Astaxanthin inhibits integrin α5 expression by suppressing activation of JAK1/STAT3 in Helicobacter pylori­stimulated gastric epithelial cells.

Integrins act as cell­matrix adhesion molecules involved in cell attachment to the extracellular matrix and generate signals that respond to cancer metastasis. Integrin α5ß1 is a heterodimer with α5 and ß1 subunits and mediates cell adhesion and migration of cancer cells. Integrins are transcriptionally regulated by the Janus kinase (JAK)/STAT signaling pathways. Our previous study revealed that Helicobacter pylori increased the levels of reactive oxygen species (ROS), which activate JAK1/STAT3 in gastric cancer AGS cells in vitro. Astaxanthin (ASX) has been reported to be an effective antioxidant and anticancer nutrient. The present study investigated whether ASX suppresses H. pylori­induced integrin α5 expression, cell adhesion and migration and whether ASX reduces ROS levels and suppresses phosphorylation of JAK1/STAT3 in gastric cancer AGS cells stimulated with H. pylori. The effect of ASX was determined using a dichlorofluorescein fluorescence assay, western blot analysis, adhesion assay and wound­healing assay in AGS cells stimulated with H. pylori. The results demonstrated that H. pylori increased the expression levels of integrin α5, without affecting integrin ß1, and increased cell adhesion and migration of AGS cells. ASX reduced ROS levels and suppressed JAK1/STAT3 activation, integrin α5 expression, cell adhesion and migration of H. pylori­stimulated AGS cells. In addition, both a JAK/STAT inhibitor, AG490, and an integrin α5ß1 antagonist, K34C, suppressed cell adhesion and migration in H. pylori­stimulated AGS cells. AG490 inhibited integrin α5 expression in AGS cells stimulated with H. pylori. In conclusion, ASX inhibited H. pylori­induced integrin α5­mediated cell adhesion and migration by decreasing the levels of ROS and suppressing JAK1/STAT3 activation in gastric epithelial cells.

Lutein Induces Reactive Oxygen Species-Mediated Apoptosis in Gastric Cancer AGS Cells via NADPH Oxidase Activation.

Disruption of apoptosis leads to cancer cell progression; thus, anticancer agents target apoptosis of cancer cells. Reactive oxygen species (ROS) induce apoptosis by activating caspases and caspase-dependent DNase, leading to DNA fragmentation. ROS increase the expression of apoptotic protein Bax, which is mediated by activation of nuclear factor-κB (NF--κB). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of endogenous ROS, and its activation is involved in apoptosis. Lutein, an oxygenated carotenoid and known antioxidant, is abundant in leafy dark green vegetables, such as spinach and kale, and in yellow-colored foods, such as corn and egg yolk. High amounts of lutein increase ROS levels and exhibit anticancer activity. However, its anticancer mechanism remains unclear. This study aimed to determine whether lutein activates NADPH oxidase to produce ROS and induce apoptosis in gastric cancer AGS cells. Lutein increased ROS levels and promoted the activation of NADPH oxidase by increasing the translocation of NADPH oxidase subunit p47 phox to the cell membrane. It increased NF-κB activation and apoptotic indices, such as Bax, caspase-3 cleavage, and DNA fragmentation, and decreased Bcl-2, cell viability, and colony formation in AGS cells. The specific NADPH oxidase inhibitor ML171, and the known antioxidant N-acetyl cysteine reversed lutein-induced cell death, DNA fragmentation, and NF-κB DNA-binding activity in AGS cells. These results suggest that lutein-induced ROS production is dependent on NADPH oxidase, which mediates NF-κB activation and apoptosis in gastric cancer AGS cells. Therefore, lutein supplementation may be beneficial for increasing ROS-mediated apoptosis in gastric cancer cells.

Astaxanthin Inhibits Oxidative Stress-Induced Ku Protein Degradation and Apoptosis in Gastric Epithelial Cells.

