39 research outputs found

    Resveratrol downregulates inflammatory pathway activated by lymphotoxin α (TNF-β) in articular chondrocytes: Comparison with TNF-α

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    <div><p>While Lymphotoxin α (TNF-β), a product of lymphocytes, is known to play a pivotal role in inflammatory joint environment, resveratrol has been shown to possess anti-inflammatory and chondroprotective effects <i>via</i> activation of the histondeacetylase Sirt1. Whether TNF-β induction of inflammatory pathways in primary human chondrocytes (PCH) can be modulated by resveratrol, was investigated. Monolayer and alginate cultures of PCH were treated with TNF-β, anti-TNF-β, nicotinamide (NAM), antisense oligonucleotides against Sirt1 (Sirt1-ASO) and/or resveratrol and co-cultured with T-lymphocytes. We found that resveratrol suppressed, similar to anti-TNF-β, TNF-β-induced increased adhesiveness in an inflammatory microenvironment of T-lymphocytes and PCH. In contrast, knockdown of Sirt1 by mRNA abolished the inhibitory effects of resveratrol on the TNF-β-induced adhesiveness, suggesting the essential role of this enzyme for resveratrol-mediated anti-inflammatory signaling. Similar results were obtained in PCH stimulated with TNF-α. Sirt1-ASO, NAM or TNF-β, similar to T-lymphocytes induced inflammatory microenvironment by down-regulation of cartilage-specific proteins, Sox9, Ki67 and enhanced NF-κB-regulated gene products involved in inflammatory and degradative processes in cartilage (MMP-9/-13, COX-2, caspase-3), NF-κB activation and its translocation to the nucleus. Moreover, resveratrol reversed the TNF-β-, NAM-, T-lymphocytes-induced up-regulation of various NF-κB-regulated gene products. Down-regulation of Sirt1 by mRNA interference abrogated the effect of resveratrol on TNF-β-induced effects. Ultrastructural and cell viability assay investigations revealed that resveratrol revoked TNF-β-induced dose-dependent degradative/apoptotic morphological changes, cell viability and proliferation in PCH. Taken together, suppression of TNF-β-induced inflammatory microenvironment in PCH by resveratrol/Sirt1 might be a novel therapeutic approach for targeting inflammation during rheumatoid arthritis.</p></div

    Effects of resveratrol and ASO against Sirt1 or TNF-β- or to TNF-α-mediated adhesiveness of T-lymphocytes to chondrocytes.

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    <p>Serum-starved human primary chondrocytes (PCH) were cultured to sub-confluence in monolayer culture and were either left untreated or co-cultured with T-lymphocytes and treated with TNF-β (a-g) or TNF-α (i-o) as described in Material and Methods. After being washed with phosphate-buffered saline, adhesion of T-lymphocytes (arrows) to chondrocytes was evaluated by light microscopy. Original magnification, x400; bar, 30nm. The number of adhered colonies of T-lymphocytes to chondrocytes was estimated and quantified by counting 10 microscopic fields per culture (h and p).</p

    Electron microscopic evaluation on cellular degeneration and apoptosis in chondrocytes in monolayer cultures after treatment with resveratrol and/or TNF-β.

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    <p>A-J: Serum-starved human primary chondrocytes (Ch) were cultured to sub-confluence in monolayer culture and were either left untreated or treated with different concentration of TNF-β (1, 5, 10 and 20ng/ml) and/or co-treated with resveratrol and evaluated with a transmission electron microscope. Untreated control (Co.), chondrocytes containing mitochondria, rough endoplasmic reticulum and many other cell organelles (A). In contrast, treatment of chondrocytes with TNF-β resulted in degenerative changes of the cells. Cells became rounded and the nucleus contained more condensed chromatin, multiple vacuoles, swelling of rough endoplasmic reticulum, and clustering of swollen mitochondria was visible. Higher concentration of TNF-β (10, 20ng/ml) led to the formation of apoptotic bodies and cell lysis (arrows). A-J: x5000; bar = 1μM. k: To quantify cellular degradation and apoptosis in chondrocytes monolayer cultures, 100 cells from 25 microscopic fields were counted and results presented are mean values with standard deviations from three independent experiments. Values were compared with the control and statistically significant values with p<0.05 were designated by (*) and p<0.01 were designated by (**).</p

    Effects of resveratrol on TNF-β-induced inflammation and apoptosis in chondrocytes in 3D-alginate cultures.

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    <p>Serum-starved chondrocytes in alginate beads were left untreated or treated with various concentrations of TNF-β or were co-stimulated with resveratrol and various concentrations of TNF-β for 14 days as described in Material and Methods. Whole cell lysates were fractionated and subjected to western blotting with indicated antibodies. Densitometric evaluation of protein expression as revealed by western blot analysis was performed in triplicate. <i>Bars</i> represent the mean values for collagen type II, Ki67, Sirt1, Sox9, MMP-9, MMP-13, COX-2, cleaved caspase-3 and p-NF-κB. Housekeeping protein β-actin served as a loading control in all experiments. The results are provided as mean values with standard deviations from at least three independent experiments. Values were compared to the control and statistically-significant values with p<0.05 are designated by (*) and p<0.01 designated by (**).</p

    Effects of resveratrol, TNF-β, NAM, ASO against Sirt-1 on extracellular matrix, Ki67, Sirt1, Sox9, NF-κB, NF-κB-dependent pro-inflammatory, matrix-degrading and apoptotic gene products in chondrocytes.

