循环内皮微粒相关microRNA在巨噬细胞炎性反应诱发小鼠动脉粥样硬化发生的机制

Mechanism of Circulating Endothelial MicroRNA Related to Atherosclerosis Induced by Inflammatory Response of Macrophages in Mouse

目的研究循环内皮微粒(EMPs)相关microRNA在巨噬细胞炎性反应诱发动脉粥样硬化(AS)发生的作用机制。方法 SPF级雄性ApoE-/-小鼠206只,对照组50只,实验组随机分为动脉粥样硬化模型(AS模型)组、NC-miRNA组和miRNA-19b抑制剂组,每组52只。采用油红染色对AS病变小鼠的组织学进行观察,计算斑块面积相对比例;检测生化指标的表达水平,主要包括小鼠血清高密度脂蛋白胆固醇(HDL-C)、胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)和甘油三酯(TG);ELISA检测血管炎症因子肿瘤坏死因子(TNF-α)、白细胞介素1(IL-1)、IL-6和IL-10的变化;Western blot检测凋亡相关蛋白B细胞淋巴瘤因子(Bcl-2)、细胞凋亡的标志蛋白PARP降解产物(cleaved-PARP)和B细胞淋巴瘤因子相关X蛋白(Bax)表达的变化,实时荧光定量PCR检测miR-19b表达;流式细胞法检测巨噬细胞的凋亡率。结果 miRNA-19b抑制剂组可减少由于AS引起的血管厚度增加,减少AS斑块面积比例;使血脂中的TC、TG和LDL-C水平降低,HDL-C水平则有升高;Bax、cleaved-PARP促相关的凋亡蛋白表达量下调,同时Bcl-2抗凋亡蛋白表达上调;血管中促炎症因子IL-1、IL-6和TNF-α水平降低,抗炎症因子IL-10的水平则升高;同时使血管内巨噬细胞的凋亡率下降。结论 EMPs相关miRNA-19b的存在可以减少巨噬细胞的凋亡,并且通过上调促炎因子促进组织周围炎症的发生,进而促进AS的发展进程。

Objective To study the mechanism of microRNA related to circulating endothelial particulates(EMPs) on atherosclerosis(AS) induced by inflammatory response of macrophages.Methods There were 206 male ApoE-/-mice of SPF grade and 50 control groups. The experimental group was randomly divided into three groups: AS model(AS) group, NC-miRNA group and miRNA-19 b inhibitor group with 52 in each group, using oil red staining of atherosclerotic lesions in the histology were observed, calculated plaque area ratio, and detect the serum cholesterol(TC), triglyceride(TG),high-density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol(LDL-C) biochemical index expression level of TNF alpha ELISA to detect vascular inflammation factors, IL-1, the change of IL-6 and IL-10, bcl-2, cleaved-PARP and Bax expression were detected by Western blot, mir-19 b expression was detected by real-time quantitative PCR, and the apoptosis rate of macrophages was detected by flow cytometry. Results Micrornas-19 b inhibitor group can decrease due to the increase in the thickness of the blood vessels of AS, reduce atherosclerotic plaque area percentage, the blood lipid level of TC, TG and LDL-C in the lower, HDL-C level has increased, promoting apoptosis related proteins Bax, cleaved-PARP expression decreased, and resistance to apoptosis related proteins increased the expression of Bcl-2 and proinflammatory factor in vascular IL-1, IL-6 and TNF-α level will be significantly lower, anti-inflammatory factor of IL-10 level is higher, At the same time, the apoptosis rate of endovascular macrophages decreased. Conclusion The presence of circulating endothelial microcore-related mirna-19 b can reduce the apoptosis of macrophages, and promote the development of peripheral inflammation by up-regulating pro-inflammatory factors, thus promoting the development of AS.