curcumin and prolinedithiocarbamate

Curcumin possesses strong anti-inflammatory, anti-rheumatoid and anti-oxidative activities, and has the potential to inhibit nuclear factor‑κB (NF‑κB) signaling. Cartilage damage in osteoarthritis (OA) is largely mediated by interleukin-1β (IL-1β) via activation of various transcription factors, including NF‑κB and activator protein‑1. The aim of the present study was to determine whether IL‑1β induces matrix metalloproteinase-13 (MMP-13) expression and inhibits type II collagen expression, as well as to examine whether cell proliferation may be inhibited by curcumin through the inhibition of NF‑κB signaling. The effects of curcumin were investigated in rat articular chondrocyte cell cultures treated with IL‑1β in the presence or absence of curcumin or the NF‑κB inhibitor pyrrolidine dithiocarbamate. Western blotting and reverse transcription‑quantitative polymerase chain reaction were conducted to evaluate protein and mRNA expression levels of type II collagen, MMP‑13, NF‑κB inhibitor α (IκBα), phosphorylated‑IκBα and NF‑κB subunit p65/RelA. Western blotting and immunofluorescence were performed to examine the effects of curcumin on the expression, phosphorylation and nuclear translocation of NF‑κB‑associated proteins. The effects of curcumin on cell proliferation were evaluated by Cell Counting Kit‑8 (CCK‑8). Curcumin was demonstrated to inhibit the IL‑1β‑induced activation of NF‑κB by suppressing IκBα phosphorylation and p65/RelA nuclear translocation. These events were associated with the downregulation of MMP‑13 expression and the upregulation of type II collagen expression, both of which are considered to be NF‑κB targets. CCK‑8 assays revealed that co‑treatment with curcumin resulted in increased proliferation in IL‑1β‑treated chondrocytes. These findings implicated curcumin as a naturally occurring anti‑inflammatory agent for the treatment of OA via inhibition of NF‑κB signaling.

Topics: Animals; Cell Nucleus; Cell Proliferation; Chondrocytes; Collagen Type II; Curcumin; Humans; Interleukin-1beta; Matrix Metalloproteinase 13; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Proline; Protein Transport; Rats; RNA, Messenger; Signal Transduction; Thiocarbamates; Transcription Factor RelA; Up-Regulation

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β2GPI/β2GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β2GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β2GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β2GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β2GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.

Topics: Animals; Antibodies; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Curcumin; E-Selectin; Immunoglobulin G; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Macrophages, Peritoneal; Male; Mice, Inbred BALB C; NF-kappa B; Phosphorylation; Proline; Real-Time Polymerase Chain Reaction; Thiocarbamates; Thromboplastin; Thrombosis; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

In the present study, we investigated the implication of NF-kappaB in the production of pro-inflammatory cytokine IL-18 by human keratinocytes stimulated by UVB. We demonstrated that NCTC 2544 keratinocyte cell line irradiated by UVB enhanced the IL-18 mRNA and protein secretion under its bioactive form. Overexpression of IL-18 by UVB irradiation was accompanied by NF-kappaB transcription factor activation using specific IL-18 gene sequence corresponding to NF-kappaB DNA binding site. The relationship between these transcription factors and IL-18 expression was confirmed using curcumin and PDTC, two inhibitors of NF-kappaB. Our results show that UVB and curcumin or PDTC co-treatment led to a down-regulation of IL-18 expression associated with an inhibition of NF-kappaB DNA binding. Hence, our results demonstrated that this transcription factor is implicated in biologically active IL-18 production by human keratinocytes irradiated by UVB.

Topics: Cells, Cultured; Curcumin; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-18; Keratinocytes; Leukocytes; NF-kappa B; Proline; Promoter Regions, Genetic; RNA, Messenger; Thiocarbamates; Ultraviolet Rays

There has been increasing evidence that tumor necrosis factor alpha (TNF-alpha) is synthesized by cardiomyoctes and contributes to their impaired function and to cardiac failure. Because the Na(+)-K(+) ATPase is a key player in the contraction of cardiomyocytes, this work was undertaken to study the effect of TNF-alpha on the Na(+)-K(+) ATPase in rat heart. Sprague Dawley rats (Rattus norvegicus) were injected with TNF-alpha (270 ng/100 g body weight) and 4 h later the ventricles were isolated, homogenized and assayed for their Na(+)-K(+) ATPase activity. The effect of TNF-alpha on the pump was studied also in isolated myocytes treated in suspension. The involvement of PGE2 was investigated by pre-treating animals or cells with indomethacin, an inhibitor of COX enzymes. The involvement of NF-kappaB and AP-1 was studied using their respective inhibitors PDTC and curcumin. A time response study showed an increase in the activity of the Na(+)-K(+) ATPase in the left and right ventricles of animals treated with the cytokine, with no change in its protein expression. This effect disappeared in the presence of indomethacin suggesting an involvement of PGE(2) in the action of TNF-alpha. Rats and cells treated directly with PGE(2) showed a dose-dependent response. A decrease in the activity of the Na(+)-K(+) ATPase was observed at a low dose and an increase at a high dose in both ventricles. Since PGE(2) is suspected to be the active mediator in TNF-alpha signaling, inhibiting its synthesis by inhibiting some suspected transcription factors was attempted. PDTC abrogated fully, and curcumin partially the effect of the cytokine. It was concluded that TNF-alpha activates NF-kappaB and AP-1 and induces PGE(2) release which alters dose-dependently the activity of the pump by activating different EP receptors with different affinities for PGE(2).

