Inhibition Of TNF-alpha Production Research Articles

Anti-inflammatory effect of the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden, the Tiger Milk mushroom.

BackgroundThe sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) is used as a traditional medicine to relieve cough, asthma and chronic hepatitis. The traditional uses of the sclerotium are presumably related to its anti-inflammatory effect. The present study was carried out to evaluate the anti-inflammatory activity of the sclerotial powder of L. rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) cultivar TM02.MethodsThe anti-acute inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 was investigated using carrageenan-induced paw edema test while the inhibition of transudative and proliferative phases of chronic inflammation were studied by cotton pellet induced granuloma model. Sprague Dawley rats were used in both studies. The anti-inflammatory activity was also measured by inhibition of lipopolysaccharide induced TNF-alpha production in RAW 264.7 macrophage cells.ResultsCold water extract (CWE), hot water extract (HWE) and methanol extract (ME) of the sclerotial powder of L. rhinocerotis cultivar TM02 possessed anti-acute inflammatory activity as was measured by carrageenan-induced paw edema test, with CWE being the most potent. The acute anti-inflammatory activity of the cold water extract (CWE) was mainly contributed by its high molecular weight (HMW) fraction isolated by Sephadex G50 gel filtration chromatography. Its protein component was very potent in the inhibition of TNF-alpha production with an IC50 of 0.76 μg/ml. CWE at 200 mg/kg did not inhibit transudative and proliferative phase of chronic inflammation as shown by using the cotton pellet induced granuloma model.ConclusionsOur results suggested that most of the bioactive substance(s) contributed to the acute-inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 appear to be in the CWE, particularly its HMW fraction. The anti-inflammatory activity of CWE was mainly contributed by the protein component of the high molecular weight (HMW) fraction and it exhibited strong inhibitory effect on TNF-alpha production but the possibility of synergistic effect between HMW, MMW and LMW fractions cannot be excluded. Future studies will provide new insights into the anti-inflammatory activity of the sclerotial powder.

Bupropion Reduces the Inflammatory Response and Intestinal Injury Due to Ischemia-Reperfusion

Intestinal ischemia-reperfusion (I/R) causes severe organ failure and intense inflammatory responses, which are mediated in part by the cytokine tumor necrosis factor-alpha (TNF-alpha). Bupropion is an antidepressant known to inhibit TNF-alpha production. We sought to examine the protective effects of bupropion on intestinal I/R injury in 15 male Sprague-Dawley rats that were randomized to sham surgery, 45 minutes of intestinal ischemia followed by 180 minutes reperfusion, or bupropion (100 mg/kg) before the intestinal I/R injury. To evaluate the systemic inflammatory response induced by intestinal I/R, we measured serum levels of TNF-alpha, interleukins-1 and -6, lipid peroxidation, and transaminases. Histologic analysis evaluated intestinal injury using the Chiu muscosal injury score. After I/R, Chiu score in control animals was 3.6 ± 1.2 vs 2.6 ± 0.53 in animals that received bupropion (P < .05). Bupropion pretreatment reduced intestinal. I/R injury and blunted serum elevations of TNF-alpha (0.96 ± 1.1 ng/mL vs 0.09 ± 0.06 ng/mL, P < .05) and interleukin-1 (0.53 ± 0.24 ng/mL vs 0.2 ± 0.11 ng/mL, P < .05). Bupropion in reduced intestinal I/R injury through immunomodulatory machanisms that involve inflammatory cytokines such as TNF-alpha.

