NF-kappaB P65 Research Articles

Liver X receptor-dependent inhibition of microglial nitric oxide synthase 2.

BackgroundThe nuclear receptor liver X receptor (LXR) exerts transcriptional control over lipid metabolism and inflammatory response in cells of the myeloid lineage, suggesting that LXR may be a potential target in a number of chronic neuroinflammatory and neurodegenerative diseases where persistent microglial activation has been implicated in the pathogenesis.MethodsThe effect of LXR activation on microglia and central nervous system (CNS) inflammation was studied using a synthetic LXR agonist in cultured microglia, a microglial cell line and experimental allergic encephalomyelitis (EAE), an animal model of CNS inflammation.ResultsLXR activation inhibited nitric oxide synthase 2, inducible (Nos2) expression and nitric oxide production in lipopolysaccharide (LPS)-stimulated microglia. Inhibition of microglial activation in response to interferon-γ was less reliable. In LPS-stimulated cells, LXR activation did not inhibit nuclear translocation of NF-kappaB1 p50. Instead, LXR-dependent Nos2 repression was associated with inhibition of histone 4 acetylation and inhibition of NF-kappaB1 p50 binding at the Nos2 promoter. Histone acetylation and NF-kappaB1 p50 binding were mechanistically linked, and histone deacetylase (HDAC) activity appeared to be important for LXR-dependent transcriptional repression of Nos2. Analysis of CNS gene expression in animals undergoing EAE showed that the expressions of Lxr and LXR-dependent genes were downregulated during CNS inflammation. Nevertheless, administration of LXR agonist GW3965 during the effector phase of EAE delayed the onset of clinical disease and reversed the diminished expression of LXR-dependent reverse cholesterol transport genes. However, the CNS expressions of Nos2 and other inflammatory genes were not significantly inhibited by LXR activation in EAE, and clinical disease severity was comparable to vehicle controls at later time points in LXR agonist treated animals.ConclusionsLXR can be targeted to modulate microglial activation. LXR-dependent repression of inflammatory genes may be stimulus-dependent and impaired by HDAC inhibition. Endogenous LXR activity does not appear to modulate CNS inflammation, but LXR activity can be partially restored in the CNS by administration of exogenous LXR agonist with an impact on clinical disease severity at early, but not late, time points in EAE.

Beta-1-Adrenergic Receptors Mediate Nrf2-HO-1-HMGB1 Axis Regulation to Attenuate Hypoxia/Reoxygenation-Induced Cardiomyocytes Injury in Vitro

Backgroud/Aims: The aim of the study was to evaluate the effects of beta1-adrenergic receptors (β1-ARs) -mediated nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1)-high mobility group box 1 protein (HMGB1) axis regulation in hypoxia/reoxygenation (H/R)-induced neonatal rat cardiomyocytes. Methods: The neonatal cultured cardiomyocytes were concentration-dependently pretreated by dobutamine (DOB), a selective β1-adrenergic receptor agonist, in the absence and/or presence of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), SB203580 (a p38mitogen-activated-protein kinase (p38MAPK) inhibitor), Nrf2siRNA and HO-1siRNA, respectively, and then treated by H/R. The effects and mechanisms by which H/R-induced cardiomyocytes injury were evaluated. Results: Significant increase of HO-1 was found in neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Significant change for Nrf2 translocation was also revealed in neonatal cultured cardiomyocytes treated with DOB. Insignificant decreases of NF-kappaB p65 activation and HMGB1 release were observed in H/R-induced neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Importantly, DOB treatment significantly increased the cell viability and decreased the levels of LDH and MDA in H/R-induced cardiomyocytes injury. However, DOB failed to increase HO-1, inhibit NF-kappaB p65 activation, prevent HMGB1 release and attenuate H/R-induced cardiomyocytes injury when the cultured cardiomyocytes were pretreated by Nrf2siRNA, HO-1siRNA, PI3K inhibitor (LY294002) and p38MAPK inhibitor (SB203580), respectively. Conclusions: β1-ARs-mediated Nrf2-HO-1-HMGB1 axis regulation plays a critical protective role in H/R-induced neonatal rat cardiomyocytes injury in vitro via PI3K/p38MAPK signaling pathway.

