NADPH Oxidase-mediated Reactive Oxygen Species Production Research Articles

Investigating the role of rotenone on human blood platelets: Molecular insights into abnormal platelet functions in Parkinson's disease.

Parkinson's disease (PD) is a predominant neuromotor disorder characterized by the selective death of dopaminergic neurons in the midbrain. The majority of PD cases are sporadic or idiopathic, with environmental toxins and pollutants potentially contributing to its development or exacerbation. However, clinical PD patients are often associated with a reduced stroke frequency, where circulating blood platelets are indispensable. Although platelet structural impairment is evident in PD, the platelet functional alterations and their underlying molecular mechanisms are still obscure. Therefore, we investigated rotenone (ROT), an environmental neurotoxin that selectively destroys dopaminergic neurons mimicking PD, on human blood platelets to explore its impact on platelet functions, thus replicating PD conditions in vitro. Our study deciphered that ROT decreased thrombin-induced platelet functions, including adhesion, activation, secretion, and aggregation in human blood platelets. As ROT is primarily responsible for generating intracellular reactive oxygen species (ROS), and ROS is a key player regulating the platelet functional parameters, we went on to check the effect of ROT on platelet ROS production. In our investigation, it became evident that ROT treatment resulted in the stimulation of ROS production in human blood platelets. Additionally, we discovered that ROT induced ROS production by augmenting Ca2+ mobilization from inositol 1,4,5-trisphosphate receptor. Apart from this, the treatment of ROT triggers protein kinase C associated NADPH oxidase-mediated ROS production in platelets. In summary, this research, for the first time, highlights ROT-induced abnormal platelet functions and may provide a mechanistic insight into the altered platelet activities observed in PD patients.

Lutein Induces Apoptosis of Gastric Cancer Cells by Producing Reactive Oxygen Species via NADPH Oxidase Activation

ObjectivesApoptosis is well known for its relationship with cancer. Disruption of apoptosis leads to tumor progression, and many cytotoxic anticancer agents induce apoptosis. As a class of phytochemicals, carotenoids are fat-soluble pigments that are abundant in fruits and vegetables and have health-promoting properties, including cancer prevention effects. The anticancer effects of carotenoids are related to their pro-oxidant activities. Lutein is a type of vitamin called a carotenoid. A recent study showed that lutein inhibits the growth of breast cancer cells. The present study was purposed to determine whether lutein induces apoptosis of gastric cancer cells through reactive oxygen species (ROS)-mediated mechanism. MethodsHuman gastric cancer cell line, AGS was treated with lutein. Cell viability was determined by counting the number of viable cells. The protein levels of Bax, Bcl-2, active caspase-3 were examined by western blot analysis. DNA fragmentation was determined by Cell Death Detection ELISA. DCF-DA fluorescence technique was used to quantify the intracellular ROS level. NADPH oxidase activity was measured by lucigenin assay. Colony formation assay was performed and colonies were counted. ResultsLutein decreased cell viability in a dose-dependent manner. Colony formation was also inhibited by lutein. Lutein increased Bax/Bcl-2 ratio, caspase-3 activity, and DNA fragmentation. The intracellular ROS level and NADPH oxidase activity were increased. The decrease in cell viability by lutein was reversed by ML171 (NADPH oxidase 1 specific inhibitor) and N-acetyl cysteine (NAC; antioxidant). ConclusionsLutein induce apoptosis of gastric cancer cells by increasing NADPH oxidase-mediated ROS production. Funding SourcesNo funding sources.

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.

Impact of small RNA RaoN on nitrosative-oxidative stress resistance and virulence of Salmonella enterica serovar Typhimurium.

RaoN is a Salmonella-specific small RNA that is encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. We previously reported that RaoN is induced under conditions of acid and oxidative stress combined with nutrient limitation, contributing to the intramacrophage growth of Salmonella enterica serovar Typhimurium. However, the role of RaoN in nitrosative stress response and virulence has not yet been elucidated. Here we show that the raoN mutant strain has increased susceptibility to nitrosative stress by using a nitric oxide generating acidified nitrite. Extending previous research on the role of RaoN in oxidative stress resistance, we found that NADPH oxidase inhibition restores the growth of the raoN mutant in LPS-treated J774A.1 macrophages. Flow cytometry analysis further revealed that the inactivation of raoN leads to an increase in the intracellular level of reactive oxygen species (ROS) in Salmonella-infected macrophages, suggesting that RaoN is involved in the inhibition of NADPH oxidase-mediated ROS production by mechanisms not yet resolved. Moreover, we evaluated the effect of raoN mutation on the virulence in murine systemic infection and determined that the raoN mutant is less virulent than the wild-type strain following oral inoculation. In conclusion, small regulatory RNA RaoN controls nitrosative-oxidative stress resistance and is required for virulence of Salmonella in mice.

