u-0126 and parthenolide

Neuropathic pain is clinically challenging because it is resistant to alleviation by morphine. The nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways may be involved in the development of neuropathic pain. The aim of our study was to examine the influence of a chronic, intrathecal administration of parthenolide (PTL, inhibitor of NF-κB) and U0126 (inhibitor of MEK1/2) on nociception and morphine effectiveness in a rat model of neuropathy.. The chronic constriction injury of the sciatic nerve in Wistar rats was performed. PTL and U0126 were injected chronic intrathecally and morphine was injected once at day 7. To evaluate allodynia and hyperalgesia, the von Frey and cold plate tests were used, respectively. The experiments were carried out according to IASP rules. Using qRT-PCR we analyzed mRNAs of μ-(mor), δ-(dor) and κ-(kor)-opioid receptors in the lumbar spinal cord after drugs administration.. The administration of PTL and U0126 decreased allodynia and hyperalgesia and significantly potentiated morphine effect. The mor, dor and kor mRNAs were down-regulated 7 days after injury in the ipsilateral spinal cord. The PTL and U0126 significantly up-regulated the mRNA levels of all opioid receptors. The levels of mor and dor mRNAs were much higher compared to those in naïve, but only the kor levels returned to control values.. These results indicate that the inhibition of the NF-κB pathway has better analgesic effects. Both inhibitors similarly potentiate morphine analgesia, which parallels the up-regulation of both mor and dor mRNAs expression spinal levels of the model of neuropathy.

Topics: Analgesics; Analgesics, Opioid; Animals; Butadienes; Disease Models, Animal; Drug Synergism; Hyperalgesia; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morphine; Neuralgia; NF-kappa B; Nitriles; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Sciatic Nerve; Sesquiterpenes; Signal Transduction

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p<0.001) and human monocyte-derived macrophages (2.3-fold increase, p<0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.

Topics: Amino Acid Chloromethyl Ketones; Anthracenes; Antithrombins; Blotting, Western; Butadienes; Cell Line; Cells, Cultured; Cyclooxygenase 2 Inhibitors; Enzyme Inhibitors; Flavonoids; Gene Expression; Glycoproteins; Hirudins; Humans; JNK Mitogen-Activated Protein Kinases; Lipoproteins, VLDL; Macrophages; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Nitrobenzenes; Reverse Transcriptase Polymerase Chain Reaction; Sesquiterpenes; Signal Transduction; Sulfonamides; Thrombin; Time Factors

In addition to elevated concentrations of cytokines, patients with congestive heart failure (CHF) show endothelial dysfunction and increased plasma concentrations of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1). Furthermore, the concentration of cardiotrophin-1 (CT-1)--a cytokine of the interleukin-6 superfamily--is increased in CHF. We tested the hypothesis whether CT-1 is able to induce ICAM-1 in human umbilical vein endothelial cells (HUVEC). Furthermore we examined the signalling mechanisms of CT-1 mediated ICAM-1 expression.. Confluent layers of HUVEC were incubated with increasing concentrations of CT-1 (5 to 100 ng/ml) for different periods. ICAM-1 mRNA was determined by real-time polymerase chain reaction (PCR) and ICAM-1 surface expression by fluorescence-activated cell sorter (FACS) analysis and soluble ICAM-1 (sICAM-1) in the culture supernatant by enzyme linked immunosorbent assay (ELISA). To clarify the signalling pathway of CT-1 induced ICAM-1 expression we used various inhibitors of possible signal transducing molecules, electromobility shift assay (EMSA) and Western blot analysis.. CT-1 induced ICAM-1 mRNA (1.8 +/- 0.8 fold increase compared to unstimulated cells after 6 hours) and protein (1.4 +/- 0.2 fold increase compared to unstimulated cells after 48 hours) in HUVEC in a time- and concentration-dependent manner. EMSA experiments show that CT-1 causes nuclear factor (NF) kappaB activation. Because parthenolide could inhibit CT-1 induced ICAM-1 expression NFkappaB activation is required in this pathway. CT-1 did not activate extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK) and p38.. CT-1 is able to induce ICAM-1 in endothelial cells by NFkappaB activation. These results may explain in part elevated ICAM-1 concentrations in patients with CHF and endothelial dysfunction.

Topics: Anthracenes; Butadienes; Cells, Cultured; Cytokines; Electrophoretic Mobility Shift Assay; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Gene Expression; Humans; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Polymerase Chain Reaction; Sesquiterpenes; Umbilical Veins

To assess the contribution of signal transducer and activator of transcription 3 (JAK-STAT3) pathway, extracellular signal-regulated kinases1/2 (ERK1/2) pathway, and phosphatidylinositol 3-kinase (PI3-K) pathway to cardiomyocytes hypertrophy induced by cardiotrophin-1 (CT-1), a new member of interleukin-6 (IL-6) family of cytokines.. STAT3, ERK1/2, and PI3-K were assessed by Western blot analysis. Activity of ERK1/2 was also confirmed by in-gel kinase assay. Hypertrophy of cardiomyocyte was evaluated by [3H]leucine incorporation and cellular protein-to-DNA ratio.. CT-1 simultaneously activated phosphorylation of STAT3, ERK1/2, and PI3-K in rat cardiomyocytes. Parthenolide, an inhibitor of STAT, suppressed CT-1-induced [3H]leucine incorporation by 88.3 % and protein-to-DNA ratio by 75.0 %. U0126, an MEK1/2 inhibitor, increased CT-1-induced the phosphorylation of STAT3 in a dose-dependent manner and, consistently, augmented CT-1-induced increase in [3H]leucine incorporation and cellular protein-to-DNA ratio by 17.6 % and 16.3 %, respectively. Wortmannin, a PI3-K inhibitor, did not influence CT-1-induced [3H]leucine incorporation and cellular protein-to-DNA ratio.. The hypertrophic effect of CT-1 was essentially mediated by STAT3, independent of PI3-K, and negatively regulated by ERK1/2 via inhibiting the phosphorylation of STAT3. The interaction between STAT3 and ERK1/2 in CT-1-induced signaling contributes to development of cardiac hypertrophy.

Topics: Androstadienes; Animals; Animals, Newborn; Butadienes; Cardiomegaly; Cell Size; Cells, Cultured; Cytokines; DNA-Binding Proteins; Heart Ventricles; Leucine; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Wortmannin