Intervention effect of quercetin on inflammatory secretion of cardiac fibroblasts

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).

Sodium butyrate protects against toxin-induced acute liver failure in rats

Acute liver failure (ALF) is a serious clinical syndrome with high mortality. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical diseases. The aim of this study was to investigate the protective effect of sodium butyrate on ALF in rats. All rats were randomly divided into control, model and sodium butyrate treatment groups. Except the control group, the rats were induced ALF animal model by subcutaneous injection of human serum albumin+ D-galactosamine+lipopolysaccharide. After induction of ALF, the rats in the treatment group received sodium butyrate (500 mg/kg) at 12-hour or 24-hour time point. Fourty-eight hours after ALF induction, the animals were sacrificed and samples were harvested. Serum endotoxin, high mobility group box-1 (HMGB1), liver function parameters, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were measured. The expression of HMGB1 and nuclear factor-kappa B (NF-kappaB) p65 protein in liver tissue was detected by Western blotting. The histological changes of liver and intestine were examined. The survival duration was also observed. Serum endotoxin, alanine aminotransferase, HMGB1, TNF-alpha and IFN-gamma were significantly increased and the liver histology showed more severe histopathological injury in the model group compared with the control group (P<0.05). Compared to the model group, sodium butyrate treatment significantly improved the histopathological changes in the liver and intestine, reduced serum endotoxin and inflammatory cytokines, suppressed HMGB1 and NF-kappaB p65 proteins in liver tissue, and prolonged the survival duration regardless of treatment at 12 hours or 24 hours after induction of ALF (P<0.05). Sodium butyrate protected the liver from toxin-induced ALF in rats. The mechanisms may be due to direct hepatoprotection and decreased intestinal permeability.

Cloning and expression of retinoic acid-induced gene-I and its effect on hepatitis C virus replication.

To explore the influence of the retinoic acid indicible gene-I (RIG-I) on hepatitis C virus (HCV) replication and the molecular mechanism of action of RIG-I. We constructed an RIG-I expression vector and co-transfected it into Huh-7 cells along with HCV-replicon RNA. We assayed HCV replication and NS5A protein synthesis via real-time polymerase chain reaction (RT-PCR) and western blotting. Also, we performed an enzyme-linked immunosorbent assay (ELISA) to measure the level of interferon (IFN)-alpha/-beta secretion. Additionally, we examined, via western blotting, the phosphorylation state of p38, Erk1/2, and nuclear factor (NF)-kappaB p65. Overexpression of RIG-1 in Huh-7 cells co-transfected with an HCV-replicon RNA significantly inhibited HCV replication and NS5A protein synthesis. Co-transfected cells had increased production of IFN-alpha/-beta production and had higher levels of phosphorylated p38, Erk1/2, and NF-kappaB p65. RIG-I significantly inhibits HCV replication and NS5A protein synthesis by inducing type I IFN production. The underlying molecular mechanism for this effect appears to be mediated by increased phosphorylation of NF-kappaB p65, p38-mitogen-activated protein kinases (MAPK), and Erk1/2.

DPP-4 inhibitor attenuates toxic effects of indoxyl sulfate on kidney tubular cells.

