Shirish Barve

Eicosapentaenoic Acid Prevents LPS-Induced TNF-α Expression by Preventing NF-κB Activation

Background: Many studies have shown that fish oil supplementation inhibits tumor necrosis factor-α (TNF-α) production in mice and human subjects; however, the mechanisms remain unclear. Nuclear factor-κB (NF-κB) is a transcription factor that plays an important role in controlling the expression of pro-inflammatory genes including TNF-α. Activation of NF-κB has been shown to mediate the maximal expression of TNF-α in human monocytes. NF-κB is kept in an inactive form in the cytoplasm by IκB, the inhibitory subunit of NF-κB complex. Phosphorylation and subsequent degradation of IκB lead to NF-κB activation. Objectives: The effect of eicosapentaenoic acid (EPA), a major n-3 fatty acid in fish oil, on the lipopolysaccharide (LPS)-induced expression of TNF-α and activation of NF-κB were investigated. The mechanism underlying EPA modulation of NF-κB activation was also studied. Methods: Human monocytic THP-1 cells were pre-incubated with EPA and stimulated with LPS. The levels of secreted TNF-α were determined by ELISA. The DNA binding activity of NF-κB was analyzed by EMSA. The degradation and phosphorylation of IκB-α were examined by Western blot analysis. Results: TNF-α production and expression induced by LPS were significantly decreased in cells pre-incubated with EPA. LPS-induced NF-κB activation, translocation of p65 subunit to the nucleus, phosphorylation and degradation of IκB-α were partially prevented by EPA. Conclusions: The results suggest that suppression of the TNF-α expression by EPA is partly attributed to its inhibitory effect on NF-κB activation. EPA appears to prevent NF-κB activation by preventing the phosphorylation of IκB-α.

Zinc attenuates tumor necrosis factor-mediated activation of transcription factors in endothelial cells

OBJECTIVE The objective of the study was to test the hypothesis that zinc can protect against endothelial dysfunction by interfering with oxidative stress-mediated cellular signaling and subsequent inhibition of an endothelial cell inflammatory response. Our approach was to compare alterations on molecular and biochemical levels with changes in endothelial barrier function that occur in zinc deficient conditions. METHODS To investigate our hypothesis, endothelial cells were exposed to zinc deficient media for 2 to 10 days to deplete cellular zinc stores. Following this, half of the groups received zinc supplementation (9.2 microM) for 48 hours. The other half served as zinc deficient controls. These cells were then challenged with tumor necrosis factor-alpha (TNF) for varying time periods. Nuclear extracts were prepared from cells and analyzed for nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding. Media from cells were analyzed for interleukin 8 (IL-8) production, and cellular proteins were determined. RESULTS Zinc supplementation resulted in a 74% increase in cellular zinc content. It was also shown that a 1.5 hour exposure to TNF (100 U/mL medium) significantly increased NF-kappa B and AP-1 binding, which was lowered considerably when cells were supplemented with physiological levels of zinc. Zinc supplementation also caused a marked attenuation in IL-8 expression by endothelial cells in response to TNF-mediated cell activation. DISCUSSION Our previous data clearly show that zinc is a protective and critical nutrient for maintenance of endothelial integrity. The present data suggest that zinc may protect against cytokine-mediated activation of oxidative stress sensitive transcription factors, upregulation of inflammatory cytokines and endothelial cell dysfunction. This may have implications in understanding mechanisms of atherosclerosis.

Antioxidants attenuate nuclear factor-kappa B activation and tumor necrosis factor-alpha production in alcoholic hepatitis patient monocytes and rat Kupffer cells, in vitro

UNLABELLED There is increased tumor necrosis factor-alpha (TNF) activity in alcoholic hepatitis (AH). OBJECTIVES To examine the effects of antioxidants and glutathione enhancing agents on NF-kappaB activation and TNF production in Kupffer cells and monocytes. DESIGN AND METHODS Isolated rat Kupffer cells and peripheral blood monocytes from AH patients were treated in vitro. NF-kappaB activation was assessed by electrophoretic mobility shift assay and TNF was measured in cell culture supernatants. RESULTS Monocytes from AH patients had greater TNF production compared to normal volunteers. Pretreatment with antioxidants or gluathione enhancing agents inhibited TNF production and NF-kappaB activation in both monocytes from normal and AH patients as well as in rat Kupffer cells. CONCLUSIONS There may be a therapeutic role for antioxidants or glutathione enhancing agents in disease states with increased TNF activity such as AH.