Oxidative stress induces DNA damage which can be repaired by DNA repair proteins, such as Ku70/80. Excess reactive oxygen species (ROS) stimulate the activation of caspase-3, which degrades Ku 70/80. Cells with decreased Ku protein levels undergo apoptosis. Astaxanthin exerts antioxidant activity by inducing the expression of catalase, an antioxidant enzyme, in gastric epithelial cells. Therefore, astaxanthin may inhibit oxidative stress-induced DNA damage by preventing Ku protein degradation and thereby suppressing apoptosis. Ku proteins can be degraded via ubiquitination and neddylation which adds ubiquitin-like protein to substrate proteins. We aimed to determine whether oxidative stress decreases Ku70/80 expression through the ubiquitin-proteasome pathway to induce apoptosis and whether astaxanthin inhibits oxidative stress-induced changes in gastric epithelial AGS cells. We induced oxidative stress caused by the treatment of ß-D-glucose (G) and glucose oxidase (GO) in the cells. As a result, the G/GO treatment increased ROS levels, decreased nuclear Ku protein levels and Ku-DNA-binding activity, and induced the ubiquitination of Ku80. G/GO increased the DNA damage marker levels (γ-H2AX; DNA fragmentation) and apoptosis marker annexin V-positive cells and cell death. Astaxanthin inhibited G/GO-induced alterations, including Ku degradation in AGS cells. MLN4924, a neddylation inhibitor, and MG132, a proteasome inhibitor, suppressed G/GO-mediated DNA fragmentation and decreased cell viability. These results indicated that G/GO-induced oxidative stress causes Ku protein loss through the ubiquitin-proteasome pathway, resulting in DNA fragmentation and apoptotic cell death. Astaxanthin inhibited oxidative stress-mediated apoptosis via the reduction of ROS levels and inhibition of Ku protein degradation. In conclusion, dietary astaxanthin supplementation or astaxanthin-rich food consumption may be effective for preventing or delaying oxidative stress-mediated cell damage by suppressing Ku protein loss and apoptosis in gastric epithelial cells.

Astaxanthin Inhibits Matrix Metalloproteinase Expression by Suppressing PI3K/AKT/mTOR Activation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) increases production of reactive oxygen species (ROS) and activates signaling pathways associated with gastric cell invasion, which are mediated by matrix metalloproteinases (MMPs). We previously demonstrated that H. pylori activated mitogen-activated protein kinase (MAPK) and increased expression of MMP-10 in gastric epithelial cells. MMPs degrade the extracellular matrix, enhancing tumor invasion and cancer progression. The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase B (AKT)/mammalian target of rapamycin (mTOR) is associated with MMP expression. ROS activates PIK3/AKT/mTOR signaling in cancer. Astaxanthin, a xanthophyll carotenoid, shows antioxidant activity by reducing ROS levels in gastric epithelial cells infected with H. pylori. This study aimed to determine whether astaxanthin inhibits MMP expression, cell invasion, and migration by reducing the PI3K/AKT/mTOR signaling in H. pylori-infected gastric epithelial AGS cells. H. pylori induced PIK3/AKT/mTOR and NF-κB activation, decreased IκBα, and induced MMP (MMP-7 and -10) expression, the invasive phenotype, and migration in AGS cells. Astaxanthin suppressed these H. pylori-induced alterations in AGS cells. Specific inhibitors of PI3K, AKT, and mTOR reversed the H. pylori-stimulated NF-κB activation and decreased IκBα levels in the cells. In conclusion, astaxanthin suppressed MMP expression, cell invasion, and migration via inhibition of PI3K/AKT/mTOR/NF-κB signaling in H. pylori-stimulated gastric epithelial AGS cells.

α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku-DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku-DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin-proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.

Lutein inhibits IL­6 expression by inducing PPAR­Î³ activation and SOCS3 expression in cerulein­stimulated pancreatic acinar cells.

