甜菜碱对载脂蛋白E^(-/-)小鼠动脉粥样硬化发生过程中铜锌超氧化物岐化酶基因甲基化及表达的影响

The Effects of Betaine on CuZnSOD Gene Methylation and Expression in Atherosclerosis of Apolipoprotein E-Deficient Mouse

目的检测载脂蛋白E^(-/-)小鼠动脉粥样硬化发生过程中肝脏铜锌超氧化物岐化酶基因启动子区CpG岛甲基化及其表达状态,探讨甜菜碱对动脉粥样硬化的作用及其可能的机制。方法将正常的C57BL/6J小鼠作为正常对照组,同品系载脂蛋白E^(-/-)小鼠分为模型组、1%、2%和4%甜菜碱组。用油红O染色法检测小鼠主动脉窦脂质斑块面积,应用甲基化特异性聚合酶链反应检测肝脏铜锌超氧化物岐化酶基因甲基化,荧光定量逆转录聚合酶链反应检测铜锌超氧化物岐化酶mRNA表达,免疫组织化学检测其蛋白表达。结果饲养至14周,2%和4%甜菜碱组小鼠主动脉窦脂质斑块面积明显少于模型组(P<0.05);各组铜锌超氧化物岐化酶基因CpG岛甲基化状态差异无显著性(P>0.05);各时间段正常对照组铜锌超氧化物岐化酶mRNA表达均高于模型组,7周时1%甜菜碱组mRNA表达高于2%和4%甜菜碱组,14周时1%甜菜碱组mRNA表达高于模型组和2%甜菜碱组;各时间段正常对照组铜锌超氧化物歧化酶蛋白表达均高于模型组、1%、2%和4%甜菜碱组(P<0.05),但模型组、1%、2%和4%甜菜碱组间无显著性差异。结论铜锌超氧化物岐化酶基因可能没有参与动脉粥样硬化过程中DNA甲基化的异常改变,补充甜菜碱可以增加肝脏铜锌超氧化物岐化酶mRNA表达,改善载脂蛋白E^(-/-)小鼠的脂质沉积,减少主动脉窦粥样斑块面积。

Aim To detect the methylation and expression of CpG island in the gene promoter region of live CuZnSOD during the occurrence of atherosclerosis （ As ） on apolipoprotein E-deficient mouse, explore the effects of betaine on atherosclerosis and its possible mechanisms. Methods Normal C57BL/6J mouse were taken as control group, apolipoprotein E-deficient mouse were randomly divided into model group, 1%, 2% and 3% betaine group. Oil red 0 staining method was used to detect the lipid plaque area in mouse aortic sinus, methylation-specific polymerase chain reaction （ MSP ） was applied to examine the gene methylation of liver CuZnSOD, quantitative fluorescence reverse transcription-polymerase chain reaction （real-time RT-PCR） was taken to detect the expression of mRNA in CuZnSOD gene and by immunohistochemical method its protein expression was detected. Results In 14th week, the lipid plaque areas of aortic sinus in 2% and 4% betaine mouse group were less than the model group, the difference was significant（ P 〈 0.05 ） ; The CpG island methylation status of CuZnSOD gene in each group had no significant difference （ P 〉 0.05 ） ; in all time the expression of mRNA in the normal groups were higher than that in the model groups ; the 7th week, the expression of mRNA in 1% betaine group was higher than that in 2% and 4% betaine group; the 14th week, the expression of mRNA in 1% betaine group was higher than that in model group and 2% betaine group. In all time the expression of CuZnSOD protein in the normal group was higher than that in the other four groups （ P 〈 0.05 ）, but the expression in the other four groups had no signifcant difference. Conclusions CuZnSOD gene may not be involved in the abnormal DNA methylation changes in the process of atherosclerosis, supplement of betaine can increase the expression of CuZnSOD gene mRNA in liver, thus improve the lipid aggradation of apolipoprotein E-deficient mouse and reduce the plaque area of aortic.