OBJECTIVECelastrol has been established as a nuclear factor-κB (NF-κB) activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that interleukin-1 receptor-associated kinases (IRAKs) are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB (e.g., the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily).

METHODSThe human hepatocellular cell line (HepG2) treated with palmitic acid (PA) was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions.

RESULTSThe results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the HepG2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation.

CONCLUSIONThe results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.

Hep G2 Cells ; Humans ; Interleukin-1 Receptor-Associated Kinases ; antagonists & inhibitors ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Phosphorylation ; Toll-Like Receptor 4 ; antagonists & inhibitors ; physiology ; Triterpenes ; pharmacology

BACKGROUNDNumerous studies have shown that reducing the level of tumor necrosis factor-alpha (TNFα) through the use of anti-TNF antibodies or soluble TNF receptor is a safe and efficacious treatment to inflammatory diseases such as rheumatoid arthritis. Therefore, novel approaches to achieve this outcome are desired. The aim of this study was to investigate the characterization of a small molecule inhibitor, Y316, which blocks TNF mRNA upregulation and TNF production by lipopolysaccharides (LPS) stimulated monocytes.

METHODSPeripheral blood mononuclear cells (PBMC) from healthy volunteers were plated in 24-well plates and stimulated with LPS (1 µg/ml), phorbol-12-myristate-13-acetate (PMA) (100 ng/ml), zymosan (10 µg/ml) and Tsst (100 ng/ml). Supernatants were collected after 4-hour culture at 37°C, and quantitative determination of TNFα, interleukin-1β (IL-1β), IL-6, IL-8, IL-10 and IL-2 production in the supernatants was performed by colorimetric enzyme-linked immunosorbent assay (ELISA). Total RNA of PBMC was isolated and cytokine mRNA quantitation was performed by using a RNA level measuring kit (R & D Systems). PBMC were pretreated with Y316 (10 µmol/L, 1 µmol/L, 0.1 µmol/L, 0.01 µol/L and 0.001 µmol/L) or dimethyl sulfoxide at 37°C for 10 minutes, and then stimulated with LPS or PMA, protein concentrations of p44.42, IKBα, P38 and Jun NH2-terminal kinase were determined by Western blotting. Cyclic adenosine-3',5'-monophosphate (cAMP) of PBMC was measured by enzyme immunoassay kit (Amersham Pharmacia Biotech).

RESULTSY316 blocked TNF production and inhibited the upregulation of TNF mRNA levels in response to LPS, and also prevented the production of IL-1 and IL-6. In contrast, Y316 augmented the production of IL-10 in LPS-stimulated monocytes. Y316 failed to prevent the production of IL-2 and TNF in antigen-stimulated T cells, suggesting that its effects may be cell-type specific. Y316 prevented the phosphorylation and activation of the MAPK, ERK, and therefore appeared to mediate its effects on TNF by acting at an early point in the signaling cascade induced in response to LPS. There was no effect of Y316 on cAMP levels either alone or in the presence of LPS.

CONCLUSIONSY316 appears to be a small molecule inhibiting TNF production, which may act via a novel mechanism. Identification of the target of Y316 may lead to the development of alternative strategies for achieving selective cytokine inhibition.

Anti-Inflammatory Agents ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Interleukin-1 ; antagonists & inhibitors ; biosynthesis ; Interleukin-6 ; antagonists & inhibitors ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; drug effects ; immunology ; Phosphorylation ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors ; biosynthesis

The present study was to investigate the effects of intracerebroventricular (i.c.v.) injection of interleukin-1beta (IL-1beta) on thermal nociception in SD rats. The rats were divided into control and drug-administration groups. The animals of control group were given vehicle solution via i.c.v. injection. The animals of drug-administered groups were given IL-1beta at different doses (5, 50 and 500 pg/kg b.w.) via i.c.v. injection. IL-1 receptor antagonist (IL-1ra, 50 ng/kg) was injected 20 min before injection of IL-1beta or vehicle solution. The nociceptive threshold, which was represented as paw withdrawal latency (PWL), to a noxious thermal stimulation was measured using an analgesiameter. I.c.v. injection of IL-1beta dose-dependently shortened the PWL. At the dose of 500 pg/kg, the shortening of the PWL occurred at 20 min, reaching a peak within 40 min, lasted 100 min after injection, then gradually returned to the baseline level. Pretreatment with IL-1ra completely blocked the effects of IL-1beta-induced shortening in PWL. The results obtained suggest that IL-1beta may induce hyperalgesia in rats through binding to IL-1 receptors in the brain.
Animals ; Hot Temperature ; Hyperalgesia ; drug therapy ; Injections, Intraventricular ; Interleukin 1 Receptor Antagonist Protein ; pharmacology ; Interleukin-1beta ; pharmacology ; Nociception ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin-1 ; antagonists & inhibitors ; metabolism ; Touch

AIMTo study the effects of human recombinant interleukin-1 receptor antagonist (IL-1ra) on isolated trachea smooth muscle (TSM) of the guinea pig.