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin­6 (IL­6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor­Î³ (PPAR­Î³) and suppressor of cytokine signaling (SOCS) 3 inhibit IL­6 expression by suppressing JAK2/STAT3 in cerulein­stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR­Î³ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR­Î³ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL­6 expression via activation of PPAR­Î³ and SOCS3 expression in cerulein­stimulated AR42J cells. The anti­inflammatory mechanism of lutein was determined using reverse transcription­quantitative PCR, western blot analysis and enzyme­linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR­Î³ antagonist GW9662 or the PPAR­Î³ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR­Î³ activation. The results indicated that lutein increased PPAR­Î³ and SOCS3 levels in unstimulated cells. Cerulein increased phospho­specific forms of JAK2 and STAT3, and mRNA and protein expression of IL­6, but decreased SOCS3 levels in AR42J cells. Cerulein­induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL­6 expression in cerulein­stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL­6 levels via PPAR­Î³­mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.

α­lipoic acid inhibits cerulein/resistin­induced expression of interleukin­6 by activating peroxisome proliferator­activated receptor­Î³ in pancreatic acinar cells.

Cerulein­induced pancreatitis resembles human acute pancreatitis in terms of pathological events, such as enzymatic activation and inflammatory cell infiltration in the pancreas. Cerulein is a cholecystokinin analog that increases levels of reactive oxygen species (ROS) and interleukin­6 (IL­6) expression level in pancreatic acinar cells. Serum levels of resistin, which is secreted from adipocytes, are reportedly higher in patients with acute pancreatitis than in healthy individuals. Previously, it was shown that the adipokine resistin can aggravate the cerulein­induced increase in ROS levels and IL­6 expression level in pancreatic acinar cells. Peroxisome proliferator­activated receptor­gamma (PPAR­Î³) is a key regulator of the transcription and expression of antioxidant enzymes, including heme oxygenase 1 (HO­1) and catalase. α­lipoic acid, a naturally occurring dithiol antioxidant, can prevent cerulein­induced pancreatic damage in rats. In the present study, it was aimed to investigate whether α­lipoic acid can attenuate the cerulein/resistin­induced increase in IL­6 expression and ROS levels via PPAR­Î³ activation in pancreatic acinar AR42J cells. The anti­inflammatory mechanism of α­lipoic acid was determined using reverse transcription­quantitative PCR, western blot analysis, enzyme­linked immunosorbent assay, immunofluorescence staining and fluorometry. Treatment with cerulein and resistin increased ROS levels and IL­6 expression level, which were inhibited by α­lipoic acid in pancreatic acinar cells. α­lipoic acid increased the nuclear translocation and expression level of PPAR­Î³ and the expression levels of its target genes: HO­1 and catalase. The PPAR­Î³ antagonist GW9662 and HO­1 inhibitor zinc protoporphyrin reversed the inhibitory effect of α­lipoic acid on cerulein/resistin­induced increase in ROS and IL­6 levels. In conclusion, α­lipoic acid inhibits the cerulein/resistin­induced increase in ROS production and IL­6 expression levels by activating PPAR­Î³ and inducing the expression of HO­1 and catalase in pancreatic acinar cells.

Lycopene Inhibits IL-6 Expression by Upregulating NQO1 and HO-1 via Activation of Nrf2 in Ethanol/Lipopolysaccharide-Stimulated Pancreatic Acinar Cells.