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    <p>Serum starved chondrocytes in monolayer culture were either left untreated or treated with resveratrol (5μM), TNF-β (10ng/ml), or with Sirt1-SO or -ASO (0.5μM) in the presence of Lipofectin for 24h, or NAM (10mM), or cells were pretreated with resveratrol (5μM) for 1h followed by co-treatment with TNF-β (10ng/ml), Sirt1-SO, or -ASO (0.5μM) in the presence of Lipofectin for 24h, NAM (10mM) as described in Material and Methods. Whole cell extracts were prepared, separated by SDS-PAGE, and subjected to western blot analysis using relevant antibodies. Densitometric evaluation of protein expression as revealed by western blot analysis was performed in triplicate. <i>Bars</i> represent the mean values for collagen type II, Ki67, Sirt1, Sox9, MMP-9, MMP-13, COX-2, cleaved caspase-3 and p-NF-κB. Housekeeping protein β-actin served as a loading control in all experiments. The results are provided as mean values with standard deviations from at least three independent experiments. Values were compared to the control and statistically-significant values with p<0.05 are designated by (*) and p<0.01 designated by (**).</p

    Effect of resveratrol on TNF-β-induced cellular degeneration and apoptosis in chondrocytes in 3-D alginate cultures.

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    <p>A-J: Serum-starved human primary chondrocytes (Ch) were cultured in alginate beads (*) and were either left untreated (Co.) or were treated with different concentrations of TNF-β (1, 5, 10 and 20ng/ml) or resveratrol (5μM) or a combination of resveratrol (5μM) and TNF-β (1, 5, 10 and 20ng/ml) for 14 days. Magnification: x5000, bar = 1μM. k: morphological features of apoptotic cell death (arrows) were quantified by counting 100 in cells from 25 different microscopic fields and results presented are mean values with standard deviations from three independent experiments. Significant values were compared with the control and statistically significant values with p<0.05 were designated by (*) and p<0.01 were designated by (**).</p

    Effects of resveratrol and/or TNF-β on the proliferation of chondrocytes in 3-D alginate culture.

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    <p>Serum-starved human articular chondrocytes were either left untreated or treated with different concentrations of TNF-β alone (0.1, 1, 5, 10, 20ng/ml) or were treated with resveratrol (5 μM) followed by treatment with different concentrations of TNF-β (0, 0.1, 1, 5, 10, 20ng/ml) for 14 days. Cell viability was examined by MTT assay. The MTT assay is a spectrophotometric measurement of the cell viability as a function of the mitochondrial activity. The results are provided as mean values with standard deviations from at least three independent experiments. Values were compared to the control and statistically-significant values with p<0.05 are designated by (*) and p<0.01 designated by (**).</p

    Schematic diagram illustrating TNF-β-induced up-regulation of pro-inflammatory, apoptotic signaling and suppression of cell viability, chondrogenic markers modulated by resveratrol-Sirt1 signaling pathways.

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    <p>Schematic diagram illustrating TNF-β-induced up-regulation of pro-inflammatory, apoptotic signaling and suppression of cell viability, chondrogenic markers modulated by resveratrol-Sirt1 signaling pathways.</p

    Resveratrol blocks TNF-β- or T-lymphocytes-induced down-regulation of extracellular matrix, Ki67, Sirt1, Sox9, expression of NF-κB and NF-κB-dependent pro-inflammatory, matrix-degrading and apoptotic gene products in chondrocytes.

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    <p>Serum starved chondrocytes were cultured in monolayer and either left untreated or treated with resveratrol, or TNF-β, or co-treated with resveratrol and TNF-β, or co-cultured with T-lymphocytes alone or additionally with TNF-β with or without resveratrol. Whole-cell extracts were prepared and then analyzed by western blotting with indicated antibodies. Densitometric evaluation of protein expression as revealed by western blot analysis was performed in triplicate. <i>Bars</i> represent the mean values for collagen type II, Ki67, Sirt1, Sox9, MMP-9, MMP-13, COX-2, cleaved caspase-3 and p-NF-κB. Housekeeping protein β-actin served as a loading control in all experiments. The results are provided as mean values with standard deviations from at least three independent experiments. Values were compared to the control and statistically-significant values with p<0.05 are designated by (*) and p<0.01 designated by (**).</p

    Ultrastructural evaluation of cytotoxicity of 5-FU, curcumin and the combinational treatment on HCT116 5-FU-chemoresistant and HCT116+ch3 5-FU-chemoresistant cell lines in high density cultures.

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    <p>A: High density cultures of HCT116R and HCT116+ch3R were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and evaluated ultrastructurally with an electron microscope. At the earliest time point when apoptosis (arrows) was first detected images are highlighted in red boxes. Micrographs shown are representative of three individual experiments. Magnification: x5000, bar = 1 µm. B: Mitochondrial changes (MC) and apoptosis were quantified by counting 100 cells with morphological features of apoptotic cell death from 25 different microscopic fields and results presented are mean values with standard deviations from three independent experiments. Significant values are marked with (*).</p
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