Zhang Jing Ge, Wang Li Zhen, Han Xiao Qun, Jiang Yi Deng, Zhang Rui Ming, Wang Shu Ren
West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University,Chengdu 610041.
Fen Zi Xi Bao Sheng Wu Xue Bao. 2007 Feb;40(1):17-23.
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.
探讨同型半胱氨酸诱导内皮型一氧化氮合酶功能障碍的致病机制以及叶酸（FA）的拮抗作用。将人脐静脉内皮细胞（HUVEC）培养至第三代。然后将HUVEC分别用不同浓度（0、10、30、100和300微摩尔/升）的同型半胱氨酸（Hcy）培养，添加或不添加叶酸（100微摩尔/升），培养72小时。分别采用逆转录聚合酶链反应（RT-PCR）和免疫组织化学法分析内皮型一氧化氮合酶（eNOS）的mRNA和蛋白水平。采用反相高效液相色谱法测定不对称二甲基精氨酸（ADMA）。同时分析二甲基精氨酸二甲胺水解酶（DDAH）、eNOS活性及一氧化氮（NO）的生成。将HUVEC暴露于不同浓度的Hcy 72小时后，与对照组相比，eNOS mRNA水平、eNOS蛋白含量、eNOS活性及一氧化氮生成均显著降低，且呈剂量依赖性（P<0.05）。DDAH活性呈平行下降，ADMA浓度相应升高。添加叶酸（100微摩尔/升）可部分拮抗Hcy对内皮细胞NOS系统的损伤，eNOS蛋白水平和eNOS活性增加，NO生成增加，DDAH活性也增加，ADMA浓度降低。但叶酸不影响eNOS mRNA表达。同型半胱氨酸诱导eNOS功能障碍的致病机制可能涉及两个层面，即eNOS蛋白和eNOS活性层面以及eNOS基因表达层面。eNOS蛋白和eNOS活性层面的损伤可能通过DDAH-ADMA途径。叶酸在eNOS蛋白和eNOS活性层面可对Hcy发挥部分保护作用，但不影响eNOS基因的表达。
