OBJECTIVETo investigate the effect of diclofenac on the expression of Kv1.3 and Kir2.1 channels in human macrophages and the membrane potential and foaming process of the macrophages.

METHODSThe effect of diclofenac on the expression of Kv1.3 and Kir2.1 channels in cultured human monocyte-derived macrophages was investigated using real-time RT-PCR and Western blotting, and its effect on the membrane potential was analyzed with optical mapping of the membrane potential with voltage-sensitive dyes. The ratio of cholesterol ester (CE) in the macrophages following intake of oxidized low-density lipoprotein (OxLDL) was analyzed by an enzymatic fluorometric method.

RESULTSThe expression of Kv1.3 and Kir2.1 channels in the macrophages were down-regulated by diclofenac (1.5 µmol/L and 15 µmol/L). Compared with those in the control group, Kv1.3 mRNA expression was reduced by over 80% and 90% (P<0.05), and Kir2.1 mRNA by over 20% and 30% (P>0.05), respectively; both their protein expression was reduced by over 10% and 60% with a dose- dependent effect (P<0.05). Diclofenac at the two doses dose-dependently reduced the surface fluorescence intensity of the macrophage, and the membrane potential was decreased by 28% and 54%, respectively (P<0.05). Incubation of the macrophages with 30 mg/L OxLDL for 60 h caused an obvious enlargement of the cell volume and deposition of numerous lipid granules in cytoplasm, resulting also in a CE/TC ratio over 50% (P<0.05). Diclofenac at 1.5 and 15 µmol/L both significantly decreased the CE/TC ratio to (23.624∓3.34)% and (13.601∓2.916)% (P<0.05), respectively, but this effect did not show a dose-response relationship (P>0.05).

CONCLUSIONDiclofenac can significant down-regulate the expression of Kv1.3 and Kir2.1 channels in human macrophages, lower their membrane potential and inhibit the process of foam cell formation.

Cells, Cultured ; Diclofenac ; pharmacology ; Foam Cells ; cytology ; drug effects ; Humans ; Kv1.3 Potassium Channel ; metabolism ; Macrophages ; drug effects ; metabolism ; physiology ; Membrane Potentials ; drug effects ; Potassium Channels, Inwardly Rectifying ; metabolism

OBJECTIVETo establish the model of bone mesenchymal stem cell-derived smooth muscle cells (BMSC-SMCs) and investigate the role of BMSC-SMCs in the development and progression of artherosclerosis.

METHODSBMSCs were isolated from the femoral bone of SD rats by adherent tissue culture method, and vascular smooth muscle cells (VSMCs) were obtained from the thoracic aorta. The differentiation of BMSCs into BMSC-SMCs was induced in the conditioned medium. The specific markers of BMSCs and BMSC-SMCs were identified by immunofluorescence (IF) staining. After treatment with 80 mg/L oxidative low-density lipoprotein (ox-LDL) for 72 h, the growth characteristics of BMSC-SMCs and VSMCs were observed. Flow cytometry was applied to analyze the cell cycle of BMSC-SMCs and VSMCs.

RESULTSBMCS-SMCs transformed into foam cells after treatment with ox-LDL, which was more obvious in comparison with VSMCs. The growth curve of BMSC-SMCs and VSMCs presented with an S-shape pattern with the cell doubling time of 20 and 32 h, which was reduced to 15 and 28 h after treatment with 80 mg/L ox-LDL, respectively. Flow cytometry showed that exposure to 80 mg/L ox-LDL significantly increased G(0)/G(1) and decreased S and G(2)/M phase cells in both BMSC-SMCs (P<0.01, n=3) and VSMCs (P<0.05, n=3) in comparison with the control cells.

CONCLUSIONBMSC-SMC might be involved in the formation of fatty core and accelerate the development of atherosclerosis.

Animals ; Atherosclerosis ; etiology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Foam Cells ; cytology ; Lipoproteins, LDL ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; Muscle, Smooth, Vascular ; cytology ; Rats ; Rats, Sprague-Dawley

Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.
Antioxidants/*pharmacology ; Cells, Cultured ; Chemokine CCL2/genetics/*metabolism ; Enzyme Activation/drug effects ; Foam Cells/*drug effects/physiology ; Forkhead Transcription Factors/metabolism ; Humans ; Lipopolysaccharides/pharmacology ; NADH, NADPH Oxidoreductases/genetics/*metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; RNA, Messenger/metabolism ; Reactive Oxygen Species/*metabolism ; Signal Transduction ; Stilbenes/*pharmacology

The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction of endoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum, and then treated with ox-LDL (25, 50 and 100 mg/L), anti-CD36 monoclonal antibody+ox-LDL and tunicamycin (TM), respectively. After incubation for 24 h, the cells were collected. The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistry assay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein 1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and 100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol were increased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expression of GRP94 in a dose-dependent manner. Furthermore, p-IRE1 and XBP1, two key components of the unfolded protein response, were also significantly induced by the treatment with ox-LDL. The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages, which is potentially mediated by scavenger receptor CD36.
Animals ; CD36 Antigens ; physiology ; Cell Line ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; drug effects ; Foam Cells ; cytology ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Protein-Serine-Threonine Kinases ; metabolism ; Regulatory Factor X Transcription Factors ; Stress, Physiological ; drug effects ; Transcription Factors ; metabolism ; X-Box Binding Protein 1