N-乙酰半胱氨酸基于miR-145/ADAM17通路缓解动脉粥样硬化氧化应激损伤的作用研究

Alleviation effect of N-acetylcysteine on oxidative stress damage of atherosclerosis based on miR-145/ADAM17 pathway

目的 探讨N-乙酰半胱氨酸(NAC)缓解动脉粥样硬化氧化应激损伤的潜在机制。方法将24只7周龄的雄性ApoE-/-小鼠随机分为3组:正常对照组(Control),喂食未经NAC处理的正常食物(n=8);动脉粥样硬化组(AS组),喂食未经NAC处理的高脂饮食(n=8);NAC处理组,喂食NAC处理(每周两次通过胃内给药50 mg/kg)的高脂饮食(n=8)。高脂饮食12周后用试剂盒检测对照组、AS组和NAC处理组中小鼠动脉的活性氧簇(ROS)和丙二醛(MDA)水平。小鼠血管内皮细胞BEND3,按照是否使用NAC处理(终浓度为10μM NAC处理细胞24 h),分为NAC处理组和不处理组,随后使用试剂盒检测两组细胞的ROS和MDA水平。miRNA高通量测序检测NAC处理后小鼠血管内皮细胞BEND3的miRNAs表达。miRDB在线分析和遗传学方法鉴定NAC调控的通路。结果 与对照组相比,AS组小鼠血管组织中MDA和ROS水平显著上升(P<0.05),NAC组小鼠血管组织中MDA和ROS水平无显著改变(P>0.05)。与AS组相比,NAC组小鼠血管组织中MDA和ROS水平均显著下降(P<0.05)。在细胞水平,与不处理组相比,处理组BEND3的MDA和ROS水平均显著下降(P<0.05),且处理组中BEND3细胞通过miRNA高通量测序发现包括miR-145在内的多种miRNAs发生上调。在BEND3细胞中过表达miR-145后,该细胞的ROS水平降低(P<0.05),去整合素金属蛋白酶17(ADAM17)的mRNA和蛋白水平亦显著下降(P<0.05)。此外,荧光素酶报告实验发现miR-145靶向ADAM17 mRNA的3’端非编码区。敲低ADAM17后,小鼠血管内皮细胞BEND3的MDA和ROS水平均显著下降(P<0.05)。结论 NAC能够调控血管内皮细胞中miR-145/ADAM17轴,降低MDA和ROS水平,缓解动脉粥样硬化氧化应激损伤。

Objective To discuss the potential mechanism of N-acetylcysteine(NAC) in alleviation of oxidative stress damage of atherosclerosis(AS). Methods Male ApoE-/-mice(n=24, aged 7 weeks) were randomly divided into normal control group(control group, given normal food without NAC treatment, n=8), AS group(given high-fat food without NAC treatment, n=8), NAC group(given food with NAC treatment, 50 mg/kg twice each week,n=8). After 12 weeks, the levels of reactive oxygen species(ROS) and malondialdehyde(MDA) in mouse artery were detected by using kit in all 3 groups. The vascular endothelial cells(BEND3) in mouse were divided, according to whether treated with NAC(final concentration=10 μM for 24 h) or not, into NAC treatment group and non-NAC treatment group. The levels of ROS and MDA were detected by using kit in 2 groups. The expression of miRNA in BEND3 was detected by using high-throughput miRNA sequencing after NAC treatment. The pathway controlled by NAC was identified by using miRDB online analysis and genetic method. Results The levels of MDA and ROS increased significantly in AS group(P<0.05), and had no significant changes in NAC group(P>0.05) compared with control group. The levels of MDA and ROS decreased significantly in NAC group(P<0.05) compared with AS group.The levels of MDA and ROS in BEND3 decreased significantly in NAC treatment group(P<0.05) compared with non-NAC treatment group. In NAC treatment group, miRNAs(including miR-145) in BEND3 were up-regulated found by high-throughput miRNA sequencing. After over-expressing of miR-145 in BEND3, ROS level decreased(P<0.05), and mRNA and protein levels of disintegrin metalloproteinase 17(ADAM17) decreased significantly(P<0.05). The results of luciferase reporter test showed that miR-145 targeted to the 3’ noncoding region of ADAM17 mRNA. After knocked down ADAM17, the levels of MDA and ROS in BEND3 decreased significantly(P<0.05). Conclusion NAC can regulate and control miR-145/ADAM17 axis in vascular endothelial cells, reduce MDA and ROS levels, and alleviate AS oxidative stress damage.