Kinase Inhibitors Reduce TNF-Alpha Over-Production in Monocytes From Fanconi Anemia Group A Patients

Abstract 2409Fanconi Anemia (FA) is a chromosomal instability syndrome with hypersensitivity to alkylating agents as principal diagnostic feature. Many laboratories have demonstrated the involvement of FA proteins in DNA repair mechanisms. Recent work has also demonstrated other functions of some FA proteins suggesting that they play alternative roles in other regulatory pathways, particularly those that influence hematopoiesis.Eighty percent of FA patients develop bone marrow failure with a high incidence of evolution in myelodysplasia and/or acute leukemia. Each of these abnormalities has been related to TNF-alpha hypersensitivity in the stem and progenitor cell pools and the toll-like receptor dependent overproduction of TNF-alpha by FA macrophages. The TNF-hypersensitive phenotype involves at least two kinases, PKR and ASK1, each of which is hyperactivated in FA cells and induce apoptotic responses both in ground state and after TNF-alpha and IFN-gamma stimulation. Recent evidence showed that R848 (a TLR8 ligand) and endotoxin (LPS, a TLR4 ligand)-induced TNF-alpha gene expression in Fancc-deficient mononuclear phagocytes cells is inhibited by kinase inhibitors dasatinib and BIRB796 We sought to evaluate the activity of these agents in primary mononuclear phagocytes obtained from children with FA-A. Objectives:To determine whether primary monocytes from the peripheral blood of FANCA-deficient patients: (a) exhibit the TNF-overproduction phenotype in response to LPS and the TLR8 ligand R848, and (b) respond to dasatinib and BIRB796 by suppressing TNF-production. Methods:Six FA patients with mild to severe marrow failure on no treatment were included in this study. Healthy subjects were recruited as normal controls that were run in parallel in each case. CD14+ monocytes freshly isolated from peripheral blood were cultured for 24 hours with LPS and R848 with or without dasatinib or BIRB796. Supernatant media were collected and frozen at −80 degrees. After thawing the samples, TNF-alpha content was quantified by ELISA. Results:Baseline TNF-alpha concentration (without any TLR stimulation) was higher in FA patients than control. After LPS or R848 stimulation FA-A monocytes produced substantially more TNF-alpha than did the control samples. Both dasatinib and BIRB796 suppressed TLR-induced (both LPS and R848) TNF-alpha production. Specifically, with R848 as the agonist, BIRB 769 suppressed TNF-alpha production by 60% and dasatinib by 42%. Both inhibitors were even more potent in suppressing LPS induced TNF-alpha expression as both reduced TNF-alpha by 75%. In the absence of TLR stimulation, the presence of BIRB or dasatinib in culture reduced TNF-alpha by >50% compared to baseline in patient samples. The inhibitory effect of kinase inhibitors was observed also in the normal control. Conclusions:These findings: (a) demonstrate for the first time that TLR-induced TNF-alpha gene expression in primary FANCA deficient mononuclear phagocytes is aberrantly regulated and (b) that in FANCA-deficient macrophages the TNF-alpha overproduction phenotype can be controlled by therapeutically achievable doses of BIRB796 and dasatinib. In addition (c), since both agents function in large part to suppress p38 MAPK activation future, our data point to the biochemical roles played by FANCA in modulating upstream pathways that govern p38 activation. Moreover (d), given that in FA patients, TNF hypersensitive stem cells are over-exposed to TNF-alpha, particularly during inflammatory events and that exposure to TNF was shown not only to suppress hematopoiesis in FA but also to favor the emergence of neoplastic clones, these results point to these two agents as potential candidates for preclinical trials seeking to enhance hematopoiesis and suppress clonal evolution. Disclosures:No relevant conflicts of interest to declare.

Anti-inflammatory compounds from Monascus pilosus-fermented rice

Investigation of the 95% ethanolic extract of red yeast rice fermented with the pink mutant of the fungus Monascus pilosus BCRC PK-1 led to the isolation of one new pyridine alkaloid, namely (E)-methyl 4-(2-acetyl-4-oxotridec-1-enyl)-6-propylnicotinate (1), together with 9 known compounds (2–10). Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. We also evaluated the inhibitory effects of some isolates on the production of tumor necrosis factor (TNF-α) induced by lipopolysaccharide (LPS). Among the isolates, compounds 1 and 2 inhibited TNF-alpha production in U937 cells in vitro, and the IC50 values are 1.52 and 2.56μg/ml, respectively.

