高同型半胱氨酸血症对 ApoE －／－鼠心脏内质网应激的影响

摘要

目的：探讨高同型半胱氨酸血症（ HHcy）对ApoE－／－鼠心脏内质网应激（ ERS）的影响及其可能机制。方法取5周龄雄鼠12只作为正常对照组；另取5周龄雄性ApoE－／－小鼠36只，随机分为3组，ApoE－／－对照组：ApoE－／－小鼠饲以普通饲料配方饮食；高蛋氨酸组：ApoE－／－小鼠饲以高蛋氨酸饮食；干预组：ApoE－／－小鼠饲以高蛋氨酸饮食同时加0.006％叶酸和0.0004％维生素B12。喂养20周后处死，行主动脉病理切片HE染色观察主动脉是否有斑块形成；取心脏，采用实时荧光定量PCR法检测GRP78、CHOP、Caspase－12 mRNA的变化；巢式降落式甲基化特异性PCR检测GRP78和CHOP启动子区DNA甲基化水平。结果正常对照组及ApoE－／－对照组斑块不明显，高蛋氨酸组动脉粥样硬化斑块面积明显增大，主动脉内膜可见灶性病变部位隆起突入动脉腔内，干预组动脉粥样硬化斑块面积较高蛋氨酸组有所减轻。与ApoE－／－对照组比较，高蛋氨酸组GRP78、CHOP、Caspase－12 mRNA水平明显升高（P＜0.05，P＜0.01），干预组GRP78、CHOP mRNA水平较高蛋氨酸组降低（P＜0.05）。与ApoE－／－对照组比较，高蛋氨酸组GRP78、CHOP启动子区DNA甲基化水平降低（P＜0.01），干预组GRP78、CHOP甲基化水平较高蛋氨酸组有所升高（ P＜0.05）。结论 HHcy可能通过影响DNA甲基化程度引起ERS相关基因表达改变，进而导致心肌细胞凋亡，补充叶酸与维生素B12可有效缓解HHcy所引起的ERS和凋亡。%Objective To investigate the effects of hyperhomocysteinemia ( HHcy ) on myocardial endoplasmic reticulum stress ( ERS) in ApoE-/-mice and its possible mechanism.Methods Thirty-six 5 -week old ApoE-/-C57BL/6J mice were randomly divided into three groups, including ApoE-/-control group, ApoE-/-HHcy group and the intervention group.Twelve 5-week old normal C57BL/6J mice were served as normal group.After 20 weeks of feed-ing, the aortae were separated for HE stain and observation of aortic plaque.GRP78, CHOP and Caspase-12 mRNA lev-els were determined by qRT-PCR.The methylation status of GRP78 and CHOP were studied using the nest touch down methylation specific polymerase chain reaction ( ntMS-MSP) .Results No significant aortic plaque was observed in ei-ther normal group or ApoE-/-control group.In the ApoE-/-HHcy group, the aortic plaque areas were greater, breaking into the aortic lumina, which were alleviated in the intervention group.The mRNA levels of GRP78, CHOP and Caspase-12 in the ApoE-/-HHcy group were significantly higher than those in ApoE-/-control group(P<0.05,P<0.01), while they were significantly down-regulated in the intervention group(P<0.05,P<0.01).GRP78 and CHOP DNA methylation levels were significantly lowered in the ApoE-/-HHcy group(P<0.01,P<0.01),while significanty higher in the intervention group(P<0.05,P<0.05).Conclusion HHcy may affect Myocardial ERS via changing DNA methy-lation levels, then lead to apoptosis.Earlier folic acid and VB12 interference can effectively reduce the myocardial ERS and apoptosis.