The redox metabolic pathways function to limit Anaplasma phagocytophilum infection and multiplication while preserving fitness in tick vector cells

Aerobic organisms evolved conserved mechanisms controlling the generation of reactive oxygen species (ROS) to maintain redox homeostasis signaling and modulate signal transduction, gene expression and cellular functional responses under physiological conditions. The production of ROS by mitochondria is essential in the oxidative stress associated with different pathologies and in response to pathogen infection. Anaplasma phagocytophilum is an intracellular pathogen transmitted by Ixodes scapularis ticks and causing human granulocytic anaplasmosis. Bacteria multiply in vertebrate neutrophils and infect first tick midgut cells and subsequently hemocytes and salivary glands from where transmission occurs. Previous results demonstrated that A. phagocytophilum does not induce the production of ROS as part of its survival strategy in human neutrophils. However, little is known about the role of ROS during pathogen infection in ticks. In this study, the role of tick oxidative stress during A. phagocytophilum infection was characterized through the function of different pathways involved in ROS production. The results showed that tick cells increase mitochondrial ROS production to limit A. phagocytophilum infection, while pathogen inhibits alternative ROS production pathways and apoptosis to preserve cell fitness and facilitate infection. The inhibition of NADPH oxidase-mediated ROS production by pathogen infection appears to occur in both neutrophils and tick cells, thus supporting that A. phagocytophilum uses common mechanisms for infection of ticks and vertebrate hosts. However, differences in ROS response to A. phagocytophilum infection between human and tick cells may reflect host-specific cell tropism that evolved during pathogen life cycle.

Mycobacterium tuberculosis PPE2 Protein Interacts with p67phox and Inhibits Reactive Oxygen Species Production.

Mycobacterium tuberculosis employs defense mechanisms to protect itself from reactive oxygen species (ROS)-mediated cytotoxicity inside macrophages. In the current study, we found that a secretory protein of M. tuberculosis PPE2 disrupted the assembly of NADPH oxidase complex. PPE2 inhibited NADPH oxidase-mediated ROS generation in RAW 264.7 macrophages and peritoneal macrophages from BALB/c mice. PPE2 interacted with the cytosolic subunit of NADPH oxidase, p67phox, and prevented translocation of p67phox and p47phox to the membrane, resulting in decreased NADPH oxidase activity. Trp236 residue present in the SH3-like domain of PPE2 was found to be critical for its interaction with p67phox Trp236Ala mutant of PPE2 did not interact with p67phox and thereby did not affect ROS generation. M. tuberculosis expressing PPE2 and PPE2-null mutants complemented with PPE2 survived better than PPE2-null mutants in infected RAW 264.7 macrophages. Altogether, this study suggests that PPE2 inhibits NADPH oxidase-mediated ROS production to favor M. tuberculosis survival in macrophages. The findings that M. tuberculosis PPE2 protein is involved in the modulation of oxidative response in macrophages will help us in improving our knowledge of host-pathogen interactions and the application of better therapeutics against tuberculosis.

Overexpression of the dominant negative nbexo70d1 mutantionconfers tolerance to salt stress in transgenic tobacco

The vesicle trafficking process, which involves exocytotic and endocytotic pathways, has been reported to play a role in regulating plant responses to different environmental stresses. The Exo70 protein is important for the localization of the exocyst in the plasma membrane; however, its role in the physiology of stress tolerance is currently unclear. In this study, we characterized NbExo70D1, an Exo70 gene from tobacco (Nicotiana benthamiana). It was shown to have a role in the plant response to salt stress. More specifically, tolerance to salt stress is conferred by the overexpression of the dominant negative nbexo70d1 domain D mutation in transgenic tobacco. In addition, a reduced accumulation of reactive oxygen species (ROS) under salt treatment was observed in the transgenic lines compared to the wild type. Treatment with diphenylene iodonium, an NADPH oxidase inhibitor, resulted in a decrease in salt stress-triggered ROS production in the roots of both wild type tobacco and transgenic tobacco. Furthermore, there was a reduction in NADPH oxidase activity in the transgenic plants under salt treatment, which indicates NbExo70D1 is involved in NADPH oxidase-mediated ROS production. We also characterized the tissue-specific expression patterns of NbExo70D1 during salt stress response by expressing the ProNbExo70D1-β-glucuronidase reporter construct in plants. Importantly, the GFP-NbExo70D1 fusion protein was localized in both the plasma membrane and the cytoplasm; expressing the dominant negative mutation disrupted the interaction between NbExo70D1 protein and the plasma membrane. Overall, our study suggests that Exo70 plays an important role in regulating the production and transmission of ROS as part of a salt stress response in plants.

