Circulating Exosomal miR-1-3p from Rats with Myocardial Infarction Plays a Protective Effect on Contrast-Induced Nephropathy via Targeting ATG13 and activating the AKT Signaling Pathway.
Circulating Exosomal miR-1-3p from Rats with Myocardial Infarction Plays a Protective Effect on Contrast-Induced Nephropathy via Targeting ATG13 and activating the AKT Signaling Pathway.
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心肌梗死大鼠循环外泌体 miR-1-3p 通过靶向 ATG13 和激活 AKT 信号通路对造影剂肾病发挥保护作用
DOI：
10.7150/ijbs.55887
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发表时间：
2021
影响因子：
9.2
通讯作者：
Zhang F
中科院分区：
文献类型：
--
作者：
Zhao P;Zhu Y;Sun L;Zhu W;Lu Y;Zhang J;Mao Y;Chen Q;Zhang F
理由：随着心血管疾病介入技术的广泛发展和造影剂（CM）的广泛应用，造影剂肾病（CIN）的发病率不断上升，与心血管疾病预后不良相关。本研究旨在探讨心肌梗死（MI）患者循环外泌体microRNA对CIN的影响及其分子机制。方法：结扎左冠状动脉前降支建立心肌梗死大鼠模型。使用商业试剂盒从对照（Exo-NC）和心肌梗死大鼠（Exo-MI）中分离循环外泌体。采用碘二醇建立CIN的体内和体外模型。采用逆转录定量PCR （RT-qPCR）检测miR-1-3p的表达。Western blot （WB）检测外泌体表面标志物、凋亡相关蛋白和自噬相关蛋白的表达。采用末端脱氧核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）染色和流式细胞术（FC）检测细胞凋亡率。透射电镜（TEM）观察外泌体和自噬体。采用苏木精和伊红（H&E）染色和肾损伤分子-1 （KIM-1）免疫组化染色评价大鼠肾损伤。采用血尿素氮（BUN）和血清肌酐（Cr）测定大鼠肾功能。采用双荧光素酶报告基因法鉴定miR-1-3p的靶基因。结果：CM处理可诱导NRK-52E细胞损伤，表现为细胞自噬增强和细胞凋亡增强。Exo-MI处理显著抑制cm诱导的NRK-52E细胞自噬和凋亡。此外，Exo-MI处理增加了Bcl-2的表达，降低了Bax的表达和LC3II/LC3I的比值。此外，TUNEL染色和FC结果显示Exo-MI可降低细胞凋亡率。通过透射电镜发现，Exo-MI减少了NRK-52E细胞中自噬体的数量。拯救实验发现Exo-MI的作用是抑制cm诱导的NRK-52E细胞的自噬和凋亡，miR-1-3p抑制剂可以抑制这一作用。此外，我们还发现过表达miR-1-3p还能抑制cm诱导的NRK-52E细胞的自噬和凋亡。通过双荧光素酶报告基因实验，发现ATG13是miR-1-3p的靶标。此外，miR-1-3p过表达显著逆转cm诱导的AKT磷酸化水平下降。此外，ATG13沉默还能抑制cm诱导的NRK-52E细胞的自噬和凋亡。在体内，Exo-MI明显减轻了CIN大鼠的肾损伤，减轻了肾纤维化，改善了肾功能。结论：心肌梗死后循环外泌体miR-1-3p通过靶向ATG13，激活AKT信号通路，抑制cm诱导的肾小管上皮细胞凋亡和自噬，改善大鼠肾功能。
Rationale: With the widespread development of the interventional technique for cardiovascular diseases and the widespread use of contrast medium (CM), the incidence of contrast-induced nephropathy (CIN) has been increasing, which is associated with poor prognosis for cardiovascular diseases. This study aims to explore the effect of circulating exosomal microRNA from patients with myocardial infarction (MI) on CIN and related molecular mechanism. Methods: A rat MI model was established by ligating the left anterior descending coronary artery. Circulating exosomes were isolated from control (Exo-NC) and MI rats (Exo-MI) using a commercial kit. The in vivo and in vitro models of CIN were created using iodixanol. Reverse transcription quantitative PCR (RT-qPCR) was utilized to detect the expression of miR-1-3p. Western blot (WB) was used to detect the expression of exosomal surface markers, and apoptosis-related and autophagy-related proteins. The apoptosis rate was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry (FC). Transmission electron microscopy (TEM) was utilized to observe the exosomes and autophagosomes. Rat kidney injury was assessed by hematoxylin and eosin (H&E) staining and kidney injury molecule-1 (KIM-1) immunohistochemical staining. Renal function of rats was assessed by detecting the levels of blood urea nitrogen (BUN) and serum creatinine (Cr). The dual luciferase reporter assay was performed to identify the target gene of miR-1-3p. Results: The treatment of CM induced NRK-52E cell damage, which manifested as enhanced cell autophagy and enhanced apoptosis. The Exo-MI treatment significantly inhibited the CM-induced autophagy and apoptosis of NRK-52E cells. Furthermore, the Exo-MI treatment increased the Bcl-2 expression, but decreased the Bax expression and the ratio of LC3II/LC3I. Furthermore, the results of the TUNEL staining and FC showed that Exo-MI can reduce apoptotic rate. Through TEM, it was found that Exo-MI reduced the number of autophagosomes in NRK-52E cells. The rescue experiments revealed that the function of Exo-MI is to inhibit the CM-induced autophagy and apoptosis of NRK-52E cells, which can be inhibited by the miR-1-3p inhibitor. Furthermore, it was found that the overexpression of miR-1-3p can also inhibit the CM-induced autophagy and apoptosis of NRK-52E cells. Through dual luciferase reporter assay, ATG13 was found to be the target of miR-1-3p. In addition, the overexpression of miR-1-3p significantly reversed the CM-induced decrease in phosphorylation level of AKT. Furthermore, ATG13 silencing can also inhibit the CM-induced autophagy and apoptosis of NRK-52E cells. In vivo, Exo-MI significantly alleviated the renal injury, reduced the renal fibrosis, and improved the renal function of CIN rats. Conclusion: The circulating exosomal miR-1-3p after MI inhibited the CM-induced apoptosis and autophagy of renal tubular epithelial cells, and improved the renal function of rats by targeting ATG13 and activating the AKT signaling pathway.
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