High-fat diet blunts activation of the nuclear factor-κB signaling pathway in lipopolysaccharide-stimulated peritoneal macrophages of Wistar rats

ObjectiveThe present study was designed to investigate the effect of a high-fat diet (HFD) on the inflammatory response of peritoneal macrophages.MethodsMale Wistar rats were fed a control diet (n = 12) or an HFD (n = 12) for 12 wk. After euthanasia, peritoneal macrophages were collected and stimulated (or not) with lipopolysaccharide (LPS). Results from the assays using peritoneal macrophages were analyzed with one-way analysis of variance or an equivalent non-parametric test. The level of significance adopted was 0.05.ResultsConsumption of the HFD was associated with significant increases in weight gain and fat depots (P < 0.05). Despite having no influence in systemic markers of inflammation, such as interleukin (IL)-6, tumor necrosis factor-α, and plasminogen activator inhibitor-1, the HFD intake significantly decreased insulin sensitivity, as evaluated by the homeostasis model assessment index (P < 0.05). A decreased production of IL-1β, IL-6, IL-10, and nitric oxide in response to the LPS stimulation was observed in peritoneal macrophages from the HFD group (P < 0.05). Also, in HFD-fed animals, LPS incubation did not increase IL-1β and IL-6 mRNA expression (P < 0.05). These effects were associated with an attenuation of IκB inhibitor kinase-β phosphorylation and nuclear factor-κB activation in response to LPS and with a failure to decrease IκB inhibitor-α expression (P < 0.05).ConclusionChronic consumption of an HFD decreased the LPS-induced inflammatory response of peritoneal macrophages, which was associated with a downregulation of the nuclear factor-κB signaling pathway.     

Zinc-suppressed inflammatory cytokines by induction of A20-mediated inhibition of nuclear factor-κB

ObjectiveChronic generation of inflammatory cytokines and reactive oxygen species are implicated in atherosclerosis, aging, cancers, and other chronic diseases. We hypothesized that zinc induces A20 in premonocytic, endothelial, and cancer cells, and A20 binds to tumor necrosis factor (TNF)-receptor associated factor, and inhibits Iκ kinase-α (IKK-α)/nuclear factor-κB (NF-κB), resulting in downregulation of TNF-α and interleukin-1β (IL-1β).MethodsTo test this hypothesis, we used HL-60, human umbilical vein endothelial cells, and SW480 cell lines under zinc-deficient and zinc-sufficient conditions in this study. We measured oxidative stress markers, inflammatory cytokines, A20 protein and mRNA, A20–FRAF-1 complex, and IKK-α/NF-κB signaling in stimulated zinc-deficient and zinc sufficient cells. We also conducted antisense A20 and siRNA studies to investigate the regulatory role of zinc in TNF-α and IL-1β via A20.ResultsWe found that zinc increased A20 and A20–tumor necrosis factor-receptor associated factor-1 complex, decreased the IKK-α/NF-κB signaling pathway, oxidative stress markers, and inflammatory cytokines in these cells compared with zinc-deficient cells. We confirmed that zinc-induced A20 contributes to downregulation of TNF-α and IL-1β by antisense and short interfering RNA A20 studies.ConclusionOur studies suggest that zinc suppresses generation of NF-κB–regulated inflammatory cytokines by induction of A20.     

Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition

Background and aims

Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine—a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis—is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice.

Methods

Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated.

Results

Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway.

Conclusions

These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.     

Indole-containing fractions of Brassica rapa inhibit inducible nitric oxide synthase and pro-inflammatory cytokine expression by inactivating nuclear factor-κB

In an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the anti-inflammatory potential of the indole-containing fraction from the roots of Brassica rapa (IBR) (Family Brassicaceae) and the underlying mechanisms. Initially, we examined the inhibitory effect of IBR on the production of pro-inflammatory mediators in vitro and then evaluated its in vivo anti-inflammatory effects. IBR was found to concentration-dependently reduce the productions of nitric oxide, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced macrophages. Consistent with these findings, IBR suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS) at the protein level and of iNOS, TNF-α, and IL-6 at the mRNA level. Furthermore, IBR attenuated LPS-induced DNA-binding activities of nuclear factor-κB (NF-κB), and this was accompanied by a parallel reduction in the degradation and phosphorylation of inhibitory κBα and, consequently, by a reduction in the nuclear translocation of the p65 subunit of NF-κB. In addition, treatment with IBR inhibited carrageenan-induced paw edema in rats and acetic acid-induced writing response in mice. Taken together, our data suggest that the expressional inhibitions of iNOS, TNF-α, and IL-6 caused by an attenuation of NF-κB activation are responsible for the anti-inflammatory and antinociceptive activity of IBR.     

