Abstract: Objective To explore the effects of cardiomyocyte derived exosomes on macrophage polarization in response to oxidative stress and related mechanisms. Methods According to the random number table method, H9C2 cardiomyocytes were divided into four groups: a 0 μmol/L group, a 100 μmol/L group, a 200 μmol/L group and a 300 μmol/L group. Each group was treated with H2O2 at indicated concentrations. Cell viability was detected by cell counting kit‑8 (CCK‑8) assay. The levels of cleaved‑caspase‑3, B‑cell lymphoma‑2 (Bcl‑2), and B‑cell lymphoma‑2‑associated X protein (Bax) were detected by Western blot. The cell apoptosis of each group was measured by flow cytometry. According to the resultant results, the optimal treatment concentration was selected for subsequent experiments. According to the classic ultracentrifugation method, H9C2 cardiomyocytes were taken to extract normal cardiomyocyte‑derived exosomes (NC‑exo) and oxidative stress cardiomyocyte‑derived exosomes (H‑Exo). The morphology of exosomes was observed by transmission electron microscopy. The levels of exosome marker proteins were examined by Western blot. Exosome uptake and internalization in macrophages were observed under a confocal laser microscope. According to the random number table method, normal cultured macrophages were selected and divided into four groups: a control group 1 (NC1 group), a lipopolysaccharide group 1 (LPS1 group), a normal cardiomyocyte‑derived exosome pretreatment+LPS group (LPS+NC‑exo group), and an oxidative stress cardiomyocyte‑derived exosome pretreatment+LPS group (LPS+H‑exo group). The LPS1 group was treated with LPS for 6 h, while the LPS+NC‑exo group and LPS+H‑exo group were pre‑treated with NC‑exo or H‑exo for 24 h, respectively, before LPS treatment for 6 h. No treatment was performed in the NC1 group. The levels of Inducible nitric oxide synthase (iNOS), arginase‑1 (Arg‑1), CD86, and CD206 were examined by Western blot, and the levels of interleukin‑6 (IL‑6), tumor necrosis factor‑α (TNF‑α), and chemokine‑10 (CXCL‑10) mRNA were detected by real‑time quantitative polymerase chain reaction (RT‑qPCR). The levels of exosome miRNA were detected by RT‑qPCR. In addition, according to the random number table method, macrophages were divided into four groups: a control group 2 (NC2 group), a lipopolysaccharide group 2 (LPS2 group), an overexpression negative control+LPS group (LPS+mimic‑NC group), and an overexpression miR‑106b‑5p+LPS group (LPS+106b‑mimic). The LPS2 group was treated with LPS for 6 h, while the LPS+mimic‑NC group and LPS+106b‑mimic group were transfected with transfection reagents mimic‑NC or 106b‑mimic for 24 h, respectively, before treatment with LPS for 6 h. No treatment was performed in the NC2 group. The levels of iNOS, Arg‑1, CD86, and CD206 as well as the levels of IL‑6, TNF‑α, and CXCL‑10 mRNA were detected. Results Compared with the 0 μmol/L group, the 100 μmol/L group, the 200 μmol/L group and the 300 μmol/L group showed increases in the levels of Bax and cleaved‑caspase‑3, decreases in the levels of Bcl‑2 (P<0.05), reduction in cell viability (P<0.05), and increases in apoptotic rate (P<0.05). After comparison, 200 μmol/L was selected as the optimal concentration for subsequent experiments. Under a microscope, normal cardiomyocytes were spindle‑shaped, but shrunk, deformed and died after H2O2 treatment. According to transmission electron microscopy, the extracted nanoparticles were round, with a double‑layer membrane structure. After the addition of fluorescently labeled exosomes to macrophages and co‑cultivation for 24 h, a large amount of exosomes were taken by macrophages. Compared with the NC1 group, the LPS+NC‑exo group and the LPS+H‑exo group showed increases in the levels of iNOS and CD86 (P<0.05), decreases in the levels of Arg‑1 and CD206 (P<0.05), and increases in the levels of IL‑6, TNF‑α, and CXCL‑10 mRNA (P<0.05). Compared with the LPS1 group, the LPS+H‑exo group presented increases in the levels of iNOS and CD86 protein (P<0.05), decreases in the levels of Arg‑1 and CD206 protein (P<0.05), and increases in the levels of IL‑6, TNF‑α, and CXCL‑10 mRNA (P<0.05). There was no statistical difference between the LPS+NC‑exo group and the LPS1 group (P>0.05). Compared with NC‑exo, the expression of miR‑106b‑5p increased in H‑exo (P<0.05). Compared with the NC2 group, the LPS2 group, the LPS+mimic‑NC group and the PLS+106b‑mimic group showed increases in the levels of iNOS and CD86 protein (P<0.05), decreases in the levels of Arg‑1 and CD206 (P<0.05), and increases in the levels of IL‑6, TNF‑α, and CXCL‑10 mRNA (P<0.05). Compared with the LPS2 group, the LPS+106b‑mimic group presented increases in the levels of iNOS and CD86 (P<0.05), decreases in the levels of Arg‑1 and CD206 (P<0.05), and increases in the levels of IL‑6, TNF‑α and CXCL‑10 mRNA (P<0.05). However, there was no statistical difference between the LPS+mimic‑NC group and the LPS2 group (P>0.05). Conclusion Oxidative stress cardiomyocyte derived exosomes with high expression of miR‑106b‑5p promote M1 polarization in macrophages.
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