Diabetic nephropathy is a common causative factor of chronic kidney disease (CKD). DPP-4 inhibitor has the ability to improve kidney function and renal microvasculature. In the present study, we investigate the deleterious effects of IS on proximal tubular cells and the protective role of DPP-4 inhibitor. Human kidney 2 (HK-2) cells were exposed to IS in the presence or absence of DPP-4 inhibitor. Effects of DPP-4 inhibitor on viability of HK-2 cells were determined by MTT assay. Reactive oxygen species (ROS) production was examined using fluorescent microscopy. Levels of cleaved caspase-3, transforming growth factor-beta (TGF-β), α-smooth muscle actin (α-SMA) and NF-kappaB p65 and phosphorylation of AKT and extracellular signal-regulated kinase (ERK) were detected by immunoblotting. Production of ROS and level of cleaved caspase-3 were increased by IS in a dose-dependent manner. The phosphorylation of AKT and ERK p65 were decreased alongside activation of NF-κB. Expression of TGF-β and α-SMA, were upregulated in IS-treated HK-2 cells. Treatment with DPP-4 inhibitor resulted in a significant increase in cell viability and a decrease of ROS production in IS-treated HK-2 cells. DPP-4 inhibitor restored IS-induced deactivations of AKT and ERK and inhibited activation of NF-κB in IS-treated HK-2 cells. Moreover, DPP-4 inhibitor could also attenuate IS-induced up-regulation of TGF-β and α-SMA expression. These findings suggest that DPP-4 inhibitor possesses anti-apoptotic activity to ameliorate the IS-induced renal damage, which may be partly attributed to regulating ROS/p38MAPK/ERK and PI3K-AKT pathways as well as downstream NF-κB signaling pathway.

Study on effect of total flavonoids of Oldenlendia difflusa on ulcerative colitis and its immunological mechanism

To observe the effect of total flavonoids of Oldenlendia difflusa (FOD) on NF-kappaB and IL-8, TNF-alpha, IL-10 expressions of ulcerative colitis (UC) model rats, and explore its immunological mechanism of anti-UC. Sixty Kunming male mice with the average weight of (20 +/- 2) g were randomly divided into six groups. The control group (cont) was orally administered with distilled water. Whereas the remaining five groups were fed with 4% dextran sulphate sodium (DSS) solution for seven days to induce acute UC, and orally administered with the following drugs: distilled water (for the DSS group), SASP at dose of 500 mg x kg(-1) x d(-1) for the DSS + SASP group, FOD at dose of 60 mg x kg(-1) x d(-1) for the DSS + FOD-H group, FOD at dose of 40 mg x kg(-1) x d(-1) for the DSS + FOD-M group, and FOD at dose of 26.7 mg x kg(-1) x d(-1) for the DSS + FOD-L group. During the modeling and drug administration, the mice were scored for DAI. Seven days later, the mice were put to death, and their colonic tissue samples were collected to evaluate colonic mucosal lesions. The NF-kappaB p65, IL-8, TNF-alpha, IL-10 expressions were tested by immunohistochemical staining and ELISA. Seven-day feeding with 4% DSS solution could successfully induce acute UC in mice. Compared with the cont group, the DSS group showed significantly higher DAI and colonic mucosal lesions, remarkable increase in NF-kappaB p65, IL-8, TNF-alpha expression in colonic tissues, and notable decrease in IL-10 expression (P < 0.05). FOD could prevent acute UC in mice included by DSS. Seven-day administration of 60 mg x kg(-1) x d(-1) or 40 mg x kg(-1) x d(-1) FOD could completely or partially resist the above mentioned changes caused by DSS. Compared with the DSS group, the DSS + FOD-H group and the DSS + FOD-M group showed reduction in colonic mucosal lesions, down-regulation in IL-8, TNF-alpha and NF-kappaB p65 expressions and up-regulation in IL-10 expression (P < 0.05). FOD could significantly resist UC in mice. Its mechanism may be related to the inhibition of NF-kappaB p65 activation, the reduction of IL-8 and TNF-alpha expressions and the increase in the anti-inflammatory factor IL-10.

