EC-SOD在同型半胱氨酸致单核细胞源性巨噬细胞氧化应激中的作用研究

Effect of extracellular superoxide dismutase on oxidative stress induced by homocysteine in monocyte-derived macrophages

目的探讨细胞外超氧化物岐化酶(EC-SOD)在同型半胱氨酸(Hcy)致巨噬细胞氧化应激中的作用及机制。方法将THP-1单核细胞用佛波酯刺激48 h后演变成巨噬细胞,用0、50、100、200、500μmol/L Hcy作用细胞72 h,并加设叶酸+维生素B12(Vit B12)干预组(100μmol/L Hcy+30μmol/L叶酸+30μmol/L Vit B12)。微板法检测氧化应激指标(H_2O_2、O_2^-、OH-)的变化;实时荧光定量PCR检测巨噬细胞中EC-SOD的mRNA表达水平;Western blot检测巨噬细胞中EC-SOD的蛋白表达水平;EC-SOD测定试剂盒检测EC-SOD活性。分别构建EC-SOD重组质粒和干扰质粒转染细胞,检测EC-SOD的mRNA及蛋白的表达水平以及超氧阴离子的表达。结果与对照组相比,100、200、500μmol/L Hcy组H_2O_2、OH-活性显著增高(P<0.01),EC-SOD mRNA和蛋白表达明显降低(P<0.01)。与对照组相比,100、200、500μmol/L Hcy组EC-SOD活性分别下降了13.92%、8.62%、10.32%(P<0.05,P<0.01)。与100μmol/L Hcy组相比,叶酸+Vit B12干预组EC-SOD mRNA的表达升高了47%。分别转染EC-SOD重组质粒和干扰质粒后,与100μmol/L Hcy组相比,EC-SOD重组组O_2^-含量降低了63.89%,干扰片段-596组O_2^-含量则增加了33.59%(P<0.05,P<0.01)。结论 EC-SOD参与了Hcy导致的单核细胞源性巨噬细胞的氧化应激。在抑制Hcy诱导动脉粥样硬化的过程中,EC-SOD可能发挥着重要的作用。

Aim To investigate the effect of extracellular superoxide dismutase（ EC-SOD） on oxidative stress induced by homocysteine（ Hcy） in macrophages and its mechanism. Methods THP-1 monocyte was stimulated by phorbol ester for 48 hours and evolved into macrophages. The macrophages were dealt with 0,50,100,200,500 μmol / L Hcy for 72 hours,and adding a folate acid＋vitamin B12（ Vit B12） intervention group（ 100 μmol / L Hcy＋30 μmol / L folate acid＋30 μmol / L Vit B12）. The changes of oxidative stress indexes（ H_2O_2,O_2^-,OH-） were detected by microplate test.The mRNA expression of EC-SOD was detected by real-time fluorescence quantitative PCR and the protein expression of EC-SOD was detected by Western blot in macrophages. EC-SOD assay kit was used for detecting EC-SOD activity. ECSOD recombinant plasmid and interfering plasmid were constructed and transfected into cells,and expressions of EC-SOD mRNA,protein and superoxide anion were detected in macrophages. Results Compared with the control group,H_2O_2 and OH-activities were significantly increased,and EC-SOD mRNA and protein expressions were significantly decreased in100,200,500 μmol / L Hcy group（ P〈0. 01）. Compared with the control group,the EC-SOD activity was respectively decreased by 13.92%,8.62%,10.32% in 100,200,500 μmol / L Hcy group（ P〈0.05 or P〈0.01）. Compared with the100 μmol / L Hcy group,the expression of EC-SOD mRNA was increased by 47% in folate acid＋VitB 12 intervention group.After transfection of EC-SOD recombinant plasmid and interfering plasmid,compared with the 100 μmol / L Hcy group,O_2^-content was decreased by 63.89% in EC-SOD recombinant group,while O_2^-content was increased by 33.59% in interfering fragment-596 group（ P〈0.05 or P〈0.01）. Conclusions EC-SOD is involved in the oxidative stress induced by Hcy in monocyte-derived macrophages. EC-SOD may play an important role in the inhibition of atherosclerosis induced by Hcy.