【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(P〈0.01)。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(P〈0.01);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(P〈0.01),0.6mmol/L NEFA处理组的GSH-Px mRNA相对表达量显著降低(P〈0.05);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(P〈0.01);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。
[Objective] This study investigated the effect of non-esterified fatty acids (NEFA) on oxi- dative stress state, activity and mRNA expression of antioxidant enzymes of dairy cow hepatocytes in vitro. [Method] Dairy cows hepatocytes were stimulated for another 12,24 and 36 h by NEFA at different con- centrations (0.0,0. 3,0. 6,1. 2,and 2. 4 mmol/L) after 72 h culture since separation. The contents of H202 ,MDA and TAC, inhibition of hydroxyl radical capacity, and activities of SOD,GSH-Px and CAT were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of GSH-Px, SOD and CAT were measured by real-time quantitative PCR (qRT-PCR). [Result] Contents of H202 and MDA increased with the increase of NEFA concentration, while TAC, activities of SOD,GSH-Px, and CAT as well as the inhibition of hydroxyl radical capacity decreased. Compared with the control group, mRNA relative expression of SOD had a dose-dependent decrease at 12 and 36 h and reduced (P〈0.01) very sig- nificantly at 24 h in 0. 6,1. 2,and 2.4 mmol/L NEFA groups. The mRNA relative expression of GSH-Px reduced (P〈0.01) very significantly at 12 h in all groups,reduced (P〈0.01) very significantly at 24 h in 1.2 and 2.4 mmol/L NEFA groups,and reduced (P〈0.05) significantly in 0.6 mmol/L NEFA group. It had dose dependently reduced effect at 36 h. The mRNA relative expression of CAT reduced (P〈0.01) very significantly at 12 h in 0.6 and 2.4 mmol/L NEFA groups,and had dose dependently reduced effect at 24 and 36 h. [Conclusion] NEFA can induce the redox imbalance in primary culture hepatocytes in vitro, especially at high concentrations.