CAJ | 학술논문

目的：探讨丙酮酸乙酯在肝细胞氧化应激损伤中的作用及机制。方法：通过对人肝细胞系L‐02进行缺氧／复氧处理各1 h建立氧化应激损伤模型。将 L‐02根据干预措施分为对照组（Control组）、乳酸钠林格液组（LR组）、丙酮酸乙酯组（EP组）。应用日本日立公司7020全自动分析仪检测细胞培养上清液中天门冬氨酸氨基转移酶（aspartate aminotransferase ， AST）和丙氨酸氨基转移酶（alanine aminotransferase ，ALT）浓度；采用 Hoechst 33342／碘化丙啶双标记荧光染色以及原位缺口末端标记法（TUNEL）检测各组细胞的凋亡和坏死情况；采用蛋白质印迹（Western blotting）法检测各组caspase‐9、caspase‐3与多聚二磷酸腺苷核糖聚合酶（PARP）蛋白的表达；采用荧光素发光法测量各组肝细胞内ATP浓度。结果：EP组细胞培养上清液中AST和ALT浓度均低于Control组和LR组（P＜0．05）；EP组坏死细胞比例低于Control组和LR组（P＜0．05），但凋亡细胞比例高于Control组和 LR组（P＜0．05）。EP组活化形式的caspase‐9、caspase‐3与 PARP蛋白表达水平高于Control组和LR组（P＜0．05）。EP组细胞内ATP浓度显著高于Control组和LR组（P＜0．05）。结论：EP对肝细胞氧化应激损伤有保护作用，能减少坏死细胞数量，增加凋亡细胞数量，改变坏死及凋亡细胞比例。EP的细胞保护作用机制可能在于改善能量代谢，提高细胞内ATP浓度，激活“凋亡／坏死共同通路”，激活凋亡过程。
목적：탐토병동산을지재간세포양화응격손상중적작용급궤제。방법：통과대인간세포계L‐02진행결양／복양처리각1 h건립양화응격손상모형。장 L‐02근거간예조시분위대조조（Control조）、유산납림격액조（LR조）、병동산을지조（EP조）。응용일본일립공사7020전자동분석의검측세포배양상청액중천문동안산안기전이매（aspartate aminotransferase ， AST）화병안산안기전이매（alanine aminotransferase ，ALT）농도；채용 Hoechst 33342／전화병정쌍표기형광염색이급원위결구말단표기법（TUNEL）검측각조세포적조망화배사정황；채용단백질인적（Western blotting）법검측각조caspase‐9、caspase‐3여다취이린산선감핵당취합매（PARP）단백적표체；채용형광소발광법측량각조간세포내ATP농도。결과：EP조세포배양상청액중AST화ALT농도균저우Control조화LR조（P＜0．05）；EP조배사세포비례저우Control조화LR조（P＜0．05），단조망세포비례고우Control조화 LR조（P＜0．05）。EP조활화형식적caspase‐9、caspase‐3여 PARP단백표체수평고우Control조화LR조（P＜0．05）。EP조세포내ATP농도현저고우Control조화LR조（P＜0．05）。결론：EP대간세포양화응격손상유보호작용，능감소배사세포수량，증가조망세포수량，개변배사급조망세포비례。EP적세포보호작용궤제가능재우개선능량대사，제고세포내ATP농도，격활“조망／배사공동통로”，격활조망과정。
Objective:To investigate the protective effect of ethyl pyruvate on oxidative stress injury in hepatocytes and its underlying molecular mechanism .Methods:Hypoxia/reoxygenation‐induced oxidative stress injury model was performed with 1 h hypoxia followed by 1 h reoxygenation .According to the different interventions ,L‐02 hepatocytes were divided into control group(Con group) ,lactate Ringer’s group(LR group) and ethyl pyruvate group(EP group) .The concentrations of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in supernatants of cell culture medium were measured by Hitachi 7020 Automatic Analyzer (Hitachi ,Tokyo ,Japan) .The percentages of necrotic cells and apoptotic cells were examined by Hoechst 33342/propidium iodide (PI) and TdT‐mediated dUTP nick end labeling(TUNEL) stalning .The expression levels of caspase‐9 ,caspase‐3 and poly (ADP‐ribose) polymerase (PARP) were determined by Western blotting .The concentrations of ATP in hepatocytes were measured by using luciferase luminescence method .Results:The concentrations of AST and ALT in EP group were lower than that in Con group and LR group(P<0 .05) .The percentage of necrotic cells was lower and the percentage of apoptotic cells was higher in EP group than those in Con group and LR group(P<0 .05) .The expression levels of cleaved caspase‐9 ,caspase‐3 and PARP in EP group were higher than that in Con group and LR group(P< 0 .05) .The concentration of ATP in EP group was higher than that in Con group and LR group(P<0 .05) .Conclusions:Ethyl pyruvate can decrease the percentage of necrotic cells but increased the percentage of apoptotic cells in hepatocytes insulted by hypoxia/reoxygenation .Ethyl pyruvate may protect hepatocytes agalnst oxidative stress injury through up‐regulating the intracellular ATP levels and consequently skewing the necrosis‐apoptosis pathway .