Rosiglitazone decreases peroxisome proliferator receptor-gamma levels in microglia and inhibits TNF-alpha production: new evidences on neuroprotection in a progressive Parkinson's disease model

Thiazolidinedione (TZD) class of peroxisome proliferator receptor gamma (PPAR-γ) agonists display neuroprotective effects in experimental Parkinson's disease (PD) models. Neurons and microglia express PPAR-γ, therefore both of them are potential targets for neuroprotection, although the role of each cell type is not clear. Moreover, receptor-dependent as well as receptor-independent mechanisms have been involved. This study further investigated mechanisms of TZD-mediated neuroprotection in PD. We investigated the rosiglitazone effect in the progressive MPTP/probenecid (MPTPp) model of PD. C57BL/6J mice received MPTP (25 mg/kg) plus probenecid (100 mg/kg) twice per week for 5 weeks. Rosiglitazone (10 mg/kg) was given daily until sacrifice, starting on the fourth week of MPTPp treatment, in presence of an ongoing neurodegeneration with microgliosis. Changes in PPAR-γ levels were measured by immunofluorescence and confocal microscopy in tyrosine hydroxylase (TH)-positive neurons and CD11b-positive microglia of the substantia nigra pars compacta (SNc). Chronic MPTPp treatment induced a PPAR-γ overexpression in both TH-positive neurons and microglia (139.9% and 121.7% over vehicle, respectively). Rosiglitazone administration to MPTPp-treated mice, reverted PPAR-γ overexpression in microglia without affecting TH-positive neurons. Thereafter, changes in CD11b and tumor necrosis factor α (TNF-α) immunoreactivity in microglia were evaluated in the SNc. MPTPp progressively increased CD11b immunoreactivity, conferring to microglia a highly activated morphology. Moreover, TNF-α levels were increased (457.38% over vehicle) after MPTPp. Rosiglitazone administration counteracted the increase in CD11b immunoreactivity caused by MPTPp. Moreover, rosiglitazone reverted TNF-α expression to control levels. Nigrostriatal degeneration was assessed by high pressure liquid chromatography (HPLC) measurement of striatal dopamine, and counting of TH-positive neurons in the SNc. MPTPp treatment caused a severe decline of striatal dopamine and a partial degeneration of the SNc. Rosiglitazone arrested the degenerative process in both areas. Results suggest that PPAR-γ expression in microglia and TNF-α production by these cells are crucial changes by which rosiglitazone exerts neuroprotection in PD.

Recent Advances in the Chemistry of Phthalimide Analogues and their Therapeutic Potential

Phthalimide analogues have been extensively used in medicinal chemistry owing to their wide range of applications as anti-convulsant, anti-inflammatory, analgesic, hypolipidimic and immunomodulatory activities. Number of anti-inflammatory phthalimide analogues have been synthesized as tumor necrosis factor-alpha (TNF-alpha) inhibitors. TNF-alpha plays a critical role in certain physiological immune systems and its over-production causes severe damage to the host. It promotes the inflammatory response leading to many of the clinical problems associated with auto-immune disorders like rheumatoid arthritis, Crohn's disease, ankylosing spondylitis, psoriasis and refractory asthma. One of the phthalimide derivatives, LASSBio-468, was recently demonstrated to inhibit TNF-alpha production induced by lipopolysaccharide (LPS), in vivo. Its potential against chronic inflammatory diseases was also witnessed. Another derivative, DIMP, showed good anti-androgenic activity. These analogues have also been employed for the synthesis of several kinds of important therapeutic synthones. However extensive research on the chemistry and biological activities of phthalimide analogues have been carried out and number of reports appeared, a compilation focusing on chemistry and biological activity is still needed. This review, concisely describes the chemical and therapeutic aspects of phthalimide derivatives.

