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目的探讨叶酸对蛋氨酸饮食诱导高同型半胱氨酸血症(Hhcy)大鼠主动脉组织Bcl-2基因甲基化水平影响。方法健康6周龄雄性wistar大鼠36只,体重(160±10)g,适应性喂养一周后随机分为对照组、高同型半胱氨酸组、叶酸干预组、每组12只。对照组给予AIN-93G配方饲料,高同型半胱氨酸组饲以含1.7%蛋氨酸的AIN-93G配方饲料,叶酸干预组饲以含1.7%蛋氨酸和0.008%叶酸的AIN-93G配方饲料。喂养18周后,全自动生化仪测定血浆Hcy浓度、叶酸浓度和维生素B12浓度;免疫组化检测大鼠主动脉组织Bcl-2表达;巢式降落式PCR联合甲基化特异性PCR检测Bcl-2基因启动子区甲基化水平,荧光RT-PCR检测主动脉组织Bcl-2基因mRNA表达。结果 18周蛋氨酸饮食可以诱导大鼠高同型半胱氨酸血症(P<0.01),叶酸摄入可以拮抗血浆同型半胱氨酸水平升高(P<0.01);与对照组比较,高Hcy组大鼠主动脉组织Bcl-2阳性细胞数目增加,Bcl-2基因启动子区甲基化水平降低(P<0.05),Bcl-2 mRNA表达增加(P<0.05)。叶酸干预后,主动脉组织Bcl-2阳性细胞数目减少,Bcl-2基因启动启动子区甲基化水平高于高Hcy组(P<0.05),Bcl-2 mRNA表达低于高Hcy组(P<0.05)。结论叶酸摄入可以拮抗高同型半胱氨酸血症大鼠主动脉组织Bcl-2低甲基化,使Bcl-2表达减弱,可能是其参与抗动脉粥样硬化形成的分子机制之一。
Objective To investigate the effects of folic acid on the methylation level of Bcl-2 gene in aorta tissue of rats with hyperhomocystinemia induced by methionine diet. Methods Thirty-six male Wistar rats, 6 weeks old, weighing 160 ± 10 g were randomly divided into control group, high homocysteine group and folic acid intervention group, with 12 rats in each group. The control group was given AIN-93G formula, the high homocysteine group was fed AIN-93G formula containing 1.7% methionine, and the folic acid intervention group was fed AIN-93G formula containing 1.7% methionine and 0.008% folic acid. After 18 weeks of feeding, plasma Hcy concentration, folic acid concentration and vitamin B12 concentration were measured by automatic biochemical analyzer; Bcl-2 expression was detected by immunohistochemistry in rat aorta; nested-type PCR combined with methylation-specific PCR detection of Bcl- 2 gene promoter region methylation level, fluorescent RT-PCR detection of aortic Bcl-2 gene mRNA expression. Results Eighteen weeks methionine diet could induce hyperhomocysteinemia in rats (P <0.01), and folic acid intake could antagonize the increase of plasma homocysteine level (P <0.01). Compared with the control group, high Hcy The number of Bcl-2 positive cells in the aorta increased, the methylation level of Bcl-2 promoter decreased (P <0.05) and the expression of Bcl-2 mRNA increased (P <0.05). After the intervention of folic acid, the number of Bcl-2 positive cells decreased, the methylation level of Bcl-2 promoter promoter region was higher than that of high Hcy group (P <0.05), and the expression of Bcl-2 mRNA was lower than that of high Hcy group <0.05). Conclusions Folic acid intake may antagonize the hypomethylation of Bcl-2 and decrease the expression of Bcl-2 in the aorta of hyperhomocysteinemia rats, which may be one of the molecular mechanisms involved in the anti-atherosclerosis formation.