Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Effects of Cinnamon extract on biochemical enzymes, TNF-α and NF-κB gene expression levels in liver of broiler chickens inoculated with Escherichia coli

Abstract: Infection with Escherichia coli (E. coli) is a common disease in poultry industry. The use of antibiotics to treat diseases is facing serious criticism and concerns. The medicinal plants may be effective alternatives because of their multiplex activities. The aim of this study was to investigate the effects of cinnamon extract on the levels of liver enzymes, tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB) gene expressions in liver of broiler chickens infected with E. coli. Ninety Ross-308 broilers were divided into healthy or E. coli-infected groups, receiving normal or cinnamon extract (in concentrations of 100 or 200mg/kg of food) supplemented diets. E. coli suspension (108cfu) was injected subcutaneously after 12 days cinnamon administration. Seventy-two hours after E. coli injection, the blood samples were taken for biochemical analysis of liver enzymes in serum (spectrophotometrically), and liver tissue samples were obtained for detection of gene expression of inflammatory markers TNF-α and NF-κB, using real-time PCR. Infection with E. coli significantly increased the levels of TNF-α and NF-κB gene expressions as well as some liver enzymes including creatine-kinase (CK), lactate-dehydrogenase (LDH), alanine-transferase (ALT) and aspartate-transferase (AST) as compared with control group (P<0.05). Pre-administration of cinnamon extract in broilers diet (in both concentrations) significantly reduced the tissue levels of TNF-α and NF-κB gene expressions and enzymes CK and ALT in serum of broiler chickens inoculated with E. coli in comparison with E. coli group (P<0.05 and P<0.01). The levels of LDH and AST were significantly decreased only by 200mg/kg cinnamon extract in infected broilers. The level of alkaline-phosphatase (ALP) was not affected in any groups. Pre-administration of cinnamon extract in diets of broiler chickens inoculated with E. coli could significantly reduce the gene expression levels of pro-inflammatory mediators and liver enzymes activities, thereby protecting the liver against this pathologic condition.

SIRT1 negatively regulates amyloid-beta-induced inflammation via the NF-κB pathway

Chronic inflammation induced by amyloid-beta (Aβ) plays a key role in the development of age-related macular degeneration (AMD), and matrix metalloproteinase-9 (MMP-9), interleukin (IL)-6, and IL-8 may be associated with chronic inflammation in AMD. Sirtuin 1 (SIRT1) regulates inflammation via inhibition of nuclear factor-kappa B (NF-κB) signaling, and resveratrol has been reported to prevent Aβ-induced retinal degeneration; therefore, we investigated whether this action was mediated via activation of SIRT1 signaling. Human adult retinal pigment epithelial (RPE) cells were exposed to Aβ, and overactivation and knockdown of SIRT1 were performed to investigate whether SIRT1 is required for abrogating Aβ-induced inflammation. We found that Aβ-induced RPE barrier disruption and expression of IL-6, IL-8, and MMP-9 were abrogated by the SIRT1 activator SRT1720, whereas alterations induced by Aβ in SIRT1-silenced RPE cells were not attenuated by SRT1720. In addition, SRT1720 inhibited Aβ-mediated NF-κB activation and decrease of the NF-κB inhibitor, IκBα. Our findings suggest a protective role for SIRT1 signaling in Aβ-dependent retinal degeneration and inflammation in AMD.

Treatment with 1,25(OH)2D3induced HDAC2 expression and reduced NF-κB p65 expression in a rat model of OVA-induced asthma

Recent evidence indicates that a deficiency of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) may influence asthma pathogenesis; however, its roles in regulating specific molecular transcription mechanisms remain unclear. We aimed to investigate the effect of 1,25(OH)2D3 on the expression and enzyme activity of histone deacetylase 2 (HDAC2) and its synergistic effects with dexamethasone (Dx) in the inhibition of inflammatory cytokine secretion in a rat asthma model. Healthy Wistar rats were randomly divided into 6 groups: control, asthma, 1,25(OH)2D3 pretreatment, 1,25(OH)2D3 treatment, Dx treatment, and Dx and 1,25(OH)2D3 treatment. Pulmonary inflammation was induced by ovalbumin (OVA) sensitization and challenge (OVA/OVA). Inflammatory cells and cytokines in the bronchoalveolar lavage (BAL) fluid and histological changes in lung tissue were examined. Nuclear factor kappa B (NF-κB) p65 and HDAC2 expression levels were assessed with Western blot analyses and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Enzyme activity measurements and immunohistochemical detection of HDAC2 were also performed. Our data demonstrated that 1,25(OH)2D3 reduced the airway inflammatory response and the level of inflammatory cytokines in BAL. Although NF-κB p65 expression was attenuated in the pretreatment and treatment groups, the expression and enzyme activity of HDAC2 were increased. In addition, 1,25(OH)2D3 and Dx had synergistic effects on the suppression of total cell infusion, cytokine release, and NF-κB p65 expression, and they also increased HDAC2 expression and activity in OVA/OVA rats. Collectively, our results indicated that 1,25(OH)2D3might be useful as a novel HDAC2 activator in the treatment of asthma.