Vibrio cholerae OmpU Mediates CD36-Dependent Reactive Oxygen Species Generation Triggering an Additional Pathway of MAPK Activation in Macrophages.

OmpU, one of the porins of Gram-negative bacteria Vibrio cholerae, induces TLR1/2-MyD88-NF-κB-dependent proinflammatory cytokine production by monocytes and macrophages of human and mouse origin. In this study, we report that in both the cell types, OmpU-induced proinflammatory responses involve activation of MAPKs (p38 and JNK). Interestingly, we observed that in OmpU-treated macrophages, p38 activation is TLR2 dependent, but JNK activation happens through a separate pathway involving reactive oxygen species (ROS) generation by NADPH oxidase complex and mitochondrial ROS. Further, we observed that OmpU-mediated mitochondrial ROS generation probably depends on OmpU translocation to mitochondria and NADPH oxidase-mediated ROS production is due to activation of scavenger receptor CD36. For the first time, to our knowledge, we are reporting that a Gram-negative bacterial protein can activate CD36 as a pattern recognition receptor. Additionally, we found that in OmpU-treated monocytes, both JNK and p38 activation is linked to the TLR2 activation only. Therefore, the ability of macrophages to employ multiple receptors such as TLR2 and CD36 to recognize a single ligand, as in this case OmpU, probably explains the very basic nature of macrophages being more proinflammatory than monocytes.

Dextromethorphan Attenuates NADPH Oxidase-Regulated Glycogen Synthase Kinase 3β and NF-κB Activation and Reduces Nitric Oxide Production in Group A Streptococcal Infection.

Group A Streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), an antitussive drug, has been demonstrated to efficiently reduce inflammatory responses, thereby contributing to an increased survival rate of GAS-infected mice. However, the anti-inflammatory mechanisms underlying DM treatment in GAS infection remain unclear. DM is known to exert neuroprotective effects through an NADPH oxidase-dependent regulated process. In the present study, membrane translocation of NADPH oxidase subunit p47phox and subsequent reactive oxygen species (ROS) generation induced by GAS infection were significantly inhibited via DM treatment in RAW264.7 murine macrophage cells. Further determination of proinflammatory mediators revealed that DM effectively suppressed inducible nitric oxide synthase (iNOS) expression and NO, tumor necrosis factor alpha, and interleukin-6 generation in GAS-infected RAW264.7 cells as well as in air-pouch-infiltrating cells from GAS/DM-treated mice. GAS infection caused AKT dephosphorylation, glycogen synthase kinase-3β (GSK-3β) activation, and subsequent NF-κB nuclear translocation, which were also markedly inhibited by treatment with DM and an NADPH oxidase inhibitor, diphenylene iodonium. These results suggest that DM attenuates GAS infection-induced overactive inflammation by inhibiting NADPH oxidase-mediated ROS production that leads to downregulation of the GSK-3β/NF-κB/NO signaling pathway.

Importance of NADPH oxidase-mediated redox signaling in the detrimental effect of CRP on pancreatic insulin secretion

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation.

Myeloid Cell-Derived Reactive Oxygen Species Externally Regulate the Proliferation of Myeloid Progenitors in Emergency Granulopoiesis

The cellular mechanisms controlling infection-induced emergency granulopoiesis are poorly defined. Here we found that reactive oxygen species (ROS) concentrations in the bone marrow (BM) were elevated during acute infection in a phagocytic NADPH oxidase-dependent manner in myeloid cells. Gr1(+) myeloid cells were uniformly distributed in the BM, and all c-kit(+) progenitor cells were adjacent to Gr1(+) myeloid cells. Inflammation-induced ROS production in the BM played a critical role in myeloid progenitor expansion during emergency granulopoiesis. ROS elicited oxidation and deactivation of phosphatase and tensin homolog (PTEN), resulting in upregulation of PtdIns(3,4,5)P3 signaling in BM myeloid progenitors. We further revealed that BM myeloid cell-produced ROS stimulated proliferation of myeloid progenitors via a paracrine mechanism. Taken together, our results establish that phagocytic NADPH oxidase-mediated ROS production by BM myeloid cells plays a critical role in mediating emergency granulopoiesis during acute infection.