DBM1285 Suppresses Tumor Necrosis Factor α Production by Blocking p38 Mitogen-Activated Protein Kinase/Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Signaling Pathway

Tumor necrosis factor alpha (TNF-alpha) is a major inflammatory cytokine that plays an important role in the development of various inflammatory diseases. TNF-alpha has been considered as a potential therapeutic target for the treatment of chronic inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. In this study, we report that cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine (DBM1285) is a novel inhibitor of TNF-alpha production. DBM1285 concentration-dependently inhibited lipopolysaccharide (LPS)-induced TNF-alpha secretion in various cells of macrophage/monocyte lineage, including mouse bone marrow macrophages, THP-1 cells, and RAW 264.7 cells. However, LPS-induced mRNA expression of TNF-alpha was not affected by DBM1285 in these cells. Further studies demonstrated that the inhibitory effect of DBM1285 on TNF-alpha production might be mediated by post-transcriptional regulation through the modulation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) signaling pathway. We also confirmed that DBM1285 directly inhibits p38 MAPK enzymatic activity. In vivo administration of DBM1285 inhibited LPS-induced increase in the plasma level of TNF-alpha in mice. Whole-blood in vivo target inhibition assay also revealed that DBM1285 attenuates p38 MAPK activity after oral administration in mice. Moreover, DBM1285 suppressed zymosan-induced inflammation and adjuvant-induced arthritis in murine models. Collectively, these results suggest that DBM1285 inhibits TNF-alpha production, at least in part, by blocking the p38 MAPK/MK2 pathway. Furthermore, in vivo results suggest that DBM1285 might be a possible therapeutic candidate for the treatment of TNF-alpha-related chronic inflammatory diseases.

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 2

We previously reported a novel pyrrole derivative 1 which possesses a tetrahydropyridine group at the beta-position with a proinflammatory cytokine TNFalpha production inhibitor. Herein, we report the synthesis and biological activity of N- and alpha-position substituted tetrahydropyridine derivatives. In this series, we found that compound 3o showed good inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS)-induced TNFalpha production in human whole blood, IC(50)=0.44 microM) and compound 3i demonstrated potent inhibitory activity in vivo (inhibition of LPS-induced TNFalpha production in mice, ID(50)=1.42 mg/kg).

Treponema denticola suppresses expression of human beta-defensin-2 in gingival epithelial cells through inhibition of TNFalpha production and TLR2 activation.

We previously reported that Treponema denticola, a periodontal pathogen, suppressed the expression of human beta-defensins (HBDs) and IL-8 in human gingival epithelial cells. To clarify the receptor(s) involved in the suppression of HBD-2, immortalized gingival epithelial (HOK-16B) cells were infected with live or heat-killed T. denticola for 24 h, and the expression of HBD-2 was examined by real-time RT-PCR. Live T. denticola, but not heat-killed bacteria, suppressed the expression of HBD-2 about 40%. Time courses of suppression revealed that T. denticola suppressed HBD-2 expression only at late time points, which was accompanied with the suppression of TNFalpha production. Neutralization of TNFalpha with an antibody abrogated the suppressive effect of T. denticola on HBD-2. Accordingly, heat-killed T. denticola did not suppress TNFalpha production. Knock-down of toll-like receptor (TLR) 2 via RNA interference reversed the suppressive effect of T. denticola on the expression of HBD-3, but not on the production of TNFalpha. Collectively, T. denticola suppresses the expression of HBD-2 in gingival epithelial cells by inhibiting the TLR2 axis and TNFalpha production, which may contribute to the pathogenesis of periodontitis by T. denticola.

The role of adenosine receptors in regulating production of tumour necrosis factor‐α and chemokines by human lung macrophages

Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A(1), A(2A), A(2B) and A(3)). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-alpha and chemokines. Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. LPS increased (about 400-fold) mRNA for A(2A) adenosine receptors, decreased mRNA for A(1) and A(2B), but had no effect on A(3) adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-alpha production concentration dependently, whereas the A(1) receptor agonist, CCPA, and the A(3) receptor agonist, AB-MECA, inhibited TNF-alpha production only at concentrations affecting A(2A) receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-alpha and chemokine production by the selective A(2A) adenosine receptor antagonist ZM 241385, but not the A(2B) receptor antagonist, MRS 1754, or the A(3) receptor antagonist, MRS 1220, indicated involvement of A(2A) receptors. LPS up-regulated A(2A) adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-alpha and of a subset of chemokines in human lung macrophages.

