微小RNA-1270靶向调控血管生成素样蛋白7抑制巨噬细胞炎症和脂质蓄积

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doi:10.6040/j.issn.1671-7554.0.2024.0787

摘要： 目的应用氧化低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)诱导的小鼠巨噬细胞株制备动脉粥样硬化模型,观察微小RNA-1270(microRNA-1270, miRNA-1270)是否经血管生成素样蛋白7(angiopoietin-like protein 7, ANGPTL7)/ p38途径干预巨噬细胞炎症及血脂代谢。方法培养小鼠单核巨噬细胞(RAW264.7),并加入ox-LDL构建动脉粥样硬化模型。根据干预条件不同分为空白组、ox-LDL组、ANGPTL7组、 p38蛋白抑制组、 p65蛋白抑制组、miR-1270模拟物组、miR-1270模拟物阴性对照组、miR-1270抑制物组和miR-1270抑制物阴性对照组。采用实时定量 PCR法检测mRNA表达水平,采用Western blotting法检测蛋白质表达水平,采用油红染色法检测脂质蓄积。结果 ox-LDL暴露的巨噬细胞中含红色脂肪微粒的巨噬细胞数量增加,ANGPTL7高表达,p38高表达,白介素-6(interleukin-6, IL-6)高表达,白介素-10(interleukin-10, IL-10)低表达,且ANGPTL7与p38及脂质蓄积呈正相关(P<0.01)。与ox-LDL组相比,ANGPTL7组ANGPTL7、p38及IL-6蛋白相对表达量增加,IL-10蛋白相对表达量减少,含红色脂肪微粒的巨噬细胞数量增加(P<0.01)。与ANGPTL7组相比,p38蛋白抑制组ANGPTL7和p38蛋白相对表达量无明显差异,IL-6蛋白相对表达量减少,IL-10蛋白相对表达量增加,含红色脂肪微粒的巨噬细胞数量减少(P<0.01)。与ANGPTL7组相比,p65蛋白抑制组各项指标表达量、含红色脂肪微粒的巨噬细胞数量差异无统计学意义(P>0.05)。与空白组相比,ox-LDL组miR-1270基因相对表达量减少,ANGPTL7基因及蛋白相对表达量增加, miR-1270与ANGPTL7基因相对表达量呈负相关(P<0.01)。与ox-LDL组相比,miR-1270模拟物组miR-1270基因相对表达量增加,ANGPTL7基因及蛋白相对表达量减少,p38及IL-6蛋白相对表达量减少,IL-10蛋白相对表达量增加,含红色脂肪微粒的巨噬细胞数量减少(P<0.01);miR-1270抑制物组miR-1270基因相对表达量减少,ANGPTL7基因及蛋白相对表达量增加,p38及IL-6蛋白相对表达量增加,IL-10蛋白相对表达量减少,含红色脂肪微粒的巨噬细胞数量增加(P<0.01);与ox-LDL组相比,阴性对照物组各项指标表达量无明显差异,含红色脂肪微粒的巨噬细胞数量差异无统计学意义(P>0.05)。结论在ox-LDL暴露的巨噬细胞模型中,ANGPTL7促进巨噬细胞内的炎症和脂质蓄积必经p38途径实现,是促进动脉粥样硬化的发生发展新机制。miR-1270作为保护性因子,可靶向抑制ANGPTL7基因转录表达,经p38途径减轻巨噬细胞炎症及脂质蓄积,可逆调控动脉粥样硬化发生发展,是动脉粥样硬化潜在的早期筛查靶点。