Activation of NADPH oxidase is essential, but not sufficient, in controlling intracellular multiplication ofBurkholderia pseudomalleiin primary human monocytes

Burkholderia pseudomallei is a Gram-negative intracellular bacterium and the causative agent of melioidosis. Innate immune mechanisms against this pathogen, which might contribute to outcomes of melioidosis, are little known. We demonstrated here that B. pseudomallei could activate NADPH oxidase in primary human monocytes as judged by production of reactive oxygen species (ROS) and p40(phox) phosphorylation after infection. However, as similar to other intracellular bacteria, this bacterium was able to resist and multiply inside monocytes despite being able to activate NADPH oxidase. In the presence of NADPH oxidase inhibitor, diphenyleneiodonium or apocynin, intracellular multiplication of B. pseudomallei was significantly increased, suggesting that NADPH oxidase-mediated ROS production is essential in suppressing intracellular multiplication of B. pseudomallei. Additionally, interferon-γ (IFN-γ)-mediated intracellular killing of B. pseudomallei requires NADPH oxidase activity, even though ROS level was not detected at higher levels in IFN-γ-treated infected monocytes. Altogether, these results imply that the activation of NADPH plays an essential role in suppressing intracellular multiplication of B. pseudomallei in human monocytes, although this enzyme is not sufficient to stop intracellular multiplication.

An S-type anion channel SLAC1 is involved in cryptogein-induced ion fluxes and modulates hypersensitive responses in tobacco BY-2 cells.

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl− and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl− efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl− efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.

Resveratrol Protects Vascular Endothelial Cells from High Glucose–Induced Apoptosis through Inhibition of NADPH Oxidase Activation–Driven Oxidative Stress

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes.

Exposure of Arabidopsis thaliana to Cd or Cu excess leads to oxidative stress mediated alterations in MAPKinase transcript levels

Metals, like cadmium (Cd) and copper (Cu), have the ability to induce the production of reactive oxygen species (ROS) at the cellular level. It is widely known that these ROS can cause irreversible damage to cellular components, like DNA, proteins and lipids. On the other hand, ROS can also act as signaling molecules and in this way they play an essential role in many normal physiological processes, but also in defense responses against stress. ROS signaling in plants uses mitogen-activated protein kinase (MAPK) pathways leading to the transcriptional control of target genes involved in the scavenging or production of ROS. Here, oxidative signaling induced by exposure to excess Cd or Cu was investigated in relation to anti-oxidative defense responses to these metals. Three-week-old Arabidopsis thaliana plants were exposed to environmentally realistic concentrations of Cu and Cd and immediate responses were measured at the level of hydrogen peroxide (H2O2) content, lipid peroxidation and transcript levels of genes involved in ROS homeostasis and signaling. Our findings show immediate (after 2h exposure) effects in the roots following Cu exposure, whereas effects in the leaves were generally more delayed. Effects of Cd exposure in leaves and roots were observed only after 24h exposure. On one hand, exposure of roots to Cu leads via activation of NADPH oxidases and Fenton reactions to H2O2 production that can induce MAPK and oxylipin signaling to control the cellular redox status. On the other hand, conversion of H2O2 to the more damaging hydroxyl radical by Fenton and Haber–Weiss reactions can initiate lipid peroxidation leading to membrane damage. In roots exposed to elevated Cd concentrations only oxidative signaling was initiated, possibly via NADPH oxidase-mediated ROS production. In leaves, time-dependent activation of MAPK and oxylipin signaling was seen after exposure to both metals, Cu or Cd, independent of changes in H2O2 content.

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22phox and p47phox expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.

The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host-defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a non-selective and redox-sensitive cation channel, inhibits ROS production in phagocytic cells and prevents endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) showed an increased inflammatory signature and decreased survival compared to controls. TRPM2 functions by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. Since ROS also activates TRPM2, our findings establish a negative feedback mechanism inactivating ROS production through inhibition of the membrane potential-sensitive NADPH oxidase.

