Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, College of Medicine, Wuhan University of Science and Technology, Wuhan, Hubei Province, China.
Department of Cardiology, Union Hospital, Huazhong University of Science and Technology, China.
Life Sci. 2021 Mar 1;268:118989. doi: 10.1016/j.lfs.2020.118989. Epub 2021 Jan 5.
The imbalance of M1/M2 macrophage ratio promotes the occurrence of diabetic cardiomyopathy (DCM), but the precise mechanisms are not fully understood. The aim of this study was to investigate whether miR-471-3p/silent information regulator 1 (SIRT1) pathway is involved in the macrophage polarization during the development of DCM.
Immunohistochemical staining was used to detect M1 and M2 macrophages infiltration in the heart tissue. Flow cytometry was used to detect the proportion of M1 and M2 macrophages. Expression of miR-471-3p was quantified by real time quantitative-PCR. Transfection of miRNA inhibitor into RAW264.7 cells was performed to investigate the underlying mechanisms. Bioinformatics methods and western blotting were used to explore the target gene of miR-471-3p and further confirmed by dual luciferase reporter assay.
We observed that M1 macrophages infiltration in the heart of tissue in DCM while M2 type was decreased. M1/M2 ratio was increased significantly in bone marrow-derived macrophages (BMDMs) from db/db mice and in RAW264.7 cells treated with advanced glycation end products (AGEs). Meanwhile, miR-471-3p was significantly upregulated in RAW264.7 cells induced by AGEs and inhibition of miR-471-3p could reduce the inflammatory polarization of macrophages. Bioinformatics analysis identified SIRT1 as a target of miR-471-3p. Both dual luciferase reporter assay and western blotting verified that miR-471-3p negatively regulated SIRT1 expression. SIRT1 agonist resveratrol could downregulate the increased proportion of M1 macrophages induced by AGEs.
Our results indicated that the development of DCM was related to AGEs-induced macrophage polarized to M1 type through a mechanism involving the miR-471-3p/SIRT1 pathway.
M1/M2 巨噬细胞比例失衡促进糖尿病心肌病（DCM）的发生，但确切机制尚不完全清楚。本研究旨在探讨 miR-471-3p/沉默信息调节因子 1（SIRT1）通路是否参与 DCM 发展过程中的巨噬细胞极化。
免疫组织化学染色检测心脏组织中 M1 和 M2 巨噬细胞浸润情况。流式细胞术检测 M1 和 M2 巨噬细胞比例。实时定量 PCR 检测 miR-471-3p 的表达。用 miRNA 抑制剂转染 RAW264.7 细胞，探讨其潜在机制。采用生物信息学方法和 Western blot 检测 miR-471-3p 的靶基因，并通过双荧光素酶报告基因实验进一步证实。
我们观察到 DCM 患者心脏组织中 M1 型巨噬细胞浸润增加，M2 型减少。db/db 小鼠骨髓来源巨噬细胞（BMDMs）和 AGEs 处理的 RAW264.7 细胞中 M1/M2 比值明显升高。同时，AGEs 诱导的 RAW264.7 细胞中 miR-471-3p 显著上调，抑制 miR-471-3p 可减少巨噬细胞的炎症极化。生物信息学分析表明 SIRT1 是 miR-471-3p 的靶基因。双荧光素酶报告基因检测和 Western blot 均证实 miR-471-3p 负调控 SIRT1 表达。SIRT1 激动剂白藜芦醇可下调 AGEs 诱导的 M1 型巨噬细胞比例增加。
本研究结果表明，DCM 的发生与 AGEs 诱导的巨噬细胞向 M1 型极化有关，其机制涉及 miR-471-3p/SIRT1 通路。