Suppression of tumor necrosis factor-α-induced nuclear factor κB activation and aromatase activity by capsaicin and its analog capsazepine

Target-specific drugs, including natural products, offer promise for the amelioration of cancer and other human ailments. Capsaicin, the pungent ingredient present in chilies (Capsicum annuum L.), and capsazepine, a synthetic analog of capsaicin (collectively referred to as vanilloids), are known to possess a variety of pharmacological and physiological properties. In our continuous effort to discover and characterize cancer chemopreventive agents from natural products, we investigated the effect of vanilloids on nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activation using stably transfected 293/NFκB-Luc human embryonic kidney cells induced by treatment with tumor necrosis factor-α (TNFα) and on aromatase activity. Capsaicin and capsazepine blocked TNFα-induced NFκB activation in a dose-dependent manner with 50% inhibitory concentration (IC(50)) values of 0.68 and 4.2 μM, respectively. No significant cytotoxicity was observed at the highest concentrations tested (53.1 μM for capsazepine and 65.5 μM for capsaicin). In addition, these vanilloids inhibited aromatase activity with IC(50) values of 13.6 and 8.8 μM, respectively. Computer-aided molecular docking studies showed docking scores indicative of good binding affinity of vanilloids with aromatase and NFκB. The highly conserved residues for capsaicin and capsazepine binding with NFκB p50 were Ser299 and Ile278 (H-bond 2.81?) and with NFκB p100 were Ser6, Arg82, Val86, Arg90 (H-bond 2.89?), Gly4, and Ser2 (H-bond 2.81?). The amino acids Trp224, Arg435, and Val373 (H-bond 2.80?) were found to be important for the binding of capsaicin and capsazepine with aromatase. Based on these findings, aromatase and NFκB are suggested as valid targets for these compounds; additional investigation of chemopreventive or chemotherapeutic potential is required.     

Dietary supplementation with geranylgeraniol suppresses lipopolysaccharide-induced inflammation via inhibition of nuclear factor-κB activation in rats

Purpose

The isoprenoid geranylgeraniol (GGOH) inhibits nuclear factor-kappa B (NF-κB) activation in the liver, yet the mechanism remains unclear. We investigated the modulation and inhibition of lipopolysaccharide (LPS)-induced NF-κB signaling in the liver of rats fed a GGOH-supplemented diet.

Methods

Rats were fed a diet supplemented with or without GGOH for 10 days. Rats were then intraperitoneally injected with 0.5 mg/kg LPS or vehicle (sterilized saline) and fasted for 18 h. Plasma levels of the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and the liver damage indicators alanine and aspartate aminotransferases (ALT and AST) were assessed. Liver mRNA and proteins were assayed for changes in NF-κB target genes and signal transduction genes.

Results

Rats fed a high-dose, GGOH-supplemented diet showed significantly lower levels of plasma inflammatory cytokines and ALT and AST activities. In the liver, GGOH significantly suppressed NF-κB activation and mRNA expression of its pro-inflammatory target genes. Furthermore, GGOH supplementation substantially suppressed mRNA expression of signal transducer genes upstream of the IκB kinase complex. Western blotting of liver extracts further demonstrated the substantial decrease in total IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), leading to lower signal transduction and inhibition of NF-κB after LPS.

Conclusion

A 10-day, high-dose, GGOH-supplemented diet was sufficient to inhibit LPS-induced inflammation and activation of NF-κB in rat livers. GGOH significantly modulated NF-κB signaling molecules, inhibiting its signal transduction and activation in the liver, thus protecting against liver damage.     

Chemoprevention by white currant is mediated by the reduction of nuclear β-catenin and NF-κB levels in Min mice adenomas

BACKGROUND: Berries are a good natural source of phenolic compounds and many berries or their compounds have been shown to be chemopreventive. White currant is an interesting berry, as it contains low levels of dominant berry phenolics such as ellagic acid, anthocyanins and other flavonoids. AIMS OF THE STUDY: To study if white currant is chemopreventive in an experimental model for intestinal tumorigenesis and further study the effects on beta-catenin and NF-kappaB signaling pathways. METHODS: Multiple intestinal neoplasia (Min) mice were fed an AIN-93G based control diet or a diet containing 10% freeze dried white currant (Ribes x pallidum) for 10 weeks. Cell signaling parameters were analysed from intestinal adenomas and surrounding mucosa by Western blotting and immunohistochemistry. RESULTS: The white currant diet reduced the number of adenomas from 81 (min-max 47-114) to 51 (36-84) in the total small intestine of Min mice (P<0.02). Most of the adenomas develop in the distal part of the small intestine, and in this area white currant reduced the number from 49 to 29.5 (P<0.01) and also the size of the adenomas from 0.88 mm to 0.70 mm (P<0.02). In the colon white currant increased the number of adenomas (0.3+/-0.6 vs. 0.8+/-0.6, mean +/- SD, P<0.05), but did not affect the size. White currant reduced nuclear beta-catenin and NF-kappaB protein levels in the adenomas (P<0.05 and P<0.02, respectively). They were correlated with the size of adenomas (P<0.01). CONCLUSIONS: This study shows that white currant is effective in preventing cancer initiation and progression in the Min mouse. Whether the positive effects are due to its special phenolic composition needs to be studied in more detail.     