2152 OXALATE STIMULATES THE PRODUCTION OF FIBRONECTIN VIA TGF- β1-DEPENDENT ACTIVATION OF NADPH OXIDASE IN RENAL EPITHELIAL CELLS

You have accessJournal of UrologyStone Disease: Basic Research1 Apr 20112152 OXALATE STIMULATES THE PRODUCTION OF FIBRONECTIN VIA TGF- β1-DEPENDENT ACTIVATION OF NADPH OXIDASE IN RENAL EPITHELIAL CELLS Vijayalakshmi Thamilselvan, Mani Menon, and Sivagnanam Thamilselvan Vijayalakshmi ThamilselvanVijayalakshmi Thamilselvan Detroit, MI More articles by this author , Mani MenonMani Menon Detroit, MI More articles by this author , and Sivagnanam ThamilselvanSivagnanam Thamilselvan Detroit, MI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2363AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hyperoxaluria is one of the major risk factors for calcium oxalate nephrolithiasis. We have previously demonstrated that oxalate induces reactive oxygen species (ROS) mediated cell injury via NADPH oxidase activation in renal epithelial cells. Generation of Inflammatory mediators and excessive accumulation of extracellular matrix proteins in the kidneys contributes to the renal fibrosis. Since there is a strong association between hyperoxaluria and tubulointerstitial scarring, we investigated whether oxalate-induced TGF- β1 can stimulate the production of fibronectin (FN) via NADPH oxidase mediated ROS production in renal epithelial cells. METHODS Confluent monolayers of LLC-PK1 cells pretreated with inhibitors of NADPH oxidase (DPI or apocynin) or neutralizing antibody to TGF- β1 were exposed to 0.75 mM oxalate for different time periods. Recombinant TGF- β1 was used as a positive control. ROS generation and LDH release were determined. Expression of TGF- β1 mRNA was analyzed by RT-PCR. Fibronectin and TGF- β1 levels were measured in culture supernatants by ELISA. TGF- β1 and FN protein expression were determined by western blot analysis. NADPH oxidase activity was determined by SOD-inhibitable cytochrome c reduction assay. RESULTS Oxalate time dependently stimulated FN expression and secretion in LLC-PK1 cells. TGF- β1 mRNA and protein expression were augmented by oxalate exposure. Oxalate significantly increased NADPH oxidase activity in LLC-PK1 cells. Treatment with TGF β1 neutralizing antibody significantly inhibited oxalate -induced NADPH oxidase activity, ROS production and LDH release. The stimulatory effect of oxalate on FN secretion was inhibited by neutralizing antibodies against TGF- β1. Recombinant TGF- β1 dose dependently increased ROS generation, LDH release and FN expression and secretion in LLC-PK1 cells. In addition, pretreatment with NADPH oxidase inhibitors significantly decreased oxalate or recombinant TGF- β1 induced ROS generation, FN expression and secretion. CONCLUSIONS Our findings demonstrate that TGF- β1-dependent activation of NADPH oxidase mediated ROS play an important role in oxalate-induced FN accumulation and development of renal interstitial fibrosis, and suggest that ROS signaling pathways may represent a novel therapeutic target to prevent the progression of tubulointerstitial fibrosis in patients with kidney stones. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e861-e862 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Vijayalakshmi Thamilselvan Detroit, MI More articles by this author Mani Menon Detroit, MI More articles by this author Sivagnanam Thamilselvan Detroit, MI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Prolonged Exposure of Cortical Neurons to Oligomeric Amyloid-β Impairs NMDA Receptor Function Via NADPH Oxidase-Mediated ROS Production: Protective Effect of Green Tea (-)-Epigallocatechin-3-Gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.

Importance of lipid rafts for lysophosphatidylcholine-induced caspase-1 activation and reactive oxygen species generation

Lipid rafts play an important role in regulating cellular processes and functions. Here, we demonstrate that in microglia stimulated with the pro-inflammatory lipid lysophosphatidylcholine (LPC), caspase-1 activation and NADPH oxidase activity depend on intact lipid rafts. Disruption of lipid rafts with methyl-β-cyclodextrin, fumonisin B1 or nystatin prevented LPC-stimulated caspase-1 activation and reactive oxygen species (ROS) production, whereas LPC-induced Na + influx remained unaffected. Since ROS regulate caspase-1 activity in LPC-stimulated microglia, the effects of lipid raft-disrupting agents on caspase-1 activation can be related to their inhibition of NADPH oxidase-mediated ROS production.