清道夫受体A启动子区DNA甲基化在同型半胱氨酸致动脉粥样硬化中的作用  

Effects of scavenger receptor A and the methylation of its promoter DNA on homocysteine-induced atherosclerosis

作　　者：张靖 张自新[2] 姜怡邓[3] ZHANG Jing;ZHANG Zi-xin;JIANG Yi-deng(Department of Cardiovascular Medicine,People's Hospital ofNingxia,Yinchuan 750002,Ningxia,China)

机构地区：[1]宁夏人民医院心血管内科,宁夏银川750002 [2]宁夏医科大学总医院放疗科,宁夏银川750004 [3]宁夏医科大学病理生理教研室,宁夏银川750004

出　　处：《广东医学》2019年第12期1678-1684,共7页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81360059);宁夏回族自治区重点研发项目(编号:2019BEG03008)

摘　　要：目的探讨清道夫受体A(SR-A)表达水平改变及其启动子区DNA甲基化在同型半胱氨酸(Hcy)致动脉粥样硬化过程中的作用及其机制,为Hcy引起动脉粥样硬化的诊疗提供理论依据。方法采用高蛋氨酸饮食饲喂ApoE^-/-小鼠16周复制高同型半胱氨酸血症动物模型,设立正常对照组(C57BL/6J小鼠饲以正常饮食)以及模型对照组(ApoE^-/-小鼠饲以正常饮食)。取小鼠主动脉制作冰冻切片,行油红O染色,观察不同组小鼠的主动脉粥样硬化病变程度;测定不同组小鼠血清中Hcy及血脂相关指标水平;用含有氧化低密度脂蛋白的培养液培养THP-1源性巨噬细胞以复制泡沫细胞模型,通过细胞油红O染色确定复制是否成功;分别以0、50、100、200、500μmol/L Hcy及50μmol/L Hcy+10μmol/L AZC干预细胞。通过实时定量荧光PCR(RT-qPCR)及Western blot检测不同组小鼠主动脉及泡沫细胞中SR-A的mRNA及蛋白表达水平;采用巢式降落式甲基化特异性PCR(nMS-PCR)测定不同组小鼠主动脉中及泡沫细胞中SR-A DNA的甲基化水平;通过泡沫细胞油红O染色观察DNA甲基化抑制剂(AZC)对泡沫细胞内脂质蓄积的影响;采用Student-Newman-Keuls检验进行相关性分析。结果与对照组小鼠相比,高蛋氨酸组小鼠血清中Hcy水平明显升高(P<0.01),小鼠主动脉冰冻切片的油红O染色结果显示其主动脉发生明显粥样病变。与对照组相比,高蛋氨酸组小鼠及Hcy刺激后的泡沫细胞中SR-A的mRNA及蛋白表达水平明显升高(P<0.01)。高蛋氨酸组小鼠及Hcy刺激的泡沫细胞中SR-ADNA甲基化水平并未发生显著改变(P<0.01)。采用DNA甲基化抑制剂AZC刺激泡沫细胞后,泡沫细胞内的脂质蓄积明显增加。结论 SR-A表达上调有可能在Hcy致动脉粥样硬化的过程中扮演了极为关键的角色,过表达DNMT1可以上调SR-A的表达,而这种调控作用的途径可能并非是通过影响SR-A启动子区的DNA甲基化程度来实现的。Objective To investigate the effects of scavenger receptor A and the methylation of its promoter DNA on homocysteine-induced atherosclerosis. Methods ApoE^-/- mice were fed with high methionine diet for 16 weeks to replicate the HHcy animal model(Meth group), while normal control group(C57 BL/6 J mouse fed with normal diet, N-control group) and model control group(ApoE^-/- mouse fed with normal diet, A-control group) were set up. Aortic frozen sections were made and performed with HE and oil red O staining to observe the degree of atherosclerosis in mice. The levels of serum Hcy and blood lipid were measured by automatic biochemical analyzer. THP-1 derived macrophages were treated with oxidized low-density lipoprotein to replicate the foam cell model. Oil red O staining was performed to determine the success of replication. The foam cells were treated with 0, 50, 100, 200, and 500 μmol/L Hcy, and 50 μmol/L Hcy+10 μmol/L AZC, respectively. The expression of SR-A in aorta of mice and foam cells was assessed by real-time quantitative PCR(RT-qPCR). The methylation level of SR-A DNA in mouse aorta and foam cells was determined by nested methylation specific PCR(nMS-PCR). The effect of DNA methylation inhibitor(AZC) on lipid accumulation foam cells was also detected. Results Compared with control groups, the serum Hcy in Methy group was significantly higher(P<0.01), with significant arthrosclerosis in aorta. Compared with control groups, significant up-regulation of SR-A mRNA was observed in Methy group and Hcy stimulation foam cells(P<0.01);however, no significant difference in SR-ARNA methylation was observed. Conclusion The up-regulation of SR-A plays an important role in Hcy-induced arthrosclerosis. The over-expression of DNMT1 induces this process, probably via the methylation of SR-A promoter.

关 键 词：清道夫受体A 同型半胱氨酸 泡沫细胞 DNA甲基化 APOE^-/-小鼠 

分 类 号：R543.5[医药卫生—心血管疾病] R363[医药卫生—内科学]