Fish oil suppressed cytokines and nuclear factor-kappaB induced by murine AIDS virus infection

Abstract The effects of dietary fish oil on pro-inflammatory cytokines, nuclear factor-kappaB (NF-κB) and viral replication were studied in a murine model of AIDS. Sixty-four female C57BL/6 mice were divided into two dietary groups and fed either a corn oil or fish oil diet. After 4 weeks, each group was divided into two subgroups. One subgroup from each group was infected with LP-BM5 murine leukemia virus. At 4 and 10 weeks post-infection, one-half of the mice were sacrificed. Blood samples, and the spleens and livers were collected. The results demonstrated that when compared to corn oil, fish oil partially suppressed the virus-induced overproduction of serum IgG and IgM and the in vitro LPS-stimulated production of leukotriene B 4 , tumor necrosis factor-α and interleukin-1β in splenocytes. Fish oil also partially suppressed NF-κB activation and viral replication in both tissues when compared to corn oil.

Cytokine Production by CAPAN-1 and CAPAN-2 Cell Lines

Recently, there has been a great deal of interest in the role of cytokines in acute pancreatitis. Serum levels of IL-1, IL-6, and TNF-α have been demonstrated to be elevated in acute pancreatitis. We hypothesized that cytokines may be produced primarily by pancreatic parenchymal cells. Reasoning that ductal epithelium is the cell type most likely to be exposed to noxious stimuli in common causes of pancreatitis, such as ERCP and passage of a gallstone, we examined the response of well differentiated pancreatic ductal adenocarcinoma cell lines to stimuli known to stimulate cytokine production in other cells. CAPAN-1 and CAPAN-2 cells were incubated with endotoxin or TNF-α. The supernatant was assayed for production of IL-1, IL-6, and IL-8 by ELISA. The cells were assayed for activation of the transcription factor NF-κB by electrophoretic mobility shift assay. There was no detectable production of IL-1 by either cell line. CAPAN-1 cells had concentration-dependent production of IL-6 and IL-8 in response to both endotoxin and TNF-α. CAPAN-2 cells had concentration-dependent production of IL-6 and IL-8 in response to TNF-α. They had low level expression of IL-8 that was unaffected by any concentration of LPS, and no detectable production of IL-6 in response to LPS. These findings suggest that pancreatic duct cells may take an active part in the pathogenesis of acute pancreatitis through the production of cytokines.

Antioxidants Inhibit Cytokine Production and Suppress NF-κB Activation in CAPAN-1 and CAPAN-2 Cell Lines

Interleukin (IL) -6 and IL-8 are cytokines that have been shown to play a role in several pancreatic diseases, including acute pancreatitis, chronic pancreatitis, and pancreatic adenocarcinoma. Previously, we have demonstrated that tumor necrosis factor-α (TNF-α) and gram-negative bacterial lipopolysaccharide stimulate production of IL-6 and IL-8 and activation of the transcription factor NF-κB in the well-differentiated pancreatic ductal adenocarcinoma cell lines CAPAN-1 and CAPAN-2. In these studies we have examined the effect of chain-breaking and glutathione-enhancing antioxidants on NF-κB activation and production of IL-6 and IL-8 in these cell lines. Generally, suppression of NF-κB activation correlated well with inhibition of IL-6 and IL-8 secretion. In the CAPAN-2 cell line, antioxidants inhibited both NF-κB activation and IL-6 and IL-8 secretion. In the CAPAN-1 cell line, antioxidants generally failed to suppress both NF-κB activation and IL-6 and IL-8 secretion. The single exception was the chain-breaking antioxidant butylated hydroxyanisole (BHA), which markedly inhibited IL-6 and IL-8 secretion, but had no effect on NF-κB activation. These findings may have implications for the treatment of acute and chronic pancreatitis and pancreatic cancer.