Zheng Wan, Li Tianfa, Wei Junping, Zhang Yuanyuan, Zuo Qi, Lin Yun
Department of Cardiovascular Internal Medicine, The First Affiliated Hospital of Hainan Medical University, Haikou, Hainan 570102, P.R. China.
Exp Ther Med. 2021 May;21(5):467. doi: 10.3892/etm.2021.9898. Epub 2021 Mar 8.
The current study aimed to explore the effects of microRNA (miR)-145 on the inflammatory response and oxidative stress (OS) in high glucose (HG)-induced cardiomyocytes, as well as the specific mechanism underlying this action. H9c2 cells were treated with 33 mmol/l glucose (HG group) or cotreated with 24.5 mmol/l mannitol and 5.5 mmol/l glucose (hypertonic group), and the expression levels of miR-145 and ADP ribosylation factor 6 (ARF6) were detected. The cells were transfected with pcDNA3.1-ARF6, miR-145 mimics or corresponding negative controls prior to the assessment of cell survival rate. Levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and the levels of IL-6, TNF-α and monocyte chemoattractant protein-1 (MCP-1) were subsequently determined. The apoptotic rate of H9c2 cells was examined by flow cytometry. The interaction between miR-145-ARF6 was predicted and confirmed by luciferase reporter assays. In the HG group, miR-145 expression was significantly decreased and ARF6 expression significantly increased compared with controls. Furthermore, the levels of inflammatory factors (IL-6, TNF-α and MCP-1), LDH, ROS and MDA were significantly elevated in the HG group compared with controls. Significantly decreased SOD, CAT and GPx activities and significantly increased numbers of apoptotic cells were observed in the HG group compared with controls. The cells transfected with miR-145 mimics exhibited significantly decreased LDH, ROS and MDA levels, significantly increased antioxidant enzyme activities and significantly decreased apoptotic rates compared with controls, while the opposite results were observed in cells transfected with pcDNA3.1-ARF6. Moreover, co-transfection with miR-145 mimics and pcDNA3.1-ARF6 exacerbated the inflammatory response and OS injury in HG-induced cardiomyocytes compared with cells transfected with miR-145 mimics alone. Furthermore, miR-145 negatively targeted ARF6. miR-145 attenuated the HG-induced inflammatory response and OS injury in cardiomyocytes by negatively regulating ARF6, which may contribute to providing a theoretical basis for the treatment of diabetic cardiomyopathy.
本研究旨在探讨微小RNA（miR）-145对高糖（HG）诱导的心肌细胞炎症反应和氧化应激（OS）的影响，以及其作用的具体机制。将H9c2细胞用33 mmol/l葡萄糖处理（HG组）或与24.5 mmol/l甘露醇和5.5 mmol/l葡萄糖共同处理（高渗组），并检测miR-145和ADP核糖基化因子6（ARF6）的表达水平。在评估细胞存活率之前，将细胞用pcDNA3.1-ARF6、miR-145模拟物或相应的阴性对照进行转染。随后测定乳酸脱氢酶（LDH）、活性氧（ROS）和丙二醛（MDA）水平，以及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx）的活性，以及白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和单核细胞趋化蛋白-1（MCP-1）的水平。通过流式细胞术检测H9c2细胞的凋亡率。通过荧光素酶报告基因检测预测并证实了miR-145与ARF6之间的相互作用。与对照组相比，HG组miR-145表达显著降低，ARF6表达显著增加。此外，与对照组相比，HG组炎症因子（IL-6、TNF-α和MCP-1）、LDH、ROS和MDA水平显著升高。与对照组相比，HG组SOD、CAT和GPx活性显著降低，凋亡细胞数量显著增加。与对照组相比，转染miR-145模拟物的细胞LDH、ROS和MDA水平显著降低，抗氧化酶活性显著增加，凋亡率显著降低，而转染pcDNA3.1-ARF6的细胞则观察到相反的结果。此外，与单独转染miR-145模拟物的细胞相比，miR-145模拟物和pcDNA3.1-ARF6共转染加剧了HG诱导的心肌细胞炎症反应和OS损伤。此外，miR-145负向靶向ARF6。miR-145通过负向调节ARF6减轻HG诱导的心肌细胞炎症反应和OS损伤，这可能为糖尿病心肌病的治疗提供理论依据。