Effect of paeoniflorin on level of glucocorticoid receptor of peripheral blood monocytes in rats of collagen-induced arthritis

The study is to explore the effect of paeoniflorin on the level of glucocorticoid receptor, including glucocorticoid receptor-alpha (GCRalpha) and glucocorticoid receptor-beta (GCRbeta), of peripheral blood mononuclear cells (PBMCs) in rats of collagen-induced arthritis (CIA). CIA is induced in Wistar rats by an intradermal injection of bovine type II collagen emulsified with complete adjuvant. From the 14th day after primary immunization, the CIA rats were intragastrically administered paeoniflorin 25, 50 and 100 mg x kg(-1) or triptolde 20 microg x kg(-1) or paeoniflorin 50 mg x kg(-1) + RU486 15 mg x kg(-1), once a day, for 28 consecutive days. After administration, apart from PF + RU486 group all experimental rats were took blood by removalling eyeball, then separated PBMCs. The level of GCRalpha, GCRbeta in PBMCs were examined by ELISA, and the mRNA expression of GCRalpha, GCRbeta was detected by RT-PCR. All rats were sacrificed and took the joint with no immunization. The expression of IL-1beta, NF-kappaB p65, TNF-alpha, PGE2 of synovial tissue was detected by immunohistochemistry. Paeoniflorin was able to inhibit the expression of IL-1beta, NF-kappaB p65, TNF-alpha, PGE2 of synovial tissue in CIA rats. While RU486, glucocorticoid receptor's blocker, could weaken the fuction of paeoniflorin. Meanwhile, paeoniflorin obviously induced the expression of GCRalpha and GCRalpha mRNA, while obviously inhibited the expression of GCRbeta and GCRbeta mRNA. These results indicat paeoniflorine suppresses inflammatory mediator production may be relating with it regulating GCR in PBMCs of CIA rats.

Ovalbumin-induced experimental allergic asthma is Toll-like receptor 2 dependent

The role of Toll-like receptor (TLR) 2 in modulating allergy-induced asthma is contradictory. We investigated the effect of TLR2 gene deletion in a murine model of ovalbumin (OVA)-challenged asthma. TLR2 wild-type (TLR2(+/+)) and TLR2-deficient (TLR2(-/-)) mice were sensitized to soluble OVA antigens and challenged with OVA, and the extent of allergic airways disease was analyzed in both groups of mice at day 8 after being challenged with OVA aerosol. At day 8 post-OVA, TLR2(-/-) mice exhibited significantly lower airway hyperresponsiveness, airway inflammation, and whole lung T-helper type 2 (Th2) cytokine levels compared with the TLR2(+/+) group. TLR2 deletion also significantly reduced mucus cell metaplasia and peribronchial fibrosis in mice at day 8 after challenged by OVA. The p38/AKT/nuclear factor kappaB (NF-kappaB) p65 and phosphate extracellular signal-regulated kinase (ERK)/p38/AKT was decreased in TLR2(-/-) mouse lungs. Thus, during OVA asthma in mice, TLR2 is a major contributor to the maintenance of the adaptive Th2-cytokine-driven inflammatory disorder and ERK/p38 as well as AKT/NF-κB playing a role in it.

Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with a 5-year survival rate less than 15%. Understanding of the molecular mechanisms involved in the pathogenesis of ESCC becomes critical to develop more effective treatments.MethodsMcl-1 expression was measured by reverse transcription (RT)-PCR and Western blotting. Human Mcl-1 promoter activity was evaluated by reporter gene assay. The interactions between DNA and transcription factors were confirmed by electrophoretic mobility shift assay (EMSA) in vitro and by chromatin immunoprecipitation (ChIP) assay in cells.ResultsFour human ESCC cell lines, TE-1, Eca109, KYSE150 and KYSE510, are revealed increased levels of Mcl-1 mRNA and protein compare with HaCaT, an immortal non-tumorigenic cell line. Results of reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of kappaB binding site, specific NF-kappaB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIkappaBalpha in TE-1 and KYSE150 cell lines. Mcl-1 protein level is also attenuated by Bay11-7082 treatment or co-transfection of DNMIkappaBalpha in TE-1 and KYSE150 cells. EMSA results indicate that NF-kappaB subunits p50 and p65 bind to human Mcl-1-kappaB probe in vitro. ChIP assay further confirm p50 and p65 directly bind to human Mcl-1 promoter in intact cells, by which regulates Mcl-1 expression and contributes to the viability of TE-1 cells.ConclusionsOur data provided evidence that one of the mechanisms of Mcl-1 expression in human ESCC is regulated by the activation of NF-kappaB signaling. The newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC.

