NEFA对体外培养奶牛肝细胞氧化还原状态的影响

发布时间：2019-05-27 20:57

【摘要】：【目的】研究高非酯化脂肪酸(NEFA)对体外培养奶牛肝细胞的氧化应激状态、氧化抗氧化酶活性及mRNA表达的影响。【方法】分离奶牛肝细胞,培养72h后,添加不同浓度(0.0,0.3,0.6,1.2和2.4mmol/L)的NEFA继续培养12,24和36h,采用酶联免疫吸附试验(ELISA)法,检测过氧化氢(H2O2)、丙二醛(MDA)含量及总抗氧化能力(TAC)、羟自由基抑制能力和超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)的活性,实时荧光定量PCR(qRT-PCR)法检测GSH-Px、SOD、CAT基因mRNA的相对表达量。【结果】随着NEFA浓度的增加,添加NEFA 12,24和36h时,各处理组肝细胞中H2O2和MDA含量增加,TAC含量、SOD、GSH-Px、CAT的活性以及羟自由基抑制能力降低。与对照组相比,添加NEFA 12和36h时,SOD mRNA相对表达量均随NEFA浓度的增加呈剂量依赖性降低;添加NEFA 24h时,0.6,1.2和2.4mmol/L NEFA处理组的SOD mRNA相对表达量均极显著降低(P0.01)。添加NEFA 12h时,各浓度NEFA处理组GSH-Px mRNA相对表达量均极显著降低(P0.01);添加NEFA 24h时,1.2和2.4mmol/L NEFA处理组的GSH-Px mRNA相对表达量均极显著降低(P0.01),0.6mmol/L NEFA处理组的GSH-Px mRNA相对表达量显著降低(P0.05);添加NEFA 36h时,GSH-Px mRNA相对表达量随NEFA浓度的增加呈剂量依赖性降低。添加NEFA 12h时,0.6和2.4mmol/L NEFA处理组的CAT mRNA相对表达量均极显著降低(P0.01);添加NEFA 24和36h时,CAT mRNA相对表达量呈剂量依赖性降低。【结论】NEFA诱导体外培养的奶牛肝细胞发生氧化还原失衡,尤其以高浓度NEFA诱导的氧化应激更为严重。
[Abstract]:[objective] to study the effects of high non-esterified fatty acid (NEFA) on oxidative stress, oxidative antioxidant enzyme activity and mRNA expression in bovine hepatocytes cultured in vitro. [methods] Dairy cow hepatocytes were isolated and cultured for 72 hours. NEFA with different concentrations (0.0, 0.3, 0.6, 1.2 and 2.4mmol/L) was cultured for 12, 24 and 36 hours. Hydrogen peroxide (H2O2), malondialdehyde (MDA) (MDA) content and total antioxidant capacity (TAC),) were detected by enzyme-linked immunosorbent assay (ELISA). The inhibitory ability of hydroxyl radical and the activities of GSH-Px and catalase (CAT) in (SOD), were detected by real-time fluorescence quantitative PCR (qRT-PCR). [results] with the increase of NEFA concentration, the contents of H2O2 and MDA in hepatocytes increased, while the content of TAC, the activity of SOD,GSH-Px,CAT and the inhibitory ability of hydroxyl radical decreased at 24 h and 36 h after the addition of NEFA. [results] with the increase of NEFA concentration, the content of H2O2 and MDA in hepatocytes increased, while the activity of SOD,GSH-Px,CAT and the inhibitory ability of hydroxyl radical decreased. Compared with the control group, the relative expression of, SOD mRNA decreased in a dose-dependent manner with the increase of NEFA concentration at 12 h and 36 h after the addition of NEFA. When NEFA was added for 24 h, the relative expression of SOD mRNA in 0.6, 1.2 and 2.4mmol/L NEFA treatment groups decreased significantly (P 0.01). When NEFA was added for 12 h, the relative expression of GSH-Px mRNA in each concentration of NEFA treatment group was significantly decreased (P 0.01). When NEFA was added for 24 h, the relative expression of GSH-Px mRNA in GSH-Px mRNA treatment group and 2.4mmol/L NEFA treatment group was significantly lower than that in 0.6mmol/L NEFA treatment group (P 0.01), and the relative expression of GSH-Px mRNA in 0.6mmol/L NEFA treatment group was significantly lower than that in 0.6mmol/L NEFA treatment group (P 0.05). When NEFA was added for 36 h, the relative expression of GSH-Px mRNA decreased in a dose-dependent manner with the increase of NEFA concentration. When NEFA was added for 12 h, the relative expression of CAT mRNA in 2.4mmol/L NEFA treatment group and 2.4mmol/L NEFA treatment group was significantly lower than that in 2.4mmol/L NEFA treatment group (P 0.01). When NEFA was added for 24 h and 36 h, the relative expression of, CAT mRNA decreased in a dose-dependent manner. [conclusion] NEFA induced redox imbalance in dairy cow hepatocytes in vitro, especially the oxidative stress induced by high concentration of NEFA.
【作者单位】：安徽农业大学动物科技学院;吉林大学动物医学学院;
【基金】：国家自然科学基金项目(31502136) 安徽省自然科学基金项目(1608085QC56) 安徽农业大学青年科学基金重点项目(2014zr008);安徽农业大学稳定和引进人才科研项目(yj2015-14) 安徽省现代农业产业技术体系项目(2016-2020)
【分类号】：S823

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