METHODSChanges of the tension of isolated trachea were measured by force-displacement transducer and MedLab recording system in vitro.

RESULTSIL-1ra showed direct relaxed effect on TSM in normal and ovalbumin sensitized guinea pig. The EC50 values were 8.06 x 10(-8) and 5.88 x 10(-7) mol.L-1 respectively. IL-1ra (1 x 10(-7)-1 x 10(-5) mol.L-1) concentration-dependently inhibited the contraction of TSM induced by 1 x 10(-3) mol.L-1 histamine (His), 1 x 10(-3) mol.L-1 acetylcholine (ACh) and 1 x 10(-6) mol.L-1 5-hydroxytryptamine (5-HT) (P < 0.05 or 0.01). When IL-1ra was given in advance, the contractile actions of His, ACh and 5-HT were antagonized by IL-1ra (1 x 10(-7)-1 x 10(-5) mol.L-1), the pD'2 value for His was 6.6 +/- 0.3. However, the contractile action of ACh was enhanced by IL-1ra at low concentration of 1 x 10(-9)-1 x 10(-8) mol.L-1. IL-1ra significantly prevented and inhibited the contraction of sensitized TSM induced by antigen ovalbumin, the IC50 value was 4.48 x 10(-7) mol.L-1.

CONCLUSIONThe results indicate that IL-1ra, within certain concentration, can relax the normal, contracted and sensitized ISM of the guinea pig in vitro.

Animals ; Female ; Guinea Pigs ; In Vitro Techniques ; Interleukin 1 Receptor Antagonist Protein ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Receptors, Interleukin-1 ; antagonists & inhibitors ; Recombinant Proteins ; pharmacology ; Sialoglycoproteins ; pharmacology ; Trachea ; cytology

OBJECTIVEThe purpose of this study was to investigate methods and expression of the recombinant human interleukin-1 receptor antagonist gene transfected into chondrocytes of temporomandibular joints (TMJ).

METHODSChondrocytes derived from the TMJ of 5-7 months old human fetus were transfected by recombinant human interleukin-1 receptor antagonist gene via cationic liposome (Lipefectine) as a medium. The expressing level of hIL-1ra protein was detected using immunocytochemistry and enzyme-linked immunosorbent assay (ELISA).

RESULTSThe positive results of the immunocytochemistry were found. The positive expression was detected in the cell plasma and the cell culture supernatant, and there was significant difference between the cells with and without gene transfection (P < 0.01).

CONCLUSIONThis feasible method provides experimented evidence for the future gene therapy of temporomandibular joint osteoarthrosis.

Chondrocytes ; cytology ; Enzyme-Linked Immunosorbent Assay ; Fetus ; Phosphatidylethanolamines ; Receptors, Interleukin-1 ; antagonists & inhibitors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Temporomandibular Joint ; cytology ; Transfection

OBJECTIVETo identify an interaction between the interleukin-1 receptor antagonist gene polymorphism and risk of Alzheimer's disease.

METHODSThe study included 117 healthy controls, 85 patients with Alzheimer's disease in a Northeastern Chinese population of Han nationality. Genotypes were determined by a polymerase chain reaction amplification of the intron 2 fragment, harbouring a variable number of short tandem nucleotide sequences. Amplification products were separated on a 2% agarose gel.

RESULTSThe allele 2 frequency was 27% in healthy controls, and 21% in patients with Alzheimer's disease. Thus for allele 2 as well as for all other alleles, genotypes, or carriage rates, no significant differences compared with controls.

CONCLUSIONSNo association of interleukin-1 receptor antagonist gene polymorphism with Alzheimer's disease was identified in this population. It is also possible that the increased risk and disease modifying effects are caused by linkage disequilibrium with other genomic variants in other nearby genes.

Aged ; Alleles ; Alzheimer Disease ; genetics ; Asian Continental Ancestry Group ; China ; Female ; Gene Frequency ; Genotype ; Humans ; Introns ; Male ; Middle Aged ; Polymorphism, Genetic ; Receptors, Interleukin-1 ; antagonists & inhibitors

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

To investigate the effect of cetirizine hydrochloride on the expression of neurokinin 1 receptor (NK-1R) and cytokines production induced by substance P (SP) in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. The effect of cetirizine on the expression of NK-1R protein was detected by flow cytometry and Western blotting analysis. The modulation of cetirizine on the production of interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6 and IL-8 in HaCaT cells and fibroblasts was measured by ELISA. The results showed that cetirizine significantly inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of IFN-gamma, IL-1beta and IL-8 in both cell types. Cetirizine 1-100 micromol x L(-1) inhibited SP-induced IL-1beta and IL-8 production in HaCaT cells and fibroblasts, while had no effect on the production of IFN-gamma in both cells. Both SP and cetirizine had no effect on the secretion of IL-6 in HaCaT cells and fibroblasts. These findings suggest that cetirizine may be involved in the treatment of SP-induced skin inflammation by inhibiting the expression of substance P receptor and regulation the production of IL-1beta and IL-8 in epidermal keratinocyte and dermal fibroblasts.
Anti-Allergic Agents ; pharmacology ; Cell Line ; Cetirizine ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Histamine H1 Antagonists, Non-Sedating ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-8 ; metabolism ; Keratinocytes ; cytology ; metabolism ; Receptors, Neurokinin-1 ; metabolism ; Substance P ; pharmacology

