过氧化氢诱导的草鱼原代肝细胞氧化损伤模型的构建及其对天然植物抗氧化能力的评价  

Establishment of Oxidative Injury Model of Grass Carp Hepatocytes Induced by Hydrogen Peroxide and Its Application in Antioxidant Ability Evaluation of Natural Plants

作　　者：石瑶瑶 叶元土[1] 蒋蓉 蔡春芳[1] 吴萍[1] SHI Yaoyao;YE Yuantu;JIANG Rong;CAI Chunfang;WU Ping(Key Laboratory of Aquatic Nutrition of Jiangsu Province,School of Biology and Basic Medical Sciences,Soochow University,Suzhou 215123,China;Wuxi Sanzhi Bio-Technology Co.,Ltd.,Wuxi 214135,China)

机构地区：[1]苏州大学基础医学与生物科学学院,江苏省水产动物与营养重点实验室,苏州215123 [2]无锡三智生物科技有限公司,无锡214135

出　　处：《动物营养学报》2021年第4期2290-2301,共12页CHINESE JOURNAL OF ANIMAL NUTRITION

摘　　要：本试验旨在探讨过氧化氢(H2 O2)诱导草鱼原代肝细胞体外氧化损伤模型的建立条件,并利用所构建的模型评价8种天然植物的抗氧化能力。采用噻唑蓝(MTT)染色法检测不同浓度[0(对照组)、100、200、400、600μmol/L]H2 O2处理草鱼原代肝细胞0.5、1.0、2.0和4.0 h后的细胞存活率,并采用微板法检测培养液中乳酸脱氢酶(LDH)活性。选择不同浓度(100、200、400、600μmol/L)H2 O2处理肝细胞1.0 h后,分光光度法检测细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,显微镜观察细胞形态学,流式细胞仪检测细胞凋亡率和细胞中活性氧(ROS)水平。通过测定清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基能力和H2 O2损伤细胞存活率,评价诃子、虎杖、荷叶、车前草、侧柏叶、甘草、大黄、肉桂这8种天然植物的抗氧化能力。结果显示:与对照组相比,200μmol/L H2 O2处理肝细胞1.0 h后,细胞存活率降低到(64.85±0.38)%;LDH和MDA含量显著或极显著升高(P<0.05或P<0.01),SOD活性显著降低(P<0.01);ROS水平和细胞凋亡率极显著升高(P<0.01)。诃子组的细胞存活率最高,比模型组高出80.17%~150.68%。清除DPPH自由基能力大小排序为诃子≥虎杖≥荷叶≥大黄≥肉桂>侧柏叶>车前草>甘草,且氧化损伤修复能力强弱与清除DPPH自由基能力的高低排序基本一致。综上可知,200μmol/L H2 O2处理1.0 h是建立草鱼原代肝细胞氧化损伤模型的最佳试验条件。8种天然植物中诃子的抗氧化能力最佳且氧化损伤修复能力最强。This experiment was to explore the conditions for the establishment of in vitro oxidative injury model of grass carp primary hepatocytes induced by hydrogen peroxide(H2 O2),and to evaluate the antioxidant abili⁃ty of 8 kinds of natural plants using this model.Methyl thiazolyl tetrazolium(MTT)staining method was used to detect the survival rate of cells treated with different concentrations(0,100,200,400 and 600μmol/L)of H2 O2 for 0.5,1.0,2.0 and 4.0 h,and the lactate dehydrogenase(LDH)activity in culture fluid was meas⁃ured by microplate method.The hepatocytes were treated with different concentrations(100,200,400 and 600μmol/L)of H2 O2 for 1.0 h.Cell morphology was observed by microscope.The content of malondialde⁃hyde(MDA)and the activity of superoxide dismutase(SOD)in cells were detected by spectrophotometry.And cell apoptosis rate and the level of reactive oxygen species(ROS)in cells were detected by flow cytome⁃try.The antioxidant ability of 8 kinds of natural plants such as Terminalia chebula Retz,Polygoni Cuspidati Rhizoma et Radix,Folium nelumbinis,Plantaginis herb,Platycladi cacumen,Glycyrrhizae Radix et Rhizoma,Rhei Radix et Rhizoma and Cinnamomi cortex was evaluated by determining the 1,1⁃diphenyl⁃2⁃trinitrophenyl⁃hydrazine(DPPH)free radical scavenging capacity and the survival rate of H2 O2 injured cells.The results showed as follows:compared with the control group,after treated with 200μmol/L H2 O2 for 1.0 h,the cell survival rate was reduced,which dropped to(64.85±0.38)%;the content of LDH and MDA was significantly or extremely significantly increased(P<0.05 or P<0.01),and the activity of SOD was extremely significantly decreased(P<0.01);the level of ROS and cell apoptosis rate were extremely significantly increased(P<0.01).The cell survival rate of Terminalia chebula Retz group was the highest,which was 80.17%to 150.68%higher than that of model group.The order of the DPPH free radical scavenging capacity was Termi⁃nalia chebula Retz≥Polygoni Cuspidati Rhizoma et Radix≥Foli

关 键 词：草鱼 原代肝细胞 过氧化氢 氧化应激 天然植物 

分 类 号：Q952[生物学—动物学] S816.7[农业科学—饲料科学]