RANK, RANKL and osteoprotegerin in arthritic bone loss

Rheumatoid arthritis is characterized by the presence of inflammatory synovitis and destruction of joint cartilage and bone. Tissue proteinases released by synovia, chondrocytes and pannus can cause cartilage destruction and cytokine-activated osteoclasts have been implicated in bone erosions. Rheumatoid arthritis synovial tissues produce a variety of cytokines and growth factors that induce monocyte differentiation to osteoclasts and their proliferation, activation and longer survival in tissues. More recently, a major role in bone erosion has been attributed to the receptor activator of nuclear factor kappa B ligand (RANKL) released by activated lymphocytes and osteoblasts. In fact, osteoclasts are markedly activated after RANKL binding to the cognate RANK expressed on the surface of these cells. RANKL expression can be upregulated by bone-resorbing factors such as glucocorticoids, vitamin D3, interleukin 1 (IL-1), IL-6, IL-11, IL-17, tumor necrosis factor-alpha, prostaglandin E2, or parathyroid hormone-related peptide. Supporting this idea, inhibition of RANKL by osteoprotegerin, a natural soluble RANKL receptor, prevents bone loss in experimental models. Tumor growth factor-ß released from bone during active bone resorption has been suggested as one feedback mechanism for upregulating osteoprotegerin and estrogen can increase its production on osteoblasts. Modulation of these systems provides the opportunity to inhibit bone loss and deformity in chronic arthritis.

Increased expression of p38 mitogen- activated protein kinase is related to the acute renal lesions induced by gentamicin

Mitogen-activated protein kinases (MAPK) may be involved in the pathogenesis of acute renal failure. This study investigated the expression of p-p38 MAPK and nuclear factor kappa B (NF-kappaB) in the renal cortex of rats treated with gentamicin. Twenty rats were injected with gentamicin, 40 mg/kg, im, twice a day for 9 days, 20 with gentamicin + pyrrolidine dithiocarbamate (PDTC, an NF-kappaB inhibitor), 14 with 0.15 M NaCl, im, twice a day for 9 days, and 14 with 0.15 M NaCl , im, twice a day for 9 days and PDTC, 50 mg kg-1 day-1, ip, twice a day for 15 days. The animals were killed 5 and 30 days after the last of the injections and the kidneys were removed for histological, immunohistochemical and Western blot analysis and for nitrate determination. The results of the immunohistochemical study were evaluated by counting the p-p38 MAPK-positive cells per area of renal cortex measuring 0.05 mm². Creatinine was measured by the Jaffé method in blood samples collected 5 and 30 days after the end of the treatments. Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. In addition, animals killed 5 days after the end of gentamicin treatment presented acute tubular necrosis and increased nitrate levels in the renal cortex. Increased expression of p-p38 MAPK and NF-kappaB was also observed in the kidneys from these animals. The animals killed 30 days after gentamicin treatment showed residual areas of interstitial fibrosis in the renal cortex, although the expression of p-p38 MAPK in their kidneys did not differ from control. Treatment with PDTC reduced the functional and structural changes induced by gentamicin as well as the expression of p-p38 MAPK and NF-kappaB. The increased expression of p-p38 MAPK and NF-kappaB observed in these rats suggests that these signaling molecules may be involved in the pathogenesis of tubulointerstitial nephritis induced by gentamicin.

Glycyrrhizin attenuates endotoxin- induced acute liver injury after partial hepatectomy in rats

Massive hepatectomy associated with infection induces liver dysfunction, or even multiple organ failure and death. Glycyrrhizin has been shown to exhibit anti-oxidant and anti-inflammatory activities. The aim of the present study was to investigate whether glycyrrhizin could attenuate endotoxin-induced acute liver injury after partial hepatectomy. Male Wistar rats (6 to 8 weeks old, weighing 200-250 g) were randomly assigned to three groups of 24 rats each: sham, saline and glycyrrhizin. Rats were injected intravenously with lipopolysaccharide (LPS) 24 h after 70% hepatectomy. Glycyrrhizin, pre-administered three times with 24 h intervals 48 h before hepatectomy, prolonged the survival of rats submitted to partial hepatectomy and LPS injection, compared with saline controls. Glycyrrhizin was shown to attenuate histological hepatic changes and significantly reduced serum levels of aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase, at all the indicated times (6 rats from each were sacrificed 1, 3, 6, and 9 h after LPS injection), compared with saline controls. Glycyrrhizin also significantly inhibited hepatocyte apoptosis by down-regulating the expression of caspase-3 and inhibiting the release of cytochrome C from mitochondria into the cytoplasm. The anti-inflammatory activity of glycyrrhizin may rely on the inhibition of release of tumor necrosis factor-a, myeloperoxidase activity, and translocation of nuclear factor-kappa B into the nuclei. Glycyrrhizin also up-regulated the expression of proliferating cell nuclear antigen, implying that it might be able to promote regeneration of livers harmed by LPS. In summary, glycyrrhizin may represent a potent drug protecting the liver against endotoxin-induced injury, especially after massive hepatectomy.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARγ, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFκB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-α also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-α activation and release, inhibitors of NFκB, specific inhibitors of iNOS and COX-2 activities and PPARγ agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Inflammation induced by increased frequency of intermittent hypoxia is attenuated by tempol administration