Паттерн нейрональной экспрессии транскрипционных факторов NF-kb при предъявлении различных режимов гипобарической гипоксии

Transcription factor NF-kappaB plays a pivotal role in mechanisms of brain neuron survival and degeneration under injurious stimuli, first of all different types of hypoxia. In the present work, using quantitative immunohystochemistry, we provide analysis of expression of different subunits of NF-kappaB (p65 and c-Rel) in the rat neocortex in response to severe injurious hypobaric hypoxia (HH) or after a single or multiple sessions of mild protective HH. Severe hypoxia (SH), resulting in loss of brain neurons, has no effect on the level of expression of p65 but suppresses expression of c-Rel. Multiple (but not single one) trials of preconditioning using mild HH which reduce neuronal damage promote p65 expression and prevent suppression of c-Rel level after SH. Triple session of mild HH itself when applied as a preconditioning stimulus upregulate expression of both subunits, while single administration or sixfold trials has no effect on the level of immunoreactivity of both subunits. The revealed peculiarities of the expression of p65 and c-Rcl implies that these subunits of NF-kappaB appear to contribute to the mechanisms of brain tolerance to SH.

Effect of formula of removing both phlegm and blood stasis on inflammatory reaction in Chinese mini-swine with coronary atherosclerosis

To observe the effect of formula of removing both phlegm and blood stasis (TYTZ) in inhibiting the inflammatory reaction in Chinese mini-swine with coronary atherosclerosis. Totally 36 Chinese mini-swine were randomly divided to six groups: the normal control group, the model group, the Shujiangzhi group and TYTZ groups with does of 2.0, 1.0 and 0.5 g x kg(-1), and six each in every group. Except for the normal control group, all of other groups were fed with high-fat diet for 2 weeks. Interventional balloons are adopted to injure their left anterior descending artery endothelium. After the operation, they were fed with high-fat diet for 8 weeks to prepare the coronary atherosclerosis model. In the 8th week after the operation and administration, the intravascular ultrasound was adopted to observe the coronary artery plaque burden of each group and the pathological morphology of coronary artery. Such inflammatory factors as high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were detected by ELISA. The expression of NF-kappaB p65 nuclear translocation was observed by the immunohistochemical method. Compared with the normal control group, the model group showed significant increase in the coronary artery plaque burden at the end of the experiment (P < 0.01), notably abnormal structural changes in atherosclerotic vascular tissues, luminal stenosis, a large number of foam cells and inflammatory cell infiltration, remarkable growth of hs-CRP, TNF-alpha and IL-6 levels (P < 0.01). The immunohistochemical staining also showed the significant increase in the NF-kappaB p65 nuclear translocation of coronary artery of Chinese mini-swine in the model group. Compared with the model group, TYTZ could significantly attenuate atherosclerotic plaque burden (P < 0.01), inhibit the coronary luminal stenosis, reduce inflammatory cell infiltration, decrease such inflammatory cell factors as hs-CRP, TNF-alpha and IL-6 in serum, and inhibit the NF-kappaB p65 nuclear translocation of coronary artery (P < 0.05 or P < 0.01). TYTZ can reduce the downstream inflammatory reaction by controlling NF-kappaB p65 nuclear translocation, so as to inhibit the occurrence and development of coronary atherosclerotic plaque in Chinese mini-swine.

