丙酮酸乙酯对脂多糖刺激下人肾小管上皮细胞线粒体动力学的影响  

Effect of ethyl pyruvate on mitochondrial dynamics of lipopolysaccharide-induced human kidney-2 cells

作　　者：刘宁[1] 江智毅[1] 聂垚[1] 刘勇军[1] 管向东[1] 陈敏英[1] Liu Ning;Jiang Zhiyi;Nie Yao;Liu Yongjun;Guan Xiangdong;Chen Minying(Department of Surgical Intensive Care Unit,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,Guangdong,China)

机构地区：[1]中山大学附属第一医院重症医学科,广州510080

出　　处：《中华危重病急救医学》2019年第12期1501-1505,共5页Chinese Critical Care Medicine

基　　金：国家临床重点专科建设项目(2011-872)。

摘　　要：目的探讨丙酮酸乙酯(EP)对内毒素处理的人肾小管上皮细胞(HK-2)线粒体动力学及细胞凋亡的影响.方法将购买的HK-2细胞分为3组:脂多糖(LPS)组使用800μg/L的LPS刺激HK-2细胞24 h;EP组使用800μg/L的LPS与0.25 mmol/L的EP混合物刺激HK-2细胞24 h;对照组(NC组)以同体积生理盐水刺激HK-2细胞24 h.采用酶联免疫吸附试验(ELISA)检测各组细胞中丙二醛(MDA)、超氧化物歧化酶(SOD)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)及细胞内三磷酸腺苷(ATP)水平;采用线粒体膜电位检测试剂盒(JC-1)检测细胞线粒体膜电位;采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)细胞凋亡检测试剂盒检测细胞凋亡率;采用蛋白质免疫印迹试验(Western Blot)检测线粒体动力学蛋白凋亡相关蛋白激酶2(DAPK-2)、线粒体融合蛋白(Mfn-1、Mfn-2)、凋亡相关蛋白天冬氨酸特异性半胱氨酸蛋白酶(caspase-3、caspase-9)、Bcl-2、Bcl-xL、细胞色素C(Cyt C)以及DNA修复酶多聚二磷酸腺苷核糖聚合酶(PARP)的表达.结果与NC组比较,LPS组细胞MDA、IL-6、TNF-α表达显著增高,SOD活性显著降低,细胞线粒体膜电位及细胞内ATP水平显著降低,细胞线粒体分裂蛋白DAPK-2表达显著增高,而线粒体融合蛋白Mfn-1、Mfn-2表达显著降低,细胞总凋亡率显著增高,同时凋亡蛋白caspase-3、caspase-9及Cyt C的表达显著增高,抗凋亡蛋白Bcl-2、Bcl-xL以及PARP的表达显著降低.与LPS组比较,EP可抑制上述氧化应激指标、炎性因子的表达〔MDA(μmol/L):12.35±2.21比45.95±1.76,SOD(kU/L):54.68±1.42比40.73±1.60,IL-6(ng/L):67.87±2.61比338.92±20.91,TNF-α(ng/L):19.23±1.80比180.69±6.51〕,细胞线粒体膜电位及细胞内ATP水平显著增高〔线粒体膜电位(正常细胞百分比):(99.43±0.25)%比(69.40±0.75)%,ATP(×10^6 RLU):0.19±0.01比0.12±0.05〕,线粒体分裂蛋白表达显著降低(DAPK-2/β-actin:0.03±0.01比0.61±0.02),融合蛋白显著增多(Mfn-1/β-actObjective To examine the effects of ethyl pyruvate(EP)on mitochondrial dynamics and cell apoptosis in lipopolysaccharide(LPS)-induced human kidney-2(HK-2)cells.Methods HK-2 cells were divided into three groups:HK-2 cells were challenged with LPS(800μg/L)for 24 hours as LPS group,or LPS mixed with EP(0.25 mmol/L)for 24 hours as EP group.Cells were incubated with normal saline for 24 hours as control group.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and intracellular adenosine triphosphate(ATP)were detected by enzyme linked immunosorbent assay(ELISA).JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide(FITC/PI)assays were used to evaluate mitochondrial membrane potential and cell apoptosis,respectively.Western Blot was used to evaluate the protein expressions of mitochondrial dynamics,including death-associated protein kinase 2(DAPK-2),mitofusin(Mfn-1 and Mfn-2),and apoptotic associated biomarkers,including caspase-3,caspase-9,Bcl-2,Bcl-xL,cytochrome C(Cyt C),and DNA repair enzyme poly ADP-ribose polymerase(PARP).Results Compared with the NC group,MDA,IL-6,TNF-αof LPS group were significantly increased,the expression of SOD,mitochondrial membrane potential and ATP level were significantly decreased,the expression of mitochondrial fission protein DAPK-2 was significantly increased,and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased,cell apoptosis and apoptotic protein caspase-3,caspase-9 and Cyt C were increased,and anti-apoptotic protein Bcl-2,Bcl-xL,PARP were significantly decreased.Compared with the LPS group,the oxidative activities and inflammatory factors above were inhibited in EP group[MDA(μmol/L):12.35±2.21 vs.45.95±1.76,SOD(kU/L):54.68±1.42 vs.40.73±1.60,IL-6(ng/L):67.87±2.61 vs.338.92±20.91,TNF-α(ng/L):19.23±1.80 vs.180.69±6.51],mitochondrial membrane potential and ATP level were significantly increased[mitochondrial membrane potential:(99.43±0.25)%vs.(69.40±0.75)%,ATP(×106 RLU

关 键 词：丙酮酸乙酯 脓毒症 线粒体动力学 人肾小管上皮细胞 

分 类 号：R45[医药卫生—治疗学]