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Intermedin Inhibits the Ox-LDL-Induced Inflammation in RAW264.7 Cells by Affecting Fatty Acid-Binding Protein 4 Through the PKA Pathway.

ABSTRACT: Objectives: Macrophages stimulated by oxidized low-density lipoprotein (ox-LDL) play an important role in the occurrence and progression of atherosclerosis. Fatty acid-binding protein 4 (FABP4), mainly existing in macrophages and adipocytes, can influence lipid metabolism and inflammation regulated by macrophages. Herein, we first established the connection between intermedin (IMD: a new peptide that has versatile biological activities in the cardiovascular system) and FABP4 and then investigated the influence of IMD on ox-LDL-induced changes in RAW264.7 macrophages line. Methods: The bioinformatics analysis, such as gene ontology enrichment and protein-protein interactions, was performed. For ox-LDL-stimulated assays, RAW264.7 was first pretreated with IMD and then exposed to ox-LDL. To explore the cell signaling pathways of IMD on inflammatory inhibition, main signaling molecules were tested and then cells were co-incubated with relevant inhibitors, and then exposed/not exposed to IMD. Finally, cells were treated with ox-LDL. The protein and gene expression of FABP4, IL-6, and TNF-α were quantified by WB/ELISA and RT-qPCR. Results: In the ox-LDL-stimulated assays, exposure of the RAW264.7 macrophages line to ox-LDL reduced cell viability and increased the expression of FABP4, as well as induced the release of IL-6 and TNF-α (all p < 0.05). On the other hand, IMD prevented ox-LDL-induced cell toxicity, FABP4 expression, and the inflammatory level in RAW264.7 (all p < 0.05) in a dose-dependent manner. The inhibition of FABP4 and the anti-inflammatory effect of IMD were partially suppressed by the protein kinase A (PKA) inhibitor H-89. Conclusion: IMD can prevent ox-LDL-induced macrophage inflammation by inhibiting FABP4, whose signaling might partially occur via the PKA pathway.

SUBMITTER: Liu K

PROVIDER: S-EPMC8671820 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Introduction: Atherosclerosis is the main cause of many cardiovascular diseases and contributes to morbidity and mortality worldwide. The formation of macrophage-derived foam cells plays a critical role in the early stage of atherosclerosis pathogenesis. Diterpenoids found in the flowers of Callicarpa rubella Lindl., a traditional Chinese medicine, have been reported to have anti-inflammatory activity. However, little is known about the effects of these diterpenoids on macrophage foam cell formation. Methods: A macrophage-derived foam cell formation model was established by treating RAW264.7 cells with oxidized low-density lipoprotein (ox-LDL) for 24 h. Oil red O staining were used to detect the intracellular lipids. The cholesterol efflux capacity was assayed by labeling cells with 22-NBD-cholesterol. Western blots and real-time PCRs were performed to quantify protein and mRNA expressions. Results: Two diterpenoid molecules, 14α-hydroxyisopimaric acid (C069002) and isopimaric acid (C069004), extracted from the flowers of Callicarpa rubella Lindl., significantly attenuated ox-LDL-induced foam cell formation in RAW264.7 macrophages. Further investigation showed that these two diterpenoids could promote cholesterol efflux from RAW264.7 macrophages to apolipoprotein A-I or high-density lipoproteins, which was associated with upregulated expression of ATP-binding cassette A1/G1 (ABCA1/G1), liver X receptor-α (LXRα), and peroxisome proliferator-activated receptor-γ (PPARγ). Unexpectedly, the diterpenoids C069002 and C069004 failed to enhance the mRNA transcription of the ABCG1 gene in macrophage-derived foam cells induced by ox-LDL. To evaluate the effects of diterpenoids on macrophage foam cell formation and determine the underlying mechanism, two drugs (lovastatin and rosiglitazone) were used as positive controls. Although both drugs could reduce macrophage foam cell formation and promote cholesterol efflux, they each had distinctive abilities to modulate the expression of cholesterol efflux-related genes. In contrast to lovastatin, rosiglitazone showed a similar influence on the expression of cholesterol efflux-related genes (including ABCA1, LXRα, and PPARγ) as the diterpenoids regardless of the presence or absence of ox-LDL, implying a similar mechanism by which they may exert atheroprotective effects. Conclusion: Our research indicates that diterpenoids effectively inhibit ox-LDL-induced macrophage foam cell formation by promoting cholesterol efflux from macrophages via the PPARγ-LXRα-ABCA1 pathway. Further investigation of diterpenoids as potential drugs for the treatment of atherosclerosis is warranted.

Project description:BackgroundAtherosclerosis (AS), a chronic inflammatory vascular disease, is a cause of heart attack and ischemic stroke. Tripartite motif-containing protein 59 (TRIM59), a member of the tripartite motif family, has been reported to be involved in inflammatory diseases. This study was to investigate the role of TRIM59 in oxidized low-density lipoprotein (ox-LDL)-induced endothelial cells and examine the mechanism of TRIM59.MethodsTo simulate a cellular model of AS in vitro, varying concentrations of ox-LDL (i.e., 20, 40, 60, 80, and 100 µg/mL) were used to treat the human umbilical vein endothelial cells (HUVECs) for 24 h. The messenger ribonucleic acid (RNA) and protein levels of TRIM59, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and annexin 2 (AnxA2) were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The transfection efficacy of overexpression (Ov)-TRIM59 and small-interfering RNA-AnxA2 was examined by RT-qPCR and western blot. Cell counting kit-8 assays, lactate dehydrogenase (LDH) assays, enzyme-linked immunosorbent assays, and terminal-deoxynucleotidyl transferase mediated nick end labeling staining were used to examine viability, LDH expression, inflammation, and apoptosis in HUVECs. The protein levels of B-cell lymphoma 2, Bcl-2-associated X (BAX), cleaved caspase3, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 were assessed by western blot. Additionally, the adhesion of THP-1 to ox-LDL-induced HUVECs was detected using monocyte adhesion assays and the binding of TRIM59 and AnxA2 was verified by co-immunoprecipitation.ResultsThis study showed that TRIM59 expression was decreased in the ox-LDL-induced HUVECs while LOX-1 expression was increased. After transfection with Ov-TRIM59, TRIM59 in ox-LDL-induced HUVECs was increased, and TRIM59 overexpression alleviated the viability damage, inflammation, and apoptosis of the ox-LDL-induced HUVECs. In addition, THP-1 adhesion to the ox-LDL-induced HUVECs was also suppressed by TRIM59 overexpression. This study also showed that TRIM59 could bind to AnxA2 and promote AnxA2 expression in ox-LDL-stimulated HUVECs. Moreover, the rescue experiments revealed that TRIM59 suppressed the viability damage, inflammation, apoptosis, and monocyte adhesion of the ox-LDL-induced HUVECs via AnxA2.ConclusionsTRIM59 protected against ox-LDL-induced AS by binding to AnxA2.

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Intermedin Inhibits the Ox-LDL-Induced Inflammation in RAW264.7 Cells by Affecting Fatty Acid-Binding Protein 4 Through the PKA Pathway.

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