rate began to rise after three hours of reoxygenation(P<0.05);the viabilities of L02 cells three,six,12 and 24 hours after reoxygenation decreased in turn while pyroptosis rates increased in turn(all P<0.05).Compared with the blank control group,the expression level of AIM2 protein in L02 cells began to rise after three hours of reoxygenation,the expression levels of Caspase-1,IL-1βand IL-18 in L02 cells began to rise after six hours of reoxygenation(all P<0.05);the expression levels of aforementioned proteins in L02 cells 24 hours after reoxygenation were higher than the levels in two or mo

关 键 词：肝缺氧复氧损伤 细胞焦亡 黑色素瘤缺乏因子2 人正常肝细胞L02 半胱氨酸天冬氨酸蛋白酶 白细胞介素1β 白细胞介素18 

分 类 号：R619.9[医药卫生—外科学]