Azelnidipine, Not Amlodipine, Induces Secretion of Vascular Endothelial Growth Factor From Smooth Muscle Cells and Promotes Endothelial Tube Formation.

BackgroundWe previously reported that the calcium channel blocker (CCB) nifedipine-induced secretion of vascular endothelial growth factor (VEGF) from human coronary smooth muscle cells (HCSMCs) promoted human coronary endothelial cell (HCEC) tube formation. Therefore, we analyzed whether other CCBs, azelnidipine and amlodipine, also induced the secretion of VEGF and promoted HCEC tube formation, and the underlying molecular mechanisms.MethodsTo evaluate the tube formation, HCECs were grown on Matrigel for 18 hours in the supernatants from HCSMCs that had been treated with different kinds of reagents. Concentrations of VEGF in cultured HCSMCs were determined by specific enzyme immunoassays. Nuclear extracts from HCSMCs were prepared, and nuclear factor-kappa B (NF-κB) activation was measured by EZ-DetectTM Transcription Factor Kits for NF-κB p50 or p65.ResultsAlthough azelnidipine dose-dependently stimulated the significant secretion of VEGF from HCSMCs and this stimulation was abolished by a protein kinase C inhibitor, amlodipine-induced secretion of VEGF was significantly lower than that induced by azelnidipine. The medium derived from azelnidipine (at up to 2 μM)-treated HCSMCs led to HCEC tube formation, whereas that obtained with amlodipine did not. Azelnidipine-induced tube formation was blocked by an inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 tyrosine kinase. Azelnidipine at up to 2 μM induced NF-κB activation.ConclusionsAzelnidipine, but not amlodipine, stimulated the secretion of VEGF from HCSMCs and induced HCEC tube formation. This secretion is mediated at least in part via the activation of NF-κB. Azelnidipine may have a novel beneficial effect in improving coronary microvascular blood flow in addition to its main effect of lowering blood pressure.

Heat Shock Stress Prior to Hyperbaric Oxygen Exposure in Rats

Heat shock proteins (HSPs) and nuclear factor-kappa B (NF-kappaB) have been established as important mediators in lung injury; however, their role in preventing pulmonary toxicity from hyperbaric oxygen (HBO) has not been evaluated. We aimed to study the effects of heat shock (HS) injury on hyperbaric hyperoxic lung injury (HHLI) in a rat model and identify a mechanism of protection by evaluating HSP 27 and HSP 70 mRNA and protein levels, NF-kappaB p65, lung injury and oxidative parameters. By varying the times between HS and exposure to HBO, the pathways of interaction between HSPs and NF-kappaB will be further clarified. Our results showed that HS exposure increases the mRNA and protein levels of HSP 27 and HSP 70; HS induced 10-fold increases of HSP 27 (9.77 +/- 0.60) and HSP 70 (10.33 +/- 2.4) within the first 10 h compared to control animals. Lesion scores were higher for the first 16 h after HS, but decreased again after 31 h (N = 7 animals; 5 lesions scores). Protein nitration showed no significant differences between groups exposed to HS or HBO; similarly there was no difference with a combination of both treatments. HBO appears to attenuate the HS response by HSP 27 and HSP 70. Histopathology results suggest that HS might mitigate pathology in animals exposed to HS and HBO. No significant effect of HS on HBO-induced HHLI was observed in animals treated with both HS and HBO.

Relationship between the expression levels of PAPP-A metalloproteinase and growth and transcriptional factors in endometrial cancer

We have examined for the first time the relationship between the expression of PAPP-A metalloproteinase and insulin-like growth factors (IGF-I, IGF-II, VEGF) and transcription factors (NF-kappaB, HIF-1) playing an important role in pathogenesis of cancer. We also demonstrated a positive association between the level of PAPP-A metalloproteinase and the level of growth (VEGF and IGF-I) and transcription factors (NF-kappaB p50, NF-kappaB p65, HIF-1alpha). The current findings suggest an important role of PAPP-A in regulation of bioavailability of IGF-I, VEGF, activated forms of NF-kappaB, and alpha-subunits of HIF-1 in endometrial tumors.

