同型半胱氨酸对内皮细胞一氧化氮合酶系统的损伤机制及叶酸的拮抗效应

本实验探讨同型半胱氨酸(Hcy)对人脐静脉内皮细胞(HUVEC)一氧化氮合酶(eNOS)的损伤机制及叶酸(FA)的拮抗效应。HUVEC原代培养,传至第3代后,将其与不同浓度Hcv(10μmol/L、30μmol/L、100μmol/L和300μmol/L)、FA(100μmol/L)或两者联合共同培养72h,用RT-PCR和免疫组织化学技术分别估测细胞eNOS mRNA水平及eNOS蛋白质量;高效液相色谱测定细胞内不对称二甲基精氨酸(ADMA)含量;并分别测定二甲基精氨酸二甲胺水解酶(DDAH)、eNOS活性及一氧化氮(NO)含量。HUVEC与不同浓度Hcy培养72h后,eNOS mRNA和蛋白质表达皆受到抑制;eNOS活性降低;NO生成减少。同时,DDAH活性降低;细胞内ADMA含量呈剂量依赖性增加。加入FA后,eNOS蛋白质水平上调;eNOS活性增强;NO生成增多。同时,DDAH活性增强,ADMA蓄积减少;但eNOS mRNA表达没有改变。Hcy对内皮细胞eNOS的损伤机制涉及eNOS酶蛋白和eNOS的基因表达两个层面,其对eNOS酶蛋白的抑制机制可能通过DDAH-ADMA通路,FA可拮抗Hcy对eNOS酶蛋白的抑制作用,显示出对HHcy有一定的保护作用。但FA对HHcy所导致的eNOS基因表达的抑制无保护效应。

THE PATHOGENIC MECHANISM OF HOMOCYSTEINE -INDUCED ENDOTHELIAL NITRIC OXIDE SYNTHASE DYSFUNCTION AND THE ANTAGONISTIC EFFECTS BY FOLIC ACID

To investigate the pathogenic mechanism of homocysteine-induced endothelial nitric oxide synthase dysfunction and the antagonistic effects by folic acid (FA). Human umbilical vein endothelial cells (HUVEC)were cultured to the third generation. Then HUVEC were cultured with Hcy at different concentrations (0,10,30,100 and 300 micromol/L),with or without FA(100 micromol/L)for 72 hours. The mRNA and protein levels of endothelial nitric oxide synthase (eNOS) were analyzed by RT-PCR and immunohistochemistry respectively. Asymmetric dimethylarginine (ADMA)was measured by reversed-phase high performance liquid chromatography. The dimethylarginine dimethylaminohydrolase(DDAH), activity of eNOS and the production of NO were analyzed simulta- neously. After HUVEC were exposed to Hcy at different concentrations for 72 hours, the level of eNOS mRNA and the content of eNOS protein, the eNOS activity, and the production of nitric oxide (NO) were all significantly and dose-dependently reduced compared with the control group (P< 0.05). The activity of DDAH has a parallel decrease and the ADMA concentration showed a cor- responding increase. The addition of folic acid (100 micromol/L)resulted in partial antagonistic effects against the injury of Hcy on NOS system of endothelial cells, the eNOS protein level and eNOS activity, and NO production increased,and so does the DDAH activity,and the ADMA concentration reduced. But the FA didn't influence the eNOS mRNA expression. The pathogenic mechanism of homocysteine-induced eNOS dysfunction may involve two levels,the level of eNOS protein and eNOS activity,and the level of the expression of eNOS gene. The injury on the level of eNOS protein and eNOS activity may go through the DDAH-ADMA pathway. Folic acid can exert partial protective roles against the Hcy in the level of eNOS protein and eNOS activity,but without impact on the expression of eNOS gene.