叶酸拮抗高同型半胱氨酸血症大鼠Bcl-2基因低甲基化研究

Folic acid prevents Bcl-2 hypomethylation in rats with hyperhomocysteinemia

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 Bcl-2 gene methylation status in rats with hyperhomocystinemia induced by ingestion of excess methionine.Methods 36 healthy 6-week-old wistar male rats,weighing（160±10）g,after being fed adaptable for one week,were randomly divided into control group（n=12）,hyperhomocysteinemia group（n=12）,folic acid treatment group（n=12）.The control group was fed with AIN-93G diet.The hyperhomocysteinemia group was fed with high-methionion diet,consisting of AIN-93G diet plus 1.7% methionion.The folic acid treatment group was fed with high-methionion plus folic acid-rich diet,consisting of AIN-93G diet plus 1.7% methionion and 0.008% folic acid.After be maintained for 18 weeks on the previously described diets,the concentrations in the plasma Hcy and folic acid and Vit B12 were measured with the IMX assays.The thoracic aorta was harvested for immunohist℃hemical analysis.The methylation status of Bcl-2 gene was determined by nest touch-down PCR combined MSP（methylation specific PCR）.Real-time RT PCR was used to detect mRNA expression of arotic Bcl-2.Results The study showed the following：（a） A high methionine diet for 18 weeks is sufficient to induce hyperhom℃ystinemia;Folic acid supplementation to the rats fed the high-methionine diet prevented an elevation homocysteine（Hcy） levels in the plasma（P0.01）.（b） Compared with the control group,the Hhcy group had a elevating Bcl-2 expression by immunohistochemical analysis in aorta,along with Bcl-2 hypomethylation（P0.05） and increased Bcl-2 mRNA expression（P0.05）.（c） Most important,after folic acid supplementation,the lowering of Hcy levels was accompanied by a marked decreased Bcl-2 expression by immunohistochemical analysis and Bcl-2 hypermethylation（P0.05）and reduced Bcl-2 mRNA expression（P0.05）.Conclusions Folic acid supplementation can prevents Bcl-2 hypomethylation in rats with hyperhomocysteinemia,resulting in a decreased Bcl-2 expression.