Suppression of IL-12 production by TTP through blocking NF-kappaB nuclear translocation (P3008)

Abstract Tristetraprolin (TTP) plays a significant role in regulating the expression of AREs containing mRNAs. TTP-/- Mice develop a systemic autoimmune inflammatory syndrome characterized by cachexia, conjunctivitis and dermatitis. IL-12 plays a crucial role in immune defense against infectious and malignant diseases. In the present study, we found increased production of IL-12 during endotoxic shock and enhanced Th1 cells in TTP knockout mice. The levels of IL-12 p70 and p40 protein as well as p40 and p35 mRNA were also increased in activated macrophages deficient of TTP. In line with these findings, overexpression of TTP suppressed IL-12 p35 and p40 expression at the mRNA and promoter level, while surprisingly had no effects on their mRNA stability. Our data showed that the inhibitory effects of TTP on p35 gene transcription were completely rescued by overexpression of NF-kappaB p65 and c-Rel but not the p50 in activated macrophages. Our data further indicated that TTP acquired its inhibition on IL-12 expression through blocking nuclear translocation of NF-kappaB p65 and c-Rel while enhancing p50 upon stimulation. In summary, our study reveals a novel pathway through which TTP suppresses IL-12 production in macrophages, resulting in suppression of Th1 cell differentiation. This study may provide us with therapeutic targets for treatment of inflammatory and autoimmune disorders

Immunohistochemical localization of selected pro-inflammatory factors in uterine myomas and myometrium in women of various ages

Uterine myomas represent one of the most frequently manifested benign tumors in women. They originate from smooth muscle cells of myometrium or its blood vessels. Many studies suggest that inflammation and pro-inflammatory factors may play a role in the carcinogenesis with an involvement of the transcription factor NF-kappaB which activity can be controlled by various environmental factors, including many cytokines. The aim of the study was to investigate the expression of NF-B, interleukin-1β (IL-1β), tumor necrosis factor a (TNF-α), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in myometrium and uterine myomas of women of various age. The expression of NF-kappaB, selected cytokines and enzymes was estimated in women of reproductive or perimenopausal age by semiquantitative immunohistochemistry. The expression of the examined proteins was higher in myomas than in control myometrium and was dependent on the size of myomas and the age of women. However, the expression of the cytoplasmic NF-kappaB observed in uterine myomas was independent on the size of myomas and no significant differences were observed in the number of stained nuclei between control and myoma groups. Thus, the expression of proinflammatory factors in myomas was not accompanied by the nuclear activation of NF-kappaB p65. The results of our study indicate that the examined factors may be involved in the pathogenesis of benign tumors and not only malignant diseases.

Suppression of NF‐kappaB p65 expression attenuates delayed xenograft rejection

Delayed xenograft rejection (DXR) involves type II vascular endothelial cell (VEC) activation including upregulation of pro-inflammatory genes, which contributes to infiltration into the graft and a complex process of cytokine production. Approaches to prevent DXR have shown limited success. In this study, we modified heart donors using siRNA in an attempt to attenuate DXR and to improve xenograft survival in the mouse-to-rat heterotopic heart transplant model. siRNA technology was used to inhibit NF-kappaB p65 gene expression in vivo in mice. After the donor was transfected with siRNA, the effects of NF-kappaB siRNA on DXR and expression of NF-kappaB and pro-inflammatory genes were evaluated in a concordant mouse-to-rat cardiac xenograft model. Treatment of NF-kappaB siRNA prolonged median heart graft survival time in the recipient rats from 1.7days in a PBS control group to 5.4days in the NF-kappaB siRNA-treated group (P<0.05). Compared with normal mouse hearts, the NF-kappaB p65 mRNA relative levels following siRNA injection in the donors decreased significantly (approximately 70% reduction) in grafts harvested 12h after transplantation. The mRNA levels of VCAM-1, ICAM-1, and interleukin-1 displayed a similar reduction. Histological evaluation using light and electron microscopy showed that damage of endothelial cells after NF-kappaB siRNA treament occured at a later time. Transfection of NF-kappaB p65 siRNA in donor animals can delay the emergence of DXR. This treatment may be used as part of strategies to minimize the complex and multi-faceted rejection responses in vascularized xenografts.

