RESUMO
PURPOSE: Inflammation associated endothelial cell (EC) dysfunction is key to atherosclerotic disease. Recent studies have demonstrated a protective role of amitriptyline in cardiomyocytes induced by hypoxia/reoxygenation. However, the mechanism by which amitriptyline regulates the inflammatory reaction in ECs remains unknown. Thus, the aim of this study was to investigate whether amitriptyline protects against inflammation in TNF-α-treated ECs. METHODS: HUVECs were incubated with amitriptyline (2.5 µM) or TNF-α (20 ng/ml) for 24 h. EdU, tube formation, transwell, DHE fluorescence staining, and monocyte adhesion assays were performed to investigate endothelial function. Thoracic aortas were isolated from mice, and vascular tone was measured with a wire myograph system. The levels of ICAM-1, VCAM-1, MCP-1, phosphorylated MAPK and NF-κB were detected using western blotting. RESULTS: Amitriptyline increased the phosphorylation of nitric oxide synthase (eNOS) and the release of NO. Amitriptyline significantly inhibited TNF-α-induced increases in ASMase activity and the release of ceramide and downregulated TNF-α-induced expression of proinflammatory proteins, including ICAM-1, VCAM-1, and MCP-1 in ECs, as well as the secretion of sICAM-1 and sVCAM-1. TNF-α treatment obviously increased monocyte adhesion and ROS production and impaired HUVEC proliferation, migration and tube formation, while amitriptyline rescued proliferation, migration, and tube formation and decreased monocyte adhesion and ROS production. Additionally, we demonstrated that amitriptyline suppressed TNF-α-induced MAPK phosphorylation as well as the activity of NF-κB in HUVECs. The results showed that the relaxation response of aortic rings to acetylcholine in the WT-TNF-α group was much lower than that in the WT group, and the sensitivity of aortic rings to acetylcholine in the WT-TNF-α group and WT-AMI-TNF-α group was significantly higher than that in the WT-TNF-α group. CONCLUSION: These results suggest that amitriptyline reduces endothelial inflammation, consequently improving vascular endothelial function. Thus, the identification of amitriptyline as a potential strategy to improve endothelial function is important for preventing vascular diseases.
RESUMO
Compound mutations in the additional sex combs-like 3 (ASXL3) gene greatly impact the expression of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in mouse myocardial tissues. Little is known about ASXL3 mutation effects on lncRNAs and mRNAs expression in the cerebrum and cerebellum. This study aims to clarify this point using quantitative real-time polymerase chain reaction and Western blotting. Transcriptome analysis based on RNA-seq followed by bioinformatics analysis were used to compare lncRNA and mRNA expression profiles. Cell proliferation, cell cycle progression, and apoptosis were evaluated after silencing of ASXL3 expression using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2 H-tetrazolium method and flow cytometry. Results showed that ASXL3 gene expression was decreased in the cerebrum and cerebellum of mice with ASXL3 P723R*P1817A mutations. We identified 319 lncRNAs and 252 mRNAs differentially expressed in the cerebrum of ASXL3 P723R*P1817A mutant mice. In the cerebellum of ASXL3 P723R*P1817A mutant mice, 5330 lncRNAs and 2204 mRNAs were differentially expressed. Differentially expressed lncRNAs and mRNAs were widely distributed across the mouse genome and were associated with various biological processes and pathways. ASXL3 silencing by siRNA transfection affected the proliferation, cell cycle progression, and apoptosis of neural cells. Therefore, the ASXL3 P723R*P1817A mutations greatly modify the lncRNA and mRNA expression profiles in the mouse cerebrum and cerebellum.