Department of Cardiology, The Second Affiliated Hospital of Zunyi Medical University.
Department of Cardiology, Guizhou Provincial People's Hospital.
Int Heart J. 2023;64(5):945-954. doi: 10.1536/ihj.23-087.
Tanshinone IIA (Tan IIA), the core ingredient of Salvia miltiorrhiza, is commonly used for treating cardiovascular diseases. However, its underlying mechanism in regulating autophagy in atherosclerosis (AS) remains unclear. An in vivo model of AS was constructed using Apolipoprotein E-deficient (ApoE) mice fed with a high-fat diet. Histopathologic changes and lipid accumulation were evaluated by hematoxylin and eosin (HE) and Oil red O staining, respectively. The inflammatory cytokine levels were evaluated by Enzyme-linked immunosorbent assay (ELISA). An oxidized low-density lipoprotein (ox-LDL) was used to induce foam cells in RAW264.7 cells. Cholesterol uptake and efflux assay were used to assess changes in intracellular and extracellular cholesterol levels. The expression levels of autophagy-related protein-16-like protein 1 (ATG16L1) and miR-214-3p in the samples and cells derived from mice were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein levels of the mitogen-activated protein kinase (MAPK)/mammalian target of rapamycin (mTOR) and autophagy-related markers were detected using western blot. The binding site of miR-214-3p on ATG16L1 was determined using a dual-luciferase reporter assay. We observed a decrease in ATG16L1 and increase in miR-214-3p expression level in the AS mice and ox-LDL stimulated RAW264.7 cells. However, the miR-214-3p and ATG16L1 expression could be reversed by Tan IIA. In vivo experiments showed that Tan IIA alleviated AS by reducing lipid accumulation and inflammatory factor levels and promoting autophagy. The in vitro assays demonstrated that Tan IIA regulated lipid levels and autophagy via the miR-214-3p/ATG16L1 axis to inhibit foam cell formation. Additionally, Tan IIA inhibited the MAPK/mTOR pathway by reducing miR-214-3p expression and promoting autophagy. Findings from this study suggested that Tan IIA regulated the MAPK/mTOR signal-mediated autophagy to alleviate AS through the miR-214-3p/ATG16L1 axis.
丹参酮 IIA（Tan IIA）是丹参的主要成分，常用于治疗心血管疾病。然而，其在调节动脉粥样硬化（AS）自噬中的作用机制尚不清楚。本研究采用载脂蛋白 E 缺陷（ApoE）小鼠高脂饮食喂养构建 AS 体内模型，通过苏木精-伊红（HE）和油红 O 染色评估组织病理学变化和脂质积聚，酶联免疫吸附试验（ELISA）评估炎症细胞因子水平。采用氧化型低密度脂蛋白（ox-LDL）诱导 RAW264.7 细胞泡沫化，胆固醇摄取和流出实验评估细胞内外胆固醇水平变化。采用实时定量聚合酶链反应（qRT-PCR）检测小鼠样本和细胞中自噬相关蛋白 16 样蛋白 1（ATG16L1）和 miR-214-3p 的表达水平，采用蛋白质印迹法检测丝裂原活化蛋白激酶（MAPK）/哺乳动物雷帕霉素靶蛋白（mTOR）和自噬相关标志物的蛋白水平。采用双荧光素酶报告基因实验检测 miR-214-3p 与 ATG16L1 的结合位点。结果显示，AS 小鼠和 ox-LDL 刺激的 RAW264.7 细胞中 ATG16L1 表达降低，miR-214-3p 表达升高，但 Tan IIA 可逆转这一变化。体内实验表明，Tan IIA 通过减少脂质积聚和炎症因子水平、促进自噬来缓解 AS。体外实验表明，Tan IIA 通过 miR-214-3p/ATG16L1 轴调节脂质水平和自噬，抑制泡沫细胞形成。此外，Tan IIA 通过降低 miR-214-3p 表达和促进自噬抑制 MAPK/mTOR 通路。综上所述，Tan IIA 通过 miR-214-3p/ATG16L1 轴调节 MAPK/mTOR 信号介导的自噬，缓解 AS。