Novel synthetic gluco-disaccharide RSCL-0409 - a lipopolysaccharide-induced Toll-like receptor-mediated signalling antagonist

The regulation of cytokines and pro-inflammatory genes is an absolute essentiality to combat inflammatory diseases. The present study investigated the effects of 4-O-chloroacetyl-2,3-di-O-acetyl-6-O-levulinoyl-beta-d-glucopyranosyl]-(1-3)-1-O-(p-methoxyphenyl)-2-deoxy-2-N-trichloroacetyl-4,6-O-benzylidene-alpha-d-glucopyranoside (RSCL-0409), a novel small molecule Toll-like receptor (TLR) signalling antagonist, and its mechanism of action in human monocytic (THP-1) cells stimulated with lipopolysaccharide (LPS). In THP-1 and RAW264.7 cells, RSCL-0409 suppressed LPS-induced production of tumour necrosis factor-alpha (TNF-alpha) with a 50% inhibitory concentration of 10.6 mum and mRNA expression of ICAM-1, Cox-2 and interleukin-8 with no evidence of cytotoxicity. RSCL-0409 also suppressed TNF-alpha production from LPS-stimulated human peripheral blood mononuclear cells. Similar results were obtained in vivo in a murine model of LPS-induced inflammation, where pretreatment with RSCL-0409 resulted in significant inhibition of TNF-alpha. It is also noteworthy that RSCL-0409 suppressed the cytokine production induced by TLR2 and -4 ligands and not for any other TLR ligands. RSCL-0409 significantly inhibited p65 nuclear translocation induced by LPS. In conclusion, RSCL-0409, a novel small molecule, is the first of its kind in the category of carbohydrate-derived TLR signalling antagonists and could definitely be a promising therapeutic agent for inflammatory diseases whose pathogenesis involves TLR2- or TLR4-mediated signalling processes.

SSeCKS promotes tumor necrosis factor-alpha autocrine via activating p38 and JNK pathways in Schwann cells.

Tumor necrosis factor-alpha (TNF-alpha) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-alpha could act as an autocrine mediator in SC activation. In this study, we found knockdown Src-suppressed protein kinase C substrate (SSeCKS) expression suppressed TNF-alpha production induced by TNF-alpha, overexpression of SSeCKS could promoted TNF-alpha autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-alpha. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.

Relationship between phosphodiesterase type 4 inhibition and anti‐inflammatory activity of CI‐1044 in rat airways

The anti-inflammatory effects of CI-1044 and of the other selective PDE4 inhibitors rolipram and cilomilast were investigated in Brown-Norway (BN) rats, against lipopolysaccharide-induced tumor necrosis factor alpha (TNFalpha) production in whole blood and antigen-induced lung eosinophilia. In vitro, CI-1044 inhibited TNFalpha production with an IC(50) of 0.31 microm being equipotent to Cilomilast (IC(50) = 0.26 microm) and rolipram (IC(50) = 0.11 microm). Given orally, CI-1044 inhibited ex vivo TNFalpha production with an ED(50) value of 0.4 mg/kg after single administration, whereas rolipram (ED(50) = 1.4 mg/kg) and cilomilast (ED(50) = 1.6 mg/kg) were less potent. In the same ex vivo setting, but given repeatedly, CI-1044 led to an ED(50) of 0.5 mg/kg corresponding to a plasma concentration of 82.6 ng/mL (0.22 microm). In vivo, CI-1044 prevented TNFalpha release with an ED(50) of 1 mg/kg p.o. and inhibited ovalbumin-induced lung eosinophilia following single or repeated oral administration with an ED(50) of 3.25 and 4.8 mg/kg p.o., respectively, suggesting the absence of pharmacological tolerance. CI-1044 in this model was equipotent to rolipram (81% inhibition at 10 mg/kg) but better than cilomilast (25% inhibition at 10 mg/kg). Finally, CI-1044 (10 mg/kg) inhibited inflammatory cell recruitment with a long duration of action (up to 8 h) and was still active when given post-challenge. Our data show that CI-1044 is an orally active PDE4 inhibitor that may be used as an anti-inflammatory therapy in lung inflammatory diseases.