In alcoholic pancreatitis, alcohol increases gut permeability, which increases the penetration of endotoxins, such as lipopolysaccharides (LPS). LPS act as clinically significant triggers to increase pancreatic damage in alcoholic pancreatitis. Ethanol or LPS treatment increases reactive oxygen species (ROS) production in pancreatic acinar cells. ROS induce inflammatory cytokine production in pancreatic acinar cells, leading to pancreatic inflammation. The nuclear erythroid-2-related factor 2 (Nrf2) pathway is activated as a cytoprotective response to oxidative stress, and induces the expression of NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Lycopene exerts anti-inflammatory and antioxidant effects in various cells. We previously showed that lycopene inhibits NADPH oxidase to reduce ROS and IL-6 levels, and zymogene activation in ethanol or palmitoleic acid-treated pancreatic acinar cells. In this study, we examined whether lycopene inhibits IL-6 expression by activating the Nrf2/NQO1-HO-1 pathway, and reducing intracellular and mitochondrial ROS levels, in ethanol and LPS-treated pancreatic AR42J cells. Lycopene increased the phosphorylated and nuclear-translocated Nrf2 levels by decreasing the amount of Nrf2 sequestered in the cytoplasm via a complex formation with Kelch-like ECH1-associated protein 1 (Keap1). Using exogenous inhibitors targeting Nrf2 and HO-1, we showed that the upregulation of activated Nrf2 and HO-1 results in lycopene-induced suppression of IL-6 expression and ROS production. The consumption of lycopene-rich foods may prevent the development of ethanol and LPS-associated pancreatic inflammation by activating Nrf2-mediated expression of NQO1 and HO-1, thereby decreasing ROS-mediated IL-6 expression in pancreatic acinar cells.

Korean Red Ginseng Extract Inhibits IL-8 Expression via Nrf2 Activation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) causes gastric diseases by increasing reactive oxygen species (ROS) and interleukin (IL)-8 expression in gastric epithelial cells. ROS and inflammatory responses are regulated by the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and the expression of Nrf2 target genes, superoxide dismutase (SOD) and heme oxygenase-1 (HO-1). We previously demonstrated that Korean red ginseng extract (RGE) decreases H. pylori-induced increases in ROS and monocyte chemoattractant protein 1 in gastric epithelial cells. We determined whether RGE suppresses the expression of IL-8 via Nrf2 activation and the expression of SOD and HO-1 in H. pylori-infected gastric epithelial AGS cells. H. pylori-infected cells were treated with RGE with or without ML385, an Nrf2 inhibitor, or zinc protoporphyrin (ZnPP), a HO-1 inhibitor. Levels of ROS and IL-8 expression; abundance of Keap1, HO-1, and SOD; levels of total, nuclear, and phosphorylated Nrf2; indices of mitochondrial dysfunction (reduction in mitochondrial membrane potential and ATP level); and SOD activity were determined. As a result, RGE disturbed Nrf2-Keap1 interactions and increased nuclear Nrf2 levels in uninfected cells. H. pylori infection decreased the protein levels of SOD-1 and HO-1, as well as SOD activity, which was reversed by RGE treatment. RGE reduced H. pylori-induced increases in ROS and IL-8 levels as well as mitochondrial dysfunction. ML385 or ZnPP reversed the inhibitory effect of RGE on the alterations caused by H. pylori. In conclusion, RGE suppressed IL-8 expression and mitochondrial dysfunction via Nrf2 activation, induction of SOD-1 and HO-1, and reduction of ROS in H. pylori-infected cells.

Astaxanthin induces NADPH oxidase activation and receptor­interacting protein kinase 1­mediated necroptosis in gastric cancer AGS cells.

Astaxanthin (ASX), a red­colored xanthophyll carotenoid, functions as an antioxidant or pro­oxidant. ASX displays anticancer effects by reducing or increasing oxidative stress. Reactive oxygen species (ROS) promote cancer cell death by necroptosis mediated by receptor­interacting protein kinase 1 (RIP1) and RIP3. NADPH oxidase is a major source of ROS that may promote necroptosis in some cancer cells. The present study aimed to investigate whether ASX induces necroptosis by increasing NADPH oxidase activity and ROS levels in gastric cancer AGS cells. AGS cells were treated with ASX with or without ML171 (NADPH oxidase 1 specific inhibitor), N­acetyl cysteine (NAC; antioxidant), z­VAD (pan­caspase inhibitor) or Necrostatin­1 (Nec­1; a specific inhibitor of RIP1). As a result, ASX increased NADPH oxidase activity, ROS levels and cell death, and these effects were suppressed by ML171 and NAC. Furthermore, ASX induced RIP1 and RIP3 activation, ultimately inducing mixed lineage kinase domain­like protein (MLKL) activation, lactate dehydrogenase (LDH) release and cell death. Moreover, the ASX­induced decrease in cell viability was reversed by Nec­1 treatment and RIP1 siRNA transfection, but not by z­VAD. ASX did not increase the ratio of apoptotic Bax/anti­apoptotic Bcl­2, the number of Annexin V­positive cells, or caspase­9 activation, which are apoptosis indices. In conclusion, ASX induced necroptotic cell death by increasing NADPH oxidase activity, ROS levels, LDH release and the number of propidium iodide­positive cells, as well as activating necroptosis­regulating proteins, RIP1/RIP3/MLKL, in gastric cancer AGS cells. The results of this study demonstrated the necroptotic effect of ASX on gastric cancer AGS cells, which required NADPH oxidase activation and RIP1/RIP3/MLKL signaling in vitro.