Grape peel powder attenuates the inflammatory and oxidative response of experimental colitis in rats by modulating the NF-κB pathway and activity of antioxidant enzymes

Most phenolic compounds and dietary fiber reach intact to the colon. We hypothesized that grape peel powder (GPP), a rich source of these bioactive compounds, modulates inflammatory and oxidative pathways collaborating to attenuate colonic damage in experimental colitis. To determine which bioactive fraction would be responsible for this effect, the aim of this study was to evaluate the effect of dietary supplementation with whole GPP or the isolated bioactive-rich fractions from GPP (extractable polyphenols [EP], dietary fiber and fiber-bound polyphenols [NEP-F], and dietary fiber) in rats with experimental colitis. Colitis was induced by intrarectal injection of 2,4,6-trinitrobenzene sulfonic acid (TNBS) after 15 days of dietary supplementation. EP diet did not reverse the decrease in feed intake and indeed worsened colon shortening and increased spleen weight; however, these effects were not observed for the GPP group, which had polyphenols associated to the matrix besides the extractable ones. Colitis impaired the activity of colonic antioxidant enzymes and increased lipid peroxidation, protein oxidation, nitric oxide (NO) levels, and proinflammatory cytokines in serum and in the colon tissue. GPP restored the activity of antioxidant enzymes and decreased colon oxidation and NO levels. All grape peel fractions reduced the protein expression of the inhibitor of kappa kinase beta and NO levels in colon tissue, but only NEP-F reduced the expression of phosphorylated nuclear factor kappa B and myeloperoxidase activity. Results demonstrated that GPP attenuates inflammatory and oxidative response in TNBS–induced colitis by downregulating the nuclear factor kappa B pathway and upregulating antioxidant enzymes, with NEP-F being the fraction most likely associated to these protective effects.     

Alcohol-fermented soybean increases the expression of receptor-interacting protein 2 and IκB kinase β in mouse peritoneal macrophages

Soybean is a useful component of traditional Korean medicine with well-documented health-promoting effects. We investigated the effects of alcohol-fermented soybean (AFS) on immune function. When AFS treatment was used in combination with recombinant interferon-γ (rIFN-γ), there was a marked cooperative induction of nitric oxide (NO) and tumor necrosis factor (TNF)-α production in mouse peritoneal macrophages. AFS increased the expression of inducible NO synthase mRNA and protein in rIFN-γ-primed macrophages. Treating macrophages with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-κB (NF-κB), decreased the synergistic effects of AFS. In addition, AFS in combination with rIFN-γ increased the phosphorylation of p38 and c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. However, AFS had no effect on phosphorylation of mitogen-activated protein kinases by itself. The p38 inhibitor SB203580 or the JNK inhibitor SP600125 inhibited the AFS-induced NO and TNF-α production. When AFS was used in combination with rIFN-γ, there was a co-operative activation of NF-κB and receptor-interacting protein 2 (Rip2)/IκB kinase (IKK)-β. Our results indicate that AFS increases the production of NO and TNF-α through the activation of Rip2/IKK-β in rIFN-γ-primed macrophages.     

Dietary bitter melon seed increases peroxisome proliferator-activated receptor-γ gene expression in adipose tissue, down-regulates the nuclear factor-κB expression, and alleviates the symptoms associated with metabolic syndrome

The objective of this study was to examine the extent to which bitter melon seed (BMS) alleviates the symptoms associated with metabolic syndrome and elucidate the mechanism by which BMS exerts beneficial effects. Three-month-old female Zucker rats were assigned to following groups: lean control (L-Ctrl), obese control (O-Ctrl), and obese + BMS (O-BMS). The control groups were fed AIN-93M purified rodent diet, and the O-BMS group was fed AIN-93M diet modified to contain 3.0% (wt/wt) ground BMS for 100 days. After 100 days of treatment, BMS supplementation in the obese rats lowered the total serum cholesterol by 38% and low-density lipoprotein-cholesterol levels by about 52% and increased the ratio of serum high-density lipoprotein-cholesterol to total cholesterol compared to the O-Ctrl group. The percentage of total liver lipids was about 32% lower and serum triglyceride levels were 71% higher in the O-BMS group compared to the O-Ctrl group. Serum glucose levels were significantly lowered partly because of the increase in the serum insulin levels in the BMS-based diet groups. BMS supplementation increased the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in the white adipose tissue of the obese rats significantly (P?相似文献   

Suppression of hypoxia-inducible factor-1α contributes to the antiangiogenic activity of red propolis polyphenols in human endothelial cells

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.