关键词: 心肌缺血再灌注, 微小RNA-378, 心肌细胞, 巨噬细胞, 缺氧/复氧

Abstract:

Objective To discuss the role of microRNA （miR）-378 in cardiomyocyte injury induced by myocardial ischemia reperfusion （I/R）， and to clarify its mechanism. Methods The human cardiomyocytes AC16 and human monocyte macrophages THP-1 were cultured. Firstly， miR-378 mimics and its negative control （miR-NC） were transfected into the THP-1 cells respectively.The cells were divided into control group， miR-NC group and miR-378 mimics group， the THP-1 cells in control group were cultured normally. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of miR-378 and the expression levels of M1 macrophage secreted factors ［inducible nitric oxide synthase （iNOS）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β） and interleukin-6 （IL-6）］ and M2 macrophage secreted factors ［arginase-1 （Arg-1）， transforming growth factor-β1 （TGF-β1）， interleukin-4 （IL-4） and interleukin-10 （IL-10）］ in the THP-1 cells after transfected； Western blotting method was used to detect the expression levels of M1 macrophage surface marker protein cluster of differentiation 86 （CD86） and M2 macrophage surface marker protein cluster of differentiation 206 （CD206）. The AC16 cells underwent hypoxia/reoxygenation （H/R） treatment to simulate I/R cardiomyocyte injury， and were co-cultured with differently treated THP-1 cells. The cells were divided into control group （upper chamber containing AC16 cells， lower chamber containing THP-1 cells）， H/R group （upper chamber containing H/R-induced AC16 cells， lower chamber containing THP-1 cells）， miR-NC+H/R group （upper chamber containing H/R-induced AC16 cells， lower chamber containing THP-1 cells transfected with miR-NC） and miR-378 mimics+H/R group （upper chamber containing H/R-induced AC16 cells， lower chamber containing THP-1 cells transfected with miR-378 mimics）. Cell counting kit-8 （CCK-8） method was used to detect the activities of the AC16 cells in various groups； kits were used to detect the levels of malondialdehyde （MDA） and reactive oxygen species （ROS） and the activity of superoxide dismutase （SOD） in the AC16 cells in various groups as well as the lactate dehydrogenase （LDH） activity in the cell supernatant. Results The RT-qPCR method and Western blotting method results showed that compared with control group and miR-NC group， the expression level of miR-378 in the THP-1 cells in miR-378 mimics group was significantly increased （P<0.05）； the expression levels of iNOS， TNF-α， IL-1β， and IL-6 mRNA and the expression level of CD86 protein in the THP-1 cells were significantly decreased （P<0.05）； the expression levels of Arg-1， TGF-β1， IL-4， and IL-10 mRNA and the expression level of CD206 protein in the THP-1 cells were significantly increased （P<0.05）. The CCK-8 method and kit method results showed that compared with control group， the activity of the AC16 cells in H/R group was significantly decreased （P<0.05）， the levels of MDA and ROS in the cells were significantly increased （P<0.05）， the activity of SOD was significantly decreased （P<0.05）， and the LDH activity in the cell supernatant was significantly increased （P<0.05）. Compared with H/R group， the activity of the AC16 cells in miR-378 mimics+H/R group was significantly increased （P<0.05）， the levels of MDA and ROS in the cells were significantly decreased （P<0.05）， the activity of SOD was significantly increased （P<0.05）， and the LDH activity in the cell supernatant was significantly decreased （P<0.05）. Conclusion miR-378 can alleviate H/R-induced cardiomyocyte injury， and its mechanism may be related to regulating macrophage polarization towards the M2 phenotype.

Key words: Myocardial ischemia-reperfusion, MicroRNA-378, Cardiomyocytes, Macrophages, Hypoxia/reoxygenation