Docosahexaenoic Acid Inhibits Cytokine Expression by Reducing Reactive Oxygen Species in Pancreatic Stellate Cells.

Pancreatic stellate cells (PSCs) are activated by inflammatory stimuli, such as TNF-α or viral infection. Activated PSCs play a crucial role in the development of chronic pancreatitis. Polyinosinic-polycytidylic acid (poly (I:C)) is structurally similar to double-stranded RNA and mimics viral infection. Docosahexaenoic acid (DHA) exhibits anti-inflammatory activity. It inhibited fibrotic mediators and reduced NF-κB activity in the pancreas of mice with chronic pancreatitis. The present study aimed to investigate whether DHA could suppress cytokine expression in PSCs isolated from rats. Cells were pre-treated with DHA or the antioxidant N-acetylcysteine (NAC) and stimulated with TNF-α or poly (I:C). Treatment with TNF-α or poly (I:C) increased the expression of monocyte chemoattractant protein 1 (MCP-1) and chemokine C-X3-C motif ligand 1 (CX3CL1), which are known chemoattractants, and enhanced intracellular and mitochondrial reactive oxygen species (ROS) production and NF-κB activity, but reduced mitochondrial membrane potential (MMP). Increased intracellular and mitochondrial ROS accumulation, cytokine expression, MMP disruption, and NF-κB activation were all prevented by DHA in TNF-α- or poly (I:C)-treated PSCs. NAC suppressed TNF-α- or poly (I:C)-induced expression of MCP-1 and CX3CL1. In conclusion, DHA inhibits poly (I:C)- or TNF-α-induced cytokine expression and NF-κB activation by reducing intracellular and mitochondrial ROS in PSCs. Consumption of DHA-rich foods may be beneficial in preventing chronic pancreatitis by inhibiting cytokine expression in PSCs.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells.

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

Lycopene Inhibits Toll-Like Receptor 4-Mediated Expression of Inflammatory Cytokines in House Dust Mite-Stimulated Respiratory Epithelial Cells.

House dust mites (HDM) are critical factors in airway inflammation. They activate respiratory epithelial cells to produce reactive oxygen species (ROS) and activate Toll-like receptor 4 (TLR4). ROS induce the expression of inflammatory cytokines in respiratory epithelial cells. Lycopene is a potent antioxidant nutrient with anti-inflammatory activity. The present study aimed to investigate whether HDM induce intracellular and mitochondrial ROS production, TLR4 activation, and pro-inflammatory cytokine expression (IL-6 and IL-8) in respiratory epithelial A549 cells. Additionally, we examined whether lycopene inhibits HDM-induced alterations in A549 cells. The treatment of A549 cells with HDM activated TLR4, induced the expression of IL-6 and IL-8, and increased intracellular and mitochondrial ROS levels. TAK242, a TLR4 inhibitor, suppressed both HDM-induced ROS production and cytokine expression. Furthermore, lycopene inhibited the HDM-induced TLR4 activation and cytokine expression, along with reducing the intracellular and mitochondrial ROS levels in HDM-treated cells. These results collectively indicated that the HDM induced TLR4 activation and increased intracellular and mitochondrial ROS levels, thus resulting in the induction of cytokine expression in respiratory epithelial cells. The antioxidant lycopene could inhibit HDM-induced cytokine expression, possibly by suppressing TLR4 activation and reducing the intracellular and mitochondrial ROS levels in respiratory epithelial cells.