A Randomized, Placebo‐Controlled Study of the Effects of the p38 MAPK Inhibitor SB‐681323 on Blood Biomarkers of Inflammation in COPD Patients

The p38 mitogen-activated protein kinase (MAPK) signaling upregulates inflammation and is known to be increased in chronic obstructive pulmonary disease (COPD). The authors assessed the pharmacology of the novel p38 MAPK inhibitor SB-681323 using blood biomarkers in COPD. Seventeen COPD patients (forced expiratory volume in 1 second 50%-80% predicted) using short-acting bronchodilators participated in a double-blind, double-dummy, randomized, crossover study. Patients received single oral doses of SB-681323 7.5 mg and 25 mg, prednisolone 10 mg and 30 mg, and placebo. Blood was obtained predose and at 1, 2, 6, and 24 hours postdose. Whole-blood sorbitol-induced phosphorylated (p) heat shock protein (HSP) 27 levels as a marker of p38 pathway activation and lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha production were assessed. Both doses of SB-681323, but not prednisolone, significantly (P < .0001) reduced weighted mean (WM) pHSP27 (0-6 hours) by 58% compared with placebo. WM TNF-alpha production (0-24 hours) was significantly reduced compared with placebo by SB-681323 25 mg (40%, P = .005) and 7.5 mg (33.4%, P = .02), while prednisolone 30 mg and 10 mg caused 81.5% and 58.2% suppression, respectively (both P < .0001). SB-681323 inhibited the p38 MAPK pathway to a greater degree than prednisolone did. SB-681323 inhibited TNF-alpha production. SB-681323 is a potent p38 MAPK inhibitor that potentially suppresses inflammation in COPD.

Adenosine Suppresses Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production by Murine Macrophages through a Protein Kinase A- and Exchange Protein Activated by cAMP-Independent Signaling Pathway

Adenosine is generated during tissue hypoxia and stress, which reduces inflammation by suppressing the activity of most immune cells. Among its various actions, adenosine suppresses the production of proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, through the cAMP-elevating A(2A) adenosine receptor (AR) subtype. In this study, we examined the signaling mechanisms by which A(2A)AR activation inhibits TNF-alpha production in thioglycollate-elicited mouse peritoneal macrophages. Pretreating murine macrophages with the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA), the A(2A)AR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), or the cAMP-elevating agent forskolin reduced TNF-alpha production in response to lipopolysaccharide (LPS) by greater than 60%. All of these agents increased cAMP production in macrophages and activated protein kinase A (PKA). However, we were surprised to find that treating macrophages with three different PKA inhibitors or small interfering RNA-mediated knockdown of the exchange protein activated by cAMP (Epac-1) failed to block the suppressive actions of NECA or forskolin on LPS-induced TNF-alpha release. Instead, okadaic acid was effective at low concentrations that selectively inhibit protein serine/threonine phosphatases. Subsequent studies showed that NECA and forskolin decreased LPS-induced steady-state TNF-alpha mRNA levels; this effect was due to a decreased rate of transcription based on assays examining the rate of generation of primary TNF-alpha transcripts. Treatment with NECA or forskolin did not interfere with LPS-induced translocation or DNA binding of the RelA/p65 subunit of nuclear factor-kappaB or phosphorylation of inhibitor of nuclear factor-kappaB-alpha, extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, or p38 kinase. Our results suggest that AR activation inhibits LPS-induced TNF-alpha production by murine macrophages at the level of gene transcription through a unique cAMP-dependent, but PKA- and Epac-independent, signaling pathway involving protein phosphatase activity.