The levels of serum inflammatory cytokines and the activation of nuclear factor kappa B (NF-κB) and hypoxia inducible factor-1α (HIF-1α) in heart tissues in response to different frequencies of intermittent hypoxia (IH) and the antioxidant tempol were evaluated. Wistar rats (64 males, 200-220 g) were randomly divided into 6 experimental groups and 2 control groups. Four groups were exposed to IH 10, 20, 30, or 40 times/h. The other 2 experimental groups were challenged with IH (30 times/h) plus tempol, either beginning on day 0 (IH30T0) or on day 29 (IH30T29). After 6 weeks of challenge, serum levels of tumor necrosis factor (TNF)-α, intracellular adhesion molecule (ICAM)-1, and interleukin-10 were measured, and western blot analysis was used to detect NF-κB p65 and HIF-1α in myocardial tissues. Serum levels of TNF-α and ICAM-1 and myocardial expression of NF-κB p65 and HIF-1α were all significantly higher in IH rats than in controls (P<0.001). Increased IH frequency resulted in more significant changes. Administration of tempol in IH rats significantly reduced levels of TNF-α, ICAM-1, NF-κB and HIF-1α compared with the non-tempol-treated group (F=16.936, P<0.001). IH induced an inflammatory response in a frequency-dependent manner. Additionally, HIF-1α and NF-κB were increased following IH administration. Importantly, tempol treatment attenuated this effect.

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein a (C/EBPa) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBPa factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBPa factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBPa factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBPa factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

Plasma cytokine response, lipid peroxidation and NF-κB activation in skeletal muscle following maximum progressive swimming

Our objective was to determine lipid peroxidation and nuclear factor-κB (NF-κB) activation in skeletal muscle and the plasma cytokine profile following maximum progressive swimming. Adult male Swiss mice (N = 15) adapted to the aquatic environment were randomly divided into three groups: immediately after exercise (EX1), 3 h after exercise (EX2) and control. Animals from the exercising groups swam until exhaustion, with an initial workload of 2% of body mass attached to the tail. Control mice did not perform any exercise but were kept immersed in water for 20 min. Maximum swimming led to reactive oxygen species (ROS) generation in skeletal muscle, as indicated by increased thiobarbituric acid reactive species (TBARS) levels (4062.67 ±1487.10 vs 19,072.48 ± 8738.16 nmol malondialdehyde (MDA)/mg protein, control vs EX1). Exercise also promoted NF-κB activation in soleus muscle. Cytokine secretion following exercise was marked by increased plasma interleukin-6 (IL-6) levels 3 h post-exercise (P < 0.05). Interleukin-10 (IL-10) levels were reduced following exercise and remained reduced 3 h post-exercise (P < 0.05). Plasma levels of other cytokines investigated, monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-12 (IL-12), were not altered by exercise. The present findings showed that maximum swimming, as well as other exercise models, led to lipid peroxidation and NF-κB activation in skeletal muscle and increased plasma IL-6 levels. The plasma cytokine response was also marked by reduced IL-10 levels. These results were attributed to exercise type and intensity.

Influence of the dopaminergic system, CREB, and transcription factor-κB on cocaine neurotoxicity

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes.

Inhibitory effect of liposomal quercetin on acute hepatitis and hepatic fibrosis induced by concanavalin A

Immune response plays an important role in the development of hepatic fibrosis. In the present study, we investigated the effects of quercetin on hepatitis and hepatic fibrosis induced by immunological mechanism. In the acute hepatitis model, quercetin (2.5 mg/kg) was injected iv into mice 30 min after concanavalin A (Con A) challenge. Mice were sacrificed 4 or 24 h after Con A injection, and aminotransferase tests and histopathological sections were performed. Treatment with quercetin significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Consistent with this observation, treatment with quercetin markedly attenuated the pathologic changes in the liver. A hepatic fibrosis model was also generated in mice by Con A challenge once a week for 6 consecutive weeks. Mice in the experimental group were treated with daily iv injections of quercetin (0.5 mg/kg). Histopathological analyses revealed that treatment with quercetin markedly decreased collagen deposition, pseudolobuli development, and hepatic stellate cells activation. We also examined the effects of quercetin on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor beta (TGF-β) pathways by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). NF-κB and TGF-β production was decreased after treatment with quercetin, indicating that the antifibrotic effect of quercetin is associated with its ability to modulate NF-κB and TGF-β production. These results suggest that quercetin may be an effective therapeutic strategy in the treatment of patients with liver damage and fibrosis.

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.