Ascorbic Acid Suppresses House Dust Mite-Induced Expression of Interleukin-8 in Human Respiratory Epithelial Cells.

House dust mite (HDM) is one of the significant causes for airway inflammation such as asthma. It induces oxidative stress and an inflammatory response in the lungs through the release of chemokines such as interleukin-8 (IL-8). Reactive oxygen species (ROS) activate inflammatory signaling mediators such as mitogen-activated protein kinases (MAPKs) and redox-sensitive transcription factors including NF-κB and AP-1. Ascorbic acid shows an antioxidant and anti-inflammatory activities in various cells. It ameliorated the symptoms of HDM-induced rhinitis. The present study was aimed to investigate whether HDM could induce IL-8 expression through activation of MAPKs, NF-κB, and AP-1 and whether ascorbic acid could inhibit HDM-stimulated IL-8 expression by reducing ROS and suppressing activation of MAPKs, NF-κB, and AP-1 in respiratory epithelial H292 cells. H292 cells were treated with HDM (5 µg/mL) in the absence or presence of ascorbic acid (100 or 200 µM). HDM treatment increased ROS levels, and activated MAPKs, NF-κB, and AP-1 and thus, induced IL-8 expression in H292 cells. Ascorbic acid reduced ROS levels and inhibited activation of MAPKs, NF-κB and AP-1 and L-8 expression in H292 cells. In conclusion, consumption of ascorbic acid-rich foods may be beneficial for prevention of HDM-mediated respiratory inflammation by suppressing oxidative stress-mediated MAPK signaling pathways and activation of NF-kB and AP-1.

ß-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells.

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell-cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. ß-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether ß-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. ß-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, ß-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.

Lycopene Inhibits Oxidative Stress-Mediated Inflammatory Responses in Ethanol/Palmitoleic Acid-Stimulated Pancreatic Acinar AR42J Cells.

High alcohol intake results in the accumulation of non-oxidative ethanol metabolites such as fatty acid ethyl esters (FAEEs) in the pancreas. High FAEE concentrations mediate pancreatic acinar cell injury and are associated with alcoholic pancreatitis. Treatment with ethanol and the fatty acid palmitoleic acid (EtOH/POA) increased the levels of palmitoleic acid ethyl ester and induced zymogen activation and cytokine expression in pancreatic acinar cells. EtOH/POA induces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production and pancreatic acinar cell injury. Lycopene, a bright-red carotenoid, is a potent antioxidant due to its high number of conjugated double bands. This study aimed to investigate whether lycopene inhibits the EtOH/POA-induced increase in ROS production, zymogen activation, and expression of the inflammatory cytokine IL-6 in EtOH/POA-stimulated pancreatic acinar AR42J cells. EtOH/POA increased the ROS levels, NADPH oxidase and NF-κB activities, zymogen activation, IL-6 expression, and mitochondrial dysfunction, which were inhibited by lycopene. The antioxidant N-acetylcysteine and NADPH oxidase 1 inhibitor ML171 suppressed the EtOH/POA-induced increases in ROS production, NF-κB activation, zymogen activation, and IL-6 expression. Therefore, lycopene inhibits EtOH/POA-induced mitochondrial dysfunction, zymogen activation, and IL-6 expression by suppressing NADPH oxidase-mediated ROS production in pancreatic acinar cells.

Docosahexaenoic Acid Induces Expression of NAD(P)H: Quinone Oxidoreductase and Heme Oxygenase-1 through Activation of Nrf2 in Cerulein-Stimulated Pancreatic Acinar Cells.