BACKGROUND: The human immune response to Mycobacterium tuberculosis is mediated by macrophages and T-lymphocytes. The alveolar macrophage phagocyting mycobacterium produces interleukin (IL) -1 as an inflammatory mediator, and IL-8 as a cytokine for leukocyte recruitment and granuloma formation. Interleukin-1 receptor antagonist (IL-1ra) is an internal antagonist of IL-1. METHODS: Plasma levels of IL-1ra and IL-8 and other serologic markers were measured in 18 patients with active tuberculosis before treatment and after 2 months and 6 months of treatment. RESULTS: During treatment with antituberculous medication, patients showed significant changes in hemoglobin, hematocrit, white blood cells (WBC), platelet, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin and plasma IL-1ra. After 2 months of treatment, ESR and CRP diminished significantly; after 6 months, hemoglobin increased while WBC, platelet, ESR, CRP and ferritin decreased significantly compared to their pre-treatment levels. There were two groups: patients with delayed therapeutic responses, and patients with early responses. At each point of observation, the former group of patients showed lower body weight and lower levels of hemoglobin and hematocrit, and higher levels of WBC, platelet, ESR, IL-8 and IL-1ra than the latter group. During the course of the treatment, we observed considerable differences in body weight, body mass index, hemoglobin, hematocrit, WBC and platelet counts, ESR, CRP and ferritin in both the early-response and delayed-response groups. CONCLUSION: We believe that the plasma concentrations of IL-1ra and IL-8, which showed different peaks during the course of treatment, reflected their different functions and patterns of secretion. Moreover the concentrations did not seem as sensitive as other inflammatory markers to evaluate disease activity during antituberculosis treatment. However, IL-1ra can be considered a marker for disease activity and response to treatment.
Adult ; Aged ; Aged, 80 and over ; Antitubercular Agents/therapeutic use ; Biological Markers/*blood ; C-Reactive Protein/analysis ; Comparative Study ; Female ; Human ; Interleukin-8/*blood ; Male ; Middle Aged ; Receptors, Interleukin-1/*antagonists & inhibitors ; Sialoglycoproteins/*blood ; Tuberculosis, Pulmonary/*blood/*drug therapy

BackgroundSerum soluble ST2 (sST2) levels are elevated early after acute myocardial infarction and are related to adverse left ventricular (LV) remodeling and cardiovascular outcomes in ST-segment elevation myocardial infarction (STEMI). Beta-blockers (BB) have been shown to improve LV remodeling and survival. However, the relationship between sST2, final therapeutic BB dose, and cardiovascular outcomes in STEMI patients remains unknown.

MethodsA total of 186 STEMI patients were enrolled at the Wuhan Asia Heart Hospital between January 2015 and June 2015. All patients received standard treatment and were followed up for 1 year. Serum sST2 was measured at baseline. Patients were divided into four groups according to their baseline sST2 values (high >56 ng/ml vs. low ≤56 ng/ml) and final therapeutic BB dose (high ≥47.5 mg/d vs. low <47.5 mg/d). Cox regression analyses were performed to determine whether sST2 and BB were independent risk factors for cardiovascular events in STEMI.

ResultsBaseline sST2 levels were positively correlated with heart rate (r = 0.327, P = 0.002), Killip class (r = 0.408, P = 0.000), lg N-terminal prohormone B-type natriuretic peptide (r = 0.467, P = 0.000), lg troponin I (r = 0.331, P = 0.000), and lg C-reactive protein (r = 0.307, P = 0.000) and negatively correlated to systolic blood pressure (r = -0.243, P = 0.009) and LV ejection fraction (r = -0.402, P = 0.000). Patients with higher baseline sST2 concentrations who were not titrated to high-dose BB therapy (P < 0.0001) had worse outcomes. Baseline high sST2 (hazard ratio [HR]: 2.653; 95% confidence interval [CI]: 1.201-8.929; P = 0.041) and final low BB dosage (HR: 1.904; 95% CI, 1.084-3.053; P = 0.035) were independent predictors of cardiovascular events in STEMI.

ConclusionsHigh baseline sST2 levels and final low BB dosage predicted cardiovascular events in STEMI. Hence, sST2 may be a useful biomarker in cardiac pathophysiology.

Adrenergic beta-Antagonists ; administration & dosage ; therapeutic use ; Adult ; Aged ; Biomarkers ; blood ; Female ; Humans ; Interleukin-1 Receptor-Like 1 Protein ; blood ; Male ; Middle Aged ; Prognosis ; Prospective Studies ; ST Elevation Myocardial Infarction ; blood ; drug therapy ; pathology