关键词: 基因表观遗传学, 动脉粥样硬化, 微小RNA-1270, 血管生成素样蛋白7, p38蛋白, 炎症反应, 脂质蓄积

Abstract: Objective To prepare an atherosclerosis(As)model employing an oxidised low-density lipoprotein(ox-LDL)-induced mouse macrophage cell line, so as to see whether microRNA-1270(miRNA-1270)interfered with macrophage inflammation and lipid metabolism via the angiopoietin-like protein 7(ANGPTL7)/p38 pathway. Methods Mouse mononuclear macrophages(RAW264.7)were cultured and ox-LDL was added to construct macrophage models. According to the different intervention conditions, the groups were as follows: blank group, ox-LDL group, ANGPTL7 group, p38 protein inhibition group, p65 protein inhibition group, miR-1270 mimic group, miR-1270 mimic negative control group, miR-1270 inhibitor group, and miR-1270 inhibitor negative control group. The mRNA expression level was detected by real-time quantitative PCR, the protein expression level was detected by Western bloting, and the lipid accumulation was detected by oil red staining. Results The number of macrophages containing red fat particles was increased in ox-LDL-exposed macrophages, with high expressions of ANGPTL7, p38, and IL-6, and low expression of IL-10, and a positive correlation between ANGPTL7 and p38 and lipid accumulation(P<0.01). Compared with the ANGPTL7 group, there was no significant difference in the relative expression of ANGPTL7 and p38 proteins in the p38 protein inhibition group, a decrease in the relative expression of IL-6 protein, an increase in the relative expression of IL-10 protein, and a decrease in the number of red adipose microparticle-containing macrophages(P<0.01). Compared with the ANGPTL7 group, there was no statistically significant difference in the expression of the indicators in the p65 protein inhibition group, and there was no statistically significant difference in the number of macrophages containing red fat particles(P>0.05). Compared with the blank group, the relative expression of miR-1270 gene was decreased and the relative expression of ANGPTL7 gene and protein was increased in the ox-LDL group, and miR-1270 was negatively correlated with the relative expressions of ANGPTL7 gene(r2=0.665 7, P<0.01). Compared with the ox-LDL group, the miR-1270 mimic group had increased relative expression of the miR-1270 gene, decreased relative expression of the ANGPTL7 gene and protein, decreased relative expression of the p38 and IL-6 proteins, increased relative expression of the IL-10 protein, and decreased number of red adipose microparticle-containing macrophages(P<0.01). The miR-1270 inhibitor group had a decrease in the relative expression of the miR-1270 gene, an increase in the relative expression of the ANGPTL7 gene and protein, an increase in the relative expressions of the p38 and IL-6 proteins, a decrease in the relative expression of the IL-10 protein, and an increase in the number of macrophages containing red adipose microparticles(P<0.01). Compared with the ox-LDL group, there was no significant difference in the expression of the indicators in the negative control group, and there was no statistically significant difference in the number of macrophages containing red fat particles(P>0.05). Conclusion In the ox-LDL-exposed macrophage model, ANGPTL7 promotes inflammation and lipid accumulation in macrophages via the p38 pathway, which is a new mechanism to promote the development of atherosclerosis. miR-1270, as a protective factor, can target and inhibit the transcriptional expression of the ANGPTL7 gene, reduce macrophage inflammation and lipid accumulation via the p38 pathway, and reversibly control the development of atherosclerosis, and is a potential early screening target for atherosclerosis. It is a potential target for early screening of atherosclerosis.

Key words: Gene epigenetics, Atherosclerosis, microRNA-1270, Angiopoietin-like protein 7, p38 protein, Inflammatory response, Lpid accumulation

中图分类号:

R543.5

杜艾家,张曼,陈鹤,王丽新,尚应殊. 微小RNA-1270靶向调控血管生成素样蛋白7抑制巨噬细胞炎症和脂质蓄积[J]. 山东大学学报 (医学版), 2025, 63(2): 1-9.

DU Aijia, ZHANG Man, CHEN He, WANG Lixin, SHANG Yingshu. miR-1270-targeted regulation of angiopoietin-like protein 7 inhibits macrophage inflammation and lipid accumulation[J]. Journal of Shandong University (Health Sciences), 2025, 63(2): 1-9.

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