Oxidative stress is a major risk factor for acute pancreatitis. Reactive oxygen species (ROS) mediate expression of inflammatory cytokines such as interleukin-6 (IL-6) which reflects the severity of acute pancreatitis. The nuclear factor erythroid-2-related factor 2 (Nrf2) pathway is activated to induce the expression of antioxidant enzymes such as NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) as a cytoprotective response to oxidative stress. In addition, binding of Kelch-like ECH-associated protein 1 (Keap1) to Nrf2 promotes degradation of Nrf2. Docosahexaenoic acid (DHA)-an omega-3 fatty acid-exerts anti-inflammatory and antioxidant effects. Oxidized omega-3 fatty acids react with Keap1 to induce Nrf2-regulated gene expression. In this study, we investigated whether DHA reduces ROS levels and inhibits IL-6 expression via Nrf2 signaling in pancreatic acinar (AR42J) cells stimulated with cerulein, as an in vitro model of acute pancreatitis. The cells were pretreated with or without DHA for 1 h and treated with cerulein (10-8 M) for 1 (ROS levels, protein levels of NQO1, HO-1, pNrf2, Nrf2, and Keap1), 6 (IL-6 mRNA expression), and 24 h (IL-6 protein level in the medium). Our results showed that DHA upregulates the expression of NQO1 and HO-1 in cerulein-stimulated AR42J cells by promoting phosphorylation and nuclear translocation of Nrf2. DHA increased interaction between Keap1 and Nrf2 in AR42J cells, which may increase Nrf2 activity by inhibiting Keap1-mediated sequestration of Nrf2. In addition, DHA-induced expression of NQO1 and HO-1 is related to reduction of ROS and IL-6 levels in cerulein-stimulated AR42J cells. In conclusion, DHA inhibits ROS-mediated IL-6 expression by upregulating Nrf2-mediated expression of NQO1 and HO-1 in cerulein-stimulated pancreatic acinar cells. DHA may exert positive modulatory effects on acute pancreatitis by inhibiting oxidative stress and inflammatory cytokine production by activating Nrf2 signaling in pancreatic acinar cells.

Effect of Astaxanthin on Activation of Autophagy and Inhibition of Apoptosis in Helicobacter pylori-Infected Gastric Epithelial Cell Line AGS.

Helicobacter pylori (H. pylori) infection leads to the massive apoptosis of the gastric epithelial cells, causing gastric ulcers, gastritis, and gastric adenocarcinoma. Autophagy is a cellular recycling process that plays important roles in cell death decisions and can protect cells by preventing apoptosis. Upon the induction of autophagy, the level of the autophagy substrate p62 is reduced and the autophagy-related ratio of microtubule-associated proteins 1A/1B light chain 3B (LC3B)-II/LC3B-I is heightened. AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are involved in the regulation of autophagy. Astaxanthin (AST) is a potent anti-oxidant that plays anti-inflammatory and anti-cancer roles in various cells. In the present study, we examined whether AST inhibits H. pylori-induced apoptosis through AMPK-mediated autophagy in the human gastric epithelial cell line AGS (adenocarcinoma gastric) in vitro. In this study, H. pylori induced apoptosis. Compound C, an AMPK inhibitor, enhanced the H. pylori-induced apoptosis of AGS cells. In contrast, metformin, an AMPK activator, suppressed H. pylori-induced apoptosis, showing that AMPK activation inhibits H. pylori-induced apoptosis. AST inhibited H. pylori-induced apoptosis by increasing the phosphorylation of AMPK and decreasing the phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt) and mTOR in H. pylori-stimulated cells. The number of LC3B puncta in H. pylori-stimulated cells increased with AST. These results suggest that AST suppresses the H. pylori-induced apoptosis of AGS cells by inducing autophagy through the activation of AMPK and the downregulation of its downstream target, mTOR. In conclusion, AST may inhibit gastric diseases associated with H. pylori infection by increasing autophagy through the activation of the AMPK pathway.