Intravenous immunoglobulin and salicylate differentially modulate pathogenic processes leading to vascular damage in a model of Kawasaki disease

Kawasaki disease (KD) is a multisystem vasculitis affecting children and is characterized by immune activation in the acute stage of disease. Systemic inflammation eventually subsides, although coronary arteritis persists, resulting in aneurysm formation. KD is the leading cause of acquired heart disease among children in North America. Accepted treatment guidelines include high-dose intravenous immunoglobulin (IVIG) and aspirin in the acute phase. Although this therapy is effective, the cellular and molecular mechanisms involved are not clear. The aim of this study was to examine the effect of IVIG and salicylate at each stage of disease development. Using a murine model of KD, we established and validated several in vitro techniques to reflect 3 key steps involved in disease pathogenesis, as follows: thymidine incorporation to evaluate T cell activation, enzyme-linked immunosorbent assay to measure tumor necrosis factor alpha (TNFalpha) production, and real-time polymerase chain reaction to examine TNFalpha-mediated expression of matrix metalloproteinase 9 (MMP-9). At therapeutic concentrations, IVIG, but not salicylate, effectively reduced the immune response leading to TNFalpha expression. Unexpectedly, pharmacologic doses of salicylate were not able to inhibit TNFalpha production and in fact enhanced its production. Neither drug directly regulated MMP-9 expression but did so only indirectly via modulating TNFalpha. TNFalpha activity was a prerequisite for local expression of MMP-9 at the coronary artery. Therapeutic concentrations of IVIG and salicylate differentially modulate the expression of TNFalpha and its downstream effects. Further dissection of the biologic effects of aspirin in acute KD is necessary for the rational design of therapy.

HMG-CoA Reductase Inhibitors (Statins) Activate Expression of PPARα/PPARγ and ABCA1 in Cultured Gallbladder Epithelial Cells

In gallbladder epithelial cells (GBEC), PPARalpha and PPARgamma ligands modulate inflammation by suppression of TNFalpha production and prevent excessive accumulation of cholesterol by ABCA1 activation. Recently, HMG-CoA reductase inhibitors (statins) were shown to activate PPARalpha and PPARgamma in various cells but no studies of their effects in GBEC have been conducted. The objective of this study was, therefore, to determine the effects of statins on PPAR and ABCA1 expression and the anti-inflammatory effect of statins in GBEC. Canine GBEC were cultured on Petri dishes. Expression of the proteins PPARalpha, PPARgamma, and ABCA1 was measured by western blotting analysis after treatment with simvastatin, pravastatin, NO-pravastatin, PPARalpha ligand, or PPARgamma ligand in the culture media. Expression of ABCA1 and LXRalpha mRNAs was estimated by RT-PCR. Expression of TNFalpha mRNA was measured by RT-PCR after 24 h pre-treatment with the statins, preceding 1 h of lipopolysaccharide (LPS) loading. Simvastatin, pravastatin, and NO-pravastatin increased expression of the proteins PPARalpha, PPARgamma, and ABCA1, and expression of the mRNA of ABCA1 and LXRalpha in GBEC. Pre-treatment with simvastatin, pravastatin, and NO-pravastatin suppressed the production of TNFalpha mRNA induced by LPS. In conclusion, statins probably contribute to the preservation of GBEC function by activation of PPARalpha and PPARgamma, which have anti-inflammatory effects by suppression of pro-inflammatory cytokines, and ABCA1 activation mediated by LXRalpha, which prevents the accumulation of cholesterol in GBEC.

