Stachowska Ewa, Dziedziejko Violetta, Safranow Krzysztof, Jakubowska Katarzyna, Olszewska Maria, Machaliñski Bogusław, Chlubek Dariusz
Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland al. Powstancow Wlkp. 72, 70-111 Szczecin, Poland.
J Agric Food Chem. 2007 Jun 27;55(13):5335-42. doi: 10.1021/jf0701077. Epub 2007 Jun 6.
Lipoxygenases are a family of non-heme enzyme dioxygenases. The role of lipoxygenases is synthesis of hydroperoxides of fatty acids, which perform signaling functions in the body. Studies on conjugated linoleic acids (CLAs) as fatty acids with a potential anti-atherosclerotic function have recently been initiated. The aim of the study was to test the effect of CLAs and linoleic acid on 5- and 15-lipoxygenase (5-LO, 15-LO-1) enzyme activity, their mRNA expression, and concentration in the cells. It was also desired to determine whether the CLAs are substrates for the enzymes. For the experiments monocytic cell line (THP-1) and monocytes obtained from human venous blood were used. Monocytes were differentiated to macrophages: THP-1 (CD14+) by PMA administration (100 nM for 24 h) and monocytes from blood (CD14+) by 7-day cultivation with the autologous serum (10%). After differentiation, macrophages were cultured with 30 microM CLAs or linoleic acid for 48 h. The 15- and 5-lipoxygenase products were measured by HPLC method. mRNA expression and protein content were analyzed by real-time PCR and Western blot analysis. The in vitro studies proved that both CLA isomers are not substrates for 15-LO-1; in ex vivo studies hydroxydecadienoic acid (HODE) concentration was significantly reduced (p = 0.019). The trans-10,cis-12 CLA isomer reduced HODE concentration by 28% (p = 0.046) and the cis-9,trans-11 CLA isomer by 35% (p = 0.028). In macrophages obtained from THP-1 fatty acids did not change significantly mRNA expression of the majority of the investigated genes. CLAs did not change the content of 5-LO and 15-LO-1 proteins in macrophages obtained from peripheral blood. Linoleic acid induced 15-LO-1 expression (2.6 times, p < 0.05). CLAs may perform the function of an inhibitor of lipoxygenase 15-LO-1 activity in macrophages.
脂氧合酶是一类非血红素酶双加氧酶。脂氧合酶的作用是合成脂肪酸氢过氧化物，这些氢过氧化物在体内发挥信号传导功能。最近已经开始了对具有潜在抗动脉粥样硬化功能的脂肪酸共轭亚油酸（CLA）的研究。该研究的目的是测试CLA和亚油酸对5-脂氧合酶和15-脂氧合酶（5-LO，15-LO-1）的酶活性、它们的mRNA表达以及细胞内浓度的影响。还希望确定CLA是否是这些酶的底物。实验使用了单核细胞系（THP-1）和从人静脉血中获得的单核细胞。单核细胞通过给予佛波酯（PMA，100 nM，作用24小时）分化为巨噬细胞（THP-1，CD14+），而来自血液的单核细胞（CD14+）则通过与自体血清（10%）培养7天进行分化。分化后，巨噬细胞用30 microM的CLA或亚油酸培养48小时。通过高效液相色谱法测量15-脂氧合酶和5-脂氧合酶的产物。通过实时聚合酶链反应和蛋白质免疫印迹分析来分析mRNA表达和蛋白质含量。体外研究证明，两种CLA异构体都不是15-LO-1的底物；在体内研究中，羟基癸二烯酸（HODE）的浓度显著降低（p = 0.019）。反式-10，顺式-12 CLA异构体使HODE浓度降低了28%（p = 0.046），顺式-9，反式-11 CLA异构体使HODE浓度降低了35%（p = 0.028）。在从THP-1获得的巨噬细胞中，脂肪酸对大多数研究基因的mRNA表达没有显著影响。CLA没有改变从外周血获得的巨噬细胞中5-LO和15-LO-1蛋白的含量。亚油酸诱导了15-LO-1的表达（2.6倍，p < 0.05）。CLA可能在巨噬细胞中发挥脂氧合酶15-LO-1活性抑制剂 的作用。
