AEE对RAW264.7细胞中iNOS表达及NO合成的影响

Effects of AEE on expression of iNOS and synthesis of NO in RAW264.7 cells

为了探讨阿司匹林丁香酚酯(AEE)对氧化型低密度脂蛋白(ox-LDL)诱导的RAW264.7细胞中诱导型一氧化氮合酶(iNOS)表达及一氧化氮(NO)合成的影响,试验首先采用CCK-8法检测细胞存活率,采用油红O染色法观察细胞内脂滴形成情况,采用实时荧光定量PCR扩增、免疫荧光技术和Western-blot技术测定iNOS mRNA和蛋白的相对表达量,最后通过硝酸盐还原酶测定法测定NO含量。结果表明:AEE和ox-LDL对RAW264.7细胞没有毒性。利用80μg/mL的ox-LDL可成功诱导RAW264.7细胞泡沫化,建立泡沫细胞模型,并且AEE可明显抑制细胞内脂滴的形成。ox-LDL可极显著上调细胞中iNOS mRNA及蛋白的相对表达量(P<0.01),并且极显著增加了细胞内NO含量(P<0.01)。经AEE预处理的RAW264.7细胞中iNOS mRNA及蛋白相对表达量显著或极显著降低(P<0.05或P<0.01),细胞内NO含量也极显著减少(P<0.01)。说明AEE可通过抑制iNOS的表达降低NO的合成,缓解RAW264.7细胞泡沫化,进而发挥抗动脉粥样硬化的作用。

To investigate the effects of Aspirin eugenol ester(AEE) on inducible nitric oxide synthase(iNOS) expression and nitric oxide(NO) synthesis in RAW264.7 cells induced by oxidized low density lipoprotein(ox-LDL),the cell survival rate was detected by CCK-8 method in the experiment.Oil red O staining was used to observe the formation of intracellular lipid droplets.The relative expression of iNOS mRNA and protein was detected by real-time fluorescent quantitative PCR,immunofluorescence and Western-blot.Finally,the content of NO was determined by nitrate reductase assay.The results showed that AEE and ox-LDL were not toxic to RAW264.7 cells.RAW264.7 macrophages were successfully induced to foam using 80 μg/mL of ox-LDL to establish a foam cell model,and AEE significantly inhibited the formation of intracellular lipid droplets.ox-LDL significantly upregulated the expression of iNOS mRNA and protein in the cells(P<0.01) and significantly increased the intracellular NO content(P<0.01).The expression of iNOS mRNA and protein in RAW264.7 cells pretreated with AEE was significantly decreased(P<0.05 or P<0.01),and the content of NO in RAW264.7 cells was also significantly decreased(P<0.01).These results indicated that AEE could inhibit the expression of iNOS and reduce the synthesis of NO,alleviate the formation of RAW264.7 foam cells,and play a role in anti-atherosclerosis.