Topics: Animals; Curcumin; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Indomethacin; Male; Myocytes, Cardiac; NF-kappa B; Proline; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Thiocarbamates; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

Chronic inflammation and fibrosis following quartz inhalation has been associated with persistent up-regulation of several "pro-inflammatory" genes, which are commonly regulated by nuclear factor kappa-B (NF-kappaB). Transcription of the NF-kappaB-inhibitor IkappaBalpha is also under NF-kappaB control, and its de novo synthesis is considered to comprise a negative feedback loop in transient inflammation. To investigate this mechanism in particle inflammation, we have studied IkappaBalpha degradation in A549 cells exposed to DQ12-quartz or TiO(2), in relation to the expression of IL-8. Although both quartz and TiO(2) were found to cause IkappaBalpha degradation, only quartz elicited a mild IkappaBalpha depletion, first appearing at 4 h. TiO(2) was found to cause a higher short-term increase in IkappaBalpha mRNA-expression compared to quartz, whereas the early enhancement of IL-8 expression and release was similar for both particles. Up-regulation of IL-8 expression was found to persist with quartz only. Cotreatment with PDTC and curcumin reduced particle-elicited IL-8 response, whereas cycloheximide caused enhancement of IL-8 mRNA expression in both the quartz- and TiO(2)-treated cells. Our results demonstrate that mineral dusts cause IkappaBalpha degradation, a transient increase in de novo synthesis of IkappaBalpha, and enhanced IL-8 expression in human pulmonary epithelial cells. While IkappaBalpha degradation and early IL-8 expression seem to be general particle phenomena, particle-specific characteristics impact on activation of IkappaBalpha gene transcription, apparently accounting for the different proinflammatory IL-8 responses seen with quartz and TiO(2) in the longer term. These observations may provide an explanation for the transient versus the persistent pulmonary inflammatory status and subsequent differences in pathogenic potency of TiO(2) and quartz.

Topics: Blotting, Western; Curcumin; Cycloheximide; DNA-Binding Proteins; Epithelial Cells; Humans; I-kappa B Proteins; Interleukin-8; Lung; NF-kappa B; NF-KappaB Inhibitor alpha; Proline; Quartz; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiocarbamates; Titanium; Tumor Cells, Cultured; Up-Regulation

Proinflammatory cytokines, a combination of IL-1beta, TNF-alpha, and IFN-gamma, caused mRNA expression of GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, and of inducible nitric oxide synthase (iNOS) in a well-characterized osteoblastic clone MC3T3-E1 cell line. We found the expression of the GTP-CH gene in osteoblasts for the first time. The expression of GTP-CH and iNOS mRNAs was found to be maximal at 3 and 9 h, respectively. The expression of both genes elicited increases in BH4 and NO levels. Pharmacological studies using 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP-CH activity, showed that BH4 is involved in the activity of iNOS, but not in the induction of iNOS mRNA. The results using an inhibitor of nuclear factor (NF)-kappaB and activating protein-1 (AP-1) activation suggested that coinduction of the two genes in response to cytokines occurred via activation of NF-kappaB and AP-1. In MC3T3-E1 cells BH4 and sepiapterin, producing BH4, could protect against apoptosis, i.e. the degradation of nuclear DNA in the cells, induced by NO derived from S-nitroso-N-acetyl-D-L-penicillamine. These results suggest that the induction of BH4 together with NO by proinflammatory cytokines could protect against NO-induced apoptosis in MC3T3-E1 cells.

Topics: 3T3 Cells; Animals; Apoptosis; Biopterins; Cell Nucleus; Cell Survival; Curcumin; Cytokines; DNA Fragmentation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; GTP Cyclohydrolase; Hypoxanthines; Interferon-gamma; Interleukin-1; Kinetics; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Osteoblasts; Penicillamine; Proline; Pteridines; Pterins; Recombinant Proteins; RNA, Messenger; S-Nitroso-N-Acetylpenicillamine; Thiocarbamates; Transcription Factor AP-1; Transcription, Genetic; Transfection; Tumor Necrosis Factor-alpha

Lipopolysaccharide (LPS) induces expression of inflammatory cytokines in monocytes/macrophages via CD14, one of the LPS receptors, which is expressed predominantly in these cells. It has been demonstrated that Porphyromonas gingivalis LPS (P-LPS) also is able to induce inflammatory cytokines in human gingival fibroblasts. Therefore, it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells. In this study, we observed unexpectedly by immunohistochemical, Western blotting (immunoblotting), and Northern (RNA) blotting assays that CD14 is expressed at high density in human gingival fibroblasts. P-LPS-induced expression of the monocyte chemoattractant protein 1 (MCP-1) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A, a potent inhibitor of tyrosine kinase. The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells. Furthermore, P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors, i.e., curcumin, an inhibitor of AP-1, and pyrolidine dithiocarbamate, an inhibitor of NF-kappaB. Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells. Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells. This study is the first to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Benzoquinones; Cells, Cultured; Chemokine CCL2; Chemotaxis, Leukocyte; Curcumin; Enzyme Inhibitors; Fibroblasts; Gene Expression; Gingiva; Humans; Lactams, Macrocyclic; Lipopolysaccharide Receptors; Lipopolysaccharides; Monocytes; NF-kappa B; Porphyromonas gingivalis; Proline; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Staurosporine; Thiocarbamates; Transcription Factor AP-1