0.5 Gy Gamma Radiation Suppresses Production of TNF-α through Up-regulation of MKP-1 in Mouse Macrophage RAW264.7 Cells

Low- or intermediate-dose gamma radiation appears to have the capacity to ameliorate certain types of diseases, including allergic conditions, when examined under specific exposure conditions and with specific animal models, though the molecular mechanisms involved remain to be fully clarified. We investigated the anti-inflammatory effects of intermediate-dose gamma radiation by examining its effects on the activation state of p38 MAPK and the production of cytokines in mouse macrophage RAW264.7 cells. Dephosphorylation of both ERK1/2 and p38 MAPK was observed at 15 min after irradiation (0.5-1 Gy from a (137)Cs source) concomitant with a significant increase in the expression of MKP-1, which dephosphorylates ERK1/2 and p38 MAPK. Since activated p38 MAPK mediates TNF-alpha production, we examined the effect of radiation on LPS-induced activation of p38 MAPK and TNF-alpha production. The activation of p38 MAPK and production of TNF-alpha induced by LPS treatment were both suppressed in preirradiated cells. LPS-induced production of TNF-alpha was enhanced by knockdown of MKP-1. These results indicate that 0.5 Gy gamma radiation would cause up-regulation of MKP-1, leading to inactivation of p38 MAPK and suppression of TNF-alpha production, in cells of mouse macrophages cell line.

Molecular Mechanisms of Alcoholic Fatty Liver

Alcoholic fatty liver is a potentially pathologic condition which can progress to steatohepatitis, fibrosis, and cirrhosis if alcohol consumption is continued. Alcohol exposure may induce fatty liver by increasing NADH/NAD(+) ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity, and increasing complement C3 hepatic levels. Alcohol may increase SREBP-1 activity by decreasing the activities of AMP-activated protein kinase and sirtuin-1. Tumor necrosis factor-alpha (TNF-alpha) produced in response to alcohol exposure may cause fatty liver by up-regulating SREBP-1 activity, whereas betaine and pioglitazone may attenuate fatty liver by down-regulating SREBP-1 activity. PPAR-alpha agonists have potentials to attenuate alcoholic fatty liver. Adiponectin and interleukin-6 may attenuate alcoholic fatty liver by up-regulating PPAR-alpha and insulin signaling pathways while down-regulating SREBP-1 activity and suppressing TNF-alpha production. Recent studies show that paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell-derived endocannabinoids also contributes to the development of alcoholic fatty liver. Furthermore, oxidative modifications and inactivation of the enzymes involved in the mitochondrial and/or peroxisomal beta-oxidation of fatty acids could contribute to fat accumulation in the liver.

RETRACTION: Preventive Effect of SA13353 [1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea], a Novel Transient Receptor Potential Vanilloid 1 Agonist, on Ischemia/Reperfusion-Induced Renal Injury in Rats

Tumor necrosis factor (TNF)-alpha plays a crucial role in the pathogenesis of ischemia/reperfusion-induced renal injury. We demonstrated recently that the preischemic treatment with resiniferatoxin, a transient receptor potential vanilloid 1 (TRPV1) agonist, attenuates renal TNF-alpha mRNA expression and improves ischemia/reperfusion-induced renal injury in rats. In addition, we found that SA13353 [1-[2-(1-adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea], a novel orally active TRPV1 agonist, inhibits TNF-alpha production through the activation of capsaicin-sensitive afferent neurons and reduces the severity of symptoms in established rat collagen-induced arthritis. In the present study, we investigated effects of treatment with SA13353 on ischemia/reperfusion-induced renal injury in rats. Ischemic acute kidney injury (AKI) was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in vehicle-treated AKI rats markedly decreased at 24 h after reperfusion. Treatment with SA13353 (3, 10, and 30 mg/kg p.o.) 30 min before ischemia dose-dependently attenuated the ischemia/reperfusion-induced renal dysfunction. Histopathological examination of the kidney of AKI rats revealed severe renal damage, which were significantly suppressed by the SA13353 treatment. In renal tissues exposed to ischemia/reperfusion, neutrophil infiltration, superoxide production, TNF-alpha mRNA expression, and cytokine-induced neutrophil chemoattractant-1 mRNA expression were augmented, but these alterations were attenuated by the treatment with SA13353. On the other hand, ischemia/reperfusion-enhanced renal interleukin-10 mRNA expression and its plasma concentration were further augmented by SA13353 treatment. These results demonstrate that the orally active TRPV1 agonist SA13353 prevents the ischemia/reperfusion-induced AKI. This renoprotective effects seem to be closely related to the inhibition of inflammatory response via TRPV1 activation.