血管软化丸调控miRNA-467b抗动脉粥样硬化的作用与初步机制研究
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河南省中医药科学研究专项重点课题(2019ZY1015);国家自然科学基金项目(81704030);河南省中医药科学研究专项重点课题(20-21ZY1023)


Anti-atherosclerotic Effect of Xueguan Ruanhua Pill and Its Preliminary Mechanism of Action by Regulating miRNA-467b
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    目的 探讨中药复方血管软化丸通过miRNA-467b靶向脂蛋白脂肪酶(lipoprotein lipase,LPL)调控巨噬细胞,减少白细胞介素(interleukin,IL)-6,IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)等炎症因子分泌和巨噬细胞脂质蓄积,从而发挥抗动脉粥样硬化的作用与初步机制。方法 将RAW264.7巨噬细胞随机分为5组,即对照组、模型组、miR-467b mimic组、中药高剂量含药血清组、中药低剂量含药血清组;经干预后,采用RT-PCR检测巨噬细胞中pri-miR-467b和LPL mRNA表达;采用高效液相色谱法检测巨噬细胞内胆固醇水平。连续4周高脂饲料(含脂肪21%、胆固醇0.15%)饲养ApoE-/-小鼠复制动脉粥样硬化模型,模型复制成功后,中药高、低剂量组每日分别灌胃血管软化丸86.4、21.6 g/kg,连续给药12周;对照组、模型组每日灌胃0.9%生理盐水86.4 g/kg 。采用免疫组织化学法检测主动脉LPL蛋白表达水平,RT-PCR检测主动脉pri-miR-467b和LPL mRNA表达水平;采用ELISA法检测血清IL-6、IL-1β、TNF-α、MCP-1水平。结果 与对照组比较,模型组巨噬细胞pri-miR-467b mRNA表达水平和巨噬细胞内游离胆固醇(free cholesterol,FC)水平均明显降低(P<0.05),巨噬细胞内LPL mRNA及其蛋白表达水平以及总胆固醇(total cholesterol,TC)、胆固醇酯(cholesterol ester,CE)水平均明显升高(P<0.05)。血管软化丸能够无剂量依赖性地升高巨噬细胞pri-miR-467b mRNA表达水平和巨噬细胞内FC水平(P<0.05),降低巨噬细胞中LPL mRNA及其蛋白表达水平以及TC、CE水平(P<0.05)。血管软化丸能够升高主动脉pri-miR-467b mRNA表达水平(P<0.05),降低主动脉中LPL mRNA及其蛋白表达水平以及血清中IL-6、IL-1β、TNF-α、 MCP-1水平(P<0.05)。结论 血管软化丸抑制动脉粥样硬化斑块形成的作用机制可能与调控miRNA-467b靶向LPL调控巨噬细胞,减少IL-6、IL-1β、TNF-α、MCP-1等炎症因子分泌和巨噬细胞脂质蓄积相关。

    Abstract:

    Objective To investigate the anti-atherosclerotic role of Xueguan Ruanhua Pill and its preliminary mechanism by targeting lipoprotein lipase (LPL) to regulate macrophages through miRNA-467b and reducing the secretion of inflammatory factors such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) and lipid accumulation in macrophages.Methods RAW264.7 macrophages were randomly divided into control group, model group, miR-467b mimic group, high-dose drug-containing serum group, and low-dose drug-containing serum group. After intervention, RT-PCR was used to measure the mRNA expression of pri-miR-467b and LPL in macrophages, and high-performance liquid chromatography was used to measure the level of cholesterol in macrophages. ApoE-/- mice were fed with high-fat diet (containing 21% fat and 0.15% cholesterol) for four consecutive weeks to establish a model of atherosclerosis; after the model was successfully established, the mice in the high- and low-dose Xueguan Ruanhua Pill groups were given Xueguan Ruanhua Pill at a dose of 86.4 and 21.6 g/kg, respectively, by gavage daily for 12 consecutive weeks, and those in the control group and the model group were given 0.9% normal saline at a dose of 86.4 g/kg by gavage daily. Immunohistochemistry was used to measure the protein expression of LPL in the aorta, RT-PCR was used to measure the mRNA expression of pri-miR-467b and LPL in the aorta, and ELISA was used to measure the serum levels of IL-6, IL-1β, TNF-α, and MCP-1. Results Compared with the control group, the model group had significant reductions in the mRNA expression of pri-miR-467b and the level of free cholesterol (FC) in macrophages (P<0.05) and significant increases in the mRNA and protein expression of LPL and the levels of total cholesterol (TC) and cholesteryl ester (CE) in macrophages (P<0.05). Xueguan Ruanhua Pill significantly increased the mRNA expression of pri-miR-467b and the level of FC in macrophages (P<0.05) and reduced the mRNA and protein expression of LPL and the levels of TC and CE in macrophages (P<0.05), without a dose-dependent manner. Xueguan Ruanhua Pill significantly increased the mRNA expression of pri-miR-467b in the aorta (P<0.05) and significantly reduced the mRNA and protein expression of LPL in the aorta and the serum levels of IL-6, IL-1β, TNF-α, and MCP-1 (P<0.05). Conclusion Xueguan Ruanhua Pill can inhibit the formation of atherosclerotic plaque, possibly by targeting LPL to regulate macrophages through miRNA-467b and reducing the secretion of inflammatory factors such as IL-6, IL-1β, TNF-α, and MCP-1 and lipid accumulation in macrophages.

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张永晨,秦合伟,赵平丽.血管软化丸调控miRNA-467b抗动脉粥样硬化的作用与初步机制研究[J].安徽中医药大学学报,2020,39(5):66-71

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  • 在线发布日期: 2020-10-15