Resveratrol Attenuates Oxidative Stress Induced by Balloon Injury in the Rat Carotid Artery Through Actions on the ERK1/2 and NF-Kappa B Pathway

Background/Aim: Oxidative stress plays a critical role in pathogenesis of the neointimal arterial hyperplasia. The aim of the study was to evaluate effects of resveratrol (RSV) on the vascular hyperplasia stimulated by oxidative damage. Methods: Balloon vascular injury was induced in rats that were intraperitonealy exposed to resveratrol (1 mg/kg) on 7 or 14 days after surgical procedure. Animals were euthanized on 7 or 14 days after operation. The blood level of 8-iso-prostaglandin F2α, arterial morphology as well as expression of monocyte chemotactic protein-1 and interleukin-6 in carotid wall were measured. Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta. Cellular proliferation and migration assays, reactive oxygen species (ROS), superoxide dismutase (SOD) and NADPH oxidative activity, protein level of β-actin, histone H3, NF-ĸB p65, IĸB, ERK1/2, phospho-ERK1/2, phospho-p38 as well as NF-ĸB transcription activity were evaluated in-vitro after angiotensin II stimulation and resveratrol (50-200 µmol/L) treatment. Results: Significant decreases in neointimal/medial area, serum prostaglandin level and genes expression were found in rats treated with resveratrol, when compared to the control group. Significant changes were also revealed for proliferation and migration rates, ROS level, as well as SOD, NADPH oxidase, ERK1/2 phosphorylation and NF-ĸB transcriptional activity in cell cultures exposed to highest dose of resveratrol. Insignificant changes were observed for NF-kappaB p65 translocation and IĸB degradation, p38 phosphorylation in MAPK pathway. Conclusion: Resveratrol significantly suppressed the neointimal hyperplasia after balloon injury through inhibition of oxidative stress and inflammation by blocking the ERK1/2/NF-kappa B pathway.

Chemopreventive properties of raisins originating from Greece in colon cancer cells

Colorectal cancer is one of the major causes of cancer-related mortality in humans in both developed and developing countries. Dietary patterns influence the risk of colon cancer development, while plant-derived foods have gained great interest, due to the high content of antioxidants. Corinthian raisins (Currants, CR) and Sultanas (S) (Vitis vinifera L., Vitaceae) are dried vine fruits produced in Greece with many culinary uses in both the Mediterranean and the Western nutrition. In the present study, we investigated the effects of CR and S on human colon cancer cells. Methanol extracts of CR and S were used at different concentrations. The total polyphenol content and anti-radical activity were measured by Folin-Ciocalteu and DPPH, respectively. Antioxidant, anti-inflammatory and anti-proliferative effects on HT29 cell culture were evaluated. All extracts exhibited DPPH˙ scavenging activity in a dose-dependent manner. Both products suppressed cell proliferation, while the levels of glutathione and cyclooxygenase 2 were significantly decreased. A significant reduction in IL-8 levels and NF-kappaB p65 activation was also observed. Both antioxidant and anti-inflammatory effects were dependent on the duration of exposure. Results indicate that the methanol extracts of CR and S exhibit anti-radical activity in vitro, as well as cancer preventive efficacy on colon cancer cells, with S having slightly higher activity. The beneficial properties of these unique dried grapes are attributed to their high content of phenolic compounds.