细胞外超氧化物歧化酶DNA 甲基化对动脉粥样硬化的诱导作用及作用靶点

Role of extracellular superoxide dismutase methylation in atherosclerosis

摘要:

目的 探讨细胞外超氧化物歧化酶DNA（EC-DNA）甲基化水平改变在动脉粥样硬化发病中的 作用。方法 16 只ApoE-/- 小鼠（ApoE-/- 组）给予高脂饲料喂养，16 只C57BL/6 小鼠（WT 组）给予不含 任何高脂成分的普通饲料喂养，分别于喂养第8、12、16 和20 周检测小鼠血脂、主动脉组织形态及丙二醛 （MDA）、活性氧（ROS）含量，采用逆转录聚合酶链反应（RT-PCR）检测主动脉组织细胞外超氧化物歧 化酶（EC-SOD）、DNMT1 mRNA 水平，巢式甲基化特异性聚合酶链反应检测主动脉组织EC-SOD DNA 甲基化程度。将THP-1 诱导为巨噬细胞，分为3 组：空白对照组（NC 组）、空白质粒组（pEGFP-N1 组） 和重组质粒组（pEGFP-N1-DNMT1 组），NC 组用不做任何处理，继续培养，pEGFP-N1 组加入10μl 脂质 体Lipofectamine 和20μg 空质粒载体pEGFP-N1，pEGFP-N1-DNMT1 组加入10μl 脂质体Lipofectamine 和 20μg pEGFP-N1-DNMT1 重组质粒载体，培养24 h 后检测细胞EC-SOD、DNMT1 蛋白表达和EC-SOD DNA 甲基化程度。结果 第8、12、16 和20 周，ApoE-/- 组TC、LDL、TG 呈升高趋势，HDL 无明显变化； 各时段ApoE-/- 组TC、LDL、TG、HDL 与WT 组比较，差异有统计学意义（P <0.05），TC、LDL、TG 高于 WT 组，HDL 低于WT 组。第16 和20 周，WT 组动脉组织内皮细胞结构规则有序，ApoE-/- 组主动脉内膜明 显增厚并伴炎症浸润。第8、12、16 及20 周，ApoE-/- 组MDA、ROS 呈升高趋势，各时段ApoE-/- 组MDA、 ROS 高于WT 组。ApoE-/- 组EC-SOD mRNA 表达水平、DNMT1 mRNA 表达水平和EC-SOD 甲基化水 平与WT 组比较，差异有统计学意义（P <0.05），EC-SOD mRNA 表达水平低于WT 组，DNMT1 mRNA 表 达水平和EC-SOD 甲基化水平高于WT 组。pEGFP-N1-DNMT1 组DNMT1 蛋白表达、EC-SOD 甲基化 水平及EC-SOD 蛋白表达水平与pEGFP-N1 组和NC 组比较，差异有统计学意义（P <0.05），pEGFP-N1- DNMT1 组DNMT1 蛋白表达、EC-SOD 甲基化水平高于pEGFP-N1 组和NC 组，EC-SOD 蛋白表达水平 低于pEGFP-N1 组和NC 组，pEGFP-N1 组和NC 组EC-SOD、DNMT1 蛋白及EC-SOD 甲基化水平比较 差异无统计学意义（P >0.05）。结论 EC-SOD 甲基化程度升高，导致EC-SOD 表达降低，机体抵抗氧化应 激反应能力减弱，最终发展成动脉粥样硬化。

Abstract:

Objective To explore the mechanism of extracellular superoxide dismutase (EC-SOD) DNA methylation changes in atherosclerosis. Methods Sixteen ApoE-/- mice (ApoE-/- group) were fed with high-fat diet,and 16 C57BL/6 mice (WT group) were fed with ordinary diet without any high-fat component. The serum lipid level, aortic morphology and tissue MDA and ROS content were detected in the 8th, 12th, 16th and 20th weeks after feeding respectively. The EC-SOD and DNA methyltransferase 1 (DNMT1) mRNA levels in the aortic tissues were detected by RT-PCR. EC-SOD DNA methylation level was detected by nested methylation specific PCR. THP-1 was induced into macrophages and divided into three groups: NC group (without any treatment, continue to cultivate), pEGFP-N1 group (added with 10 μl liposome Lipofectamine and 20 μg of empty plasmid vector pEGFP-N1), pEGFP-N1-DNMT1 group (added with 10 μl liposome Lipofectamine and 20 μg recombinant plasmid vector pEGFP-N1-DNMT1), The EC-SOD and DNMT1 protein expressions and EC-SOD DNA methylation level were detected after culture for 24 h. Results In the 8th, 12th, 16th and 20th weeks, TC, LDL and TG of the ApoE-/- group were in increasing trends, and HDL had no significant change, the TC, LDL and TG of the ApoE-/- group were significantly higher than those of the WT group, HDL was significantly lower than that of the WT group (P < 0.05). In the 16th and 20th weeks, the endothelial cells of artery tissue were regularly and orderly arranged in the WT group, while in the ApoE-/- group aortic intima was obviously thickened with inflammatory infiltration. In the 8th, 12th, 16th and 20th weeks, MDA and ROS of the ApoE-/- group were in increasing trends, the MDA and ROS content of the ApoE-/- group was significantly higher than that of the WT group (P < 0.05). EC-SOD mRNA expression level of the ApoE-/- group was significantly lower than that of the WT group, DNMT1 mRNA level and EC-SOD methylation level were significantly higher than those of the WT group (P < 0.05). DNMT1 protein expression and EC-SOD methylation level of the pEGFP-N1-DNMT1 group were significantly higher than those of the pEGFP-N1 group and the NC group, EC-SOD protein level was significantly lower than that of the pEGFP-N1 group and the NC group (P < 0.05). EC-SOD and DNMT1 protein levels and EC-SOD methylation level had no statistically differences between the pEGFP-N1 group and the NC group (P > 0.05). Conclusions Increase of EC-SOD methylation can lead to decrease of EC-SOD expression and weakening of body resistance to oxidative stress reaction, and ultimately results in development of atherosclerosis.