Docosahexaenoic acid inhibits zymogen activation by suppressing vacuolar ATPase activation in cerulein-stimulated pancreatic acinar cells.

BACKGROUND: The premature activation of digestive enzyme zymogens within pancreatic acinar cells is an important early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause intrapancreatic zymogen activation and acute pancreatitis. Stimulation of vacuolar ATPase (vATPase) activity is required for zymogen activation in pancreatic acinar cells. Parkin, a multiprotein E3 ubiquitin ligase complex, promotes vATPase ubiquitination and degradation, which inhibits vATPase activity. Docosahexaenoic acid (DHA), an omega-3 fatty acid, exerts anti-inflammatory effects. It is reported to bind to G-protein coupled receptor 120 (GPR120) and GPR40. DHA induces the degradation of certain proteins by activating ubiquitin-proteasome system in various cells. This study aimed to investigate whether DHA induces Parkin and inhibits vATPase activity, resulting in zymogen inactivation in pancreatic acinar AR42J cells stimulated with cerulein, a CCK analog. RESULTS: Cerulein induced the translocation of the cytosolic V1 domain (E subunit) of vATPase to the membrane, which indicated vATPase activation, and zymogen activation in AR42J cells. DHA suppressed the association of the vATPase with membranes, and zymogen activation (increased trypsin activity and amylase release) induced by cerulein. Pretreatment with a GPR120 antagonist AH-7614, a GPR40 antagonist DC260126, or an ubiquitination inhibitor PYR-41 reduced the effect of DHA on cerulein-induced zymogen activation. Treatment with PYR-41 reversed the DHA-induced decrease in vATPase activation in cerulein-treated cells. Furthermore, DHA increased the level of Parkin in membranes of cerulein-treated cells. CONCLUSIONS: DHA upregulates Parkin which inhibits vATPase-mediated zymogen activation, via GPR120 and GPR40, in cerulein-stimulated pancreatic acinar cells.

Inhibitory effect of Korean Red Ginseng extract on DNA damage response and apoptosis in Helicobacter pylori-infected gastric epithelial cells.

BACKGROUND: Helicobacter pylori increases reactive oxygen species (ROS) and induces oxidative DNA damage and apoptosis in gastric epithelial cells. DNA damage activates DNA damage response (DDR) which includes ataxia-telangiectasia-mutated (ATM) activation. ATM increases alternative reading frame (ARF) but decreases mouse double minute 2 (Mdm2). Because p53 interacts with Mdm2, H. pylori-induced loss of Mdm2 stabilizes p53 and induces apoptosis. Previous study showed that Korean Red Ginseng extract (KRG) reduces ROS and prevents cell death in H. pylori-infected gastric epithelial cells. METHODS: We determined whether KRG inhibits apoptosis by suppressing DDRs and apoptotic indices in H. pylori-infected gastric epithelial AGS cells. The infected cells were treated with or without KRG or an ATM kinase inhibitor KU-55933. ROS levels, apoptotic indices (cell death, DNA fragmentation, Bax/Bcl-2 ratio, caspase-3 activity) and DDRs (activation and levels of ATM, checkpoint kinase 2, Mdm2, ARF, and p53) were determined. RESULTS: H. pylori induced apoptosis by increasing apoptotic indices and ROS levels. H. pylori activated DDRs (increased p-ATM, p-checkpoint kinase 2, ARF, p-p53, and p53, but decreased Mdm2) in gastric epithelial cells. KRG reduced ROS and inhibited increase in apoptotic indices and DDRs in H. pylori-infected gastric epithelial cells. KU-55933 suppressed DDRs and apoptosis in H. pylori-infected gastric epithelial cells, similar to KRG. CONCLUSION: KRG suppressed ATM-mediated DDRs and apoptosis by reducing ROS in H. pylori-infected gastric epithelial cells. Supplementation with KRG may prevent the oxidative stress-mediated gastric impairment associated with H. pylori infection.