Reduced Reactive Oxygen Species Levels Research Articles

β-carotene Inhibits Expression of c-Myc and Cyclin E in Helicobacter pylori-infected Gastric Epithelial Cells.

BackgroundHelicobacter pylori infection is a major risk factor in the development of gastric cancer. H. pylori infection of gastric epithelial cells increases the levels of reactive oxygen species (ROS), activates oncogenes, and leads to β-catenin-mediated hyper-proliferation. β-Carotene reduces ROS levels, inhibits oxidant-mediated activation of inflammatory signaling and exhibits anticancer properties. The present study was carried out to determine if β-carotene inhibits H. pylori-induced cell proliferation and the expression of oncogenes c-myc and cyclin E by reducing the levels of β-catenin and phosphorylated glycogen synthase kinase 3β (p-GSK3β).MethodsGastric epithelial AGS cells were pre-treated with β-carotene (5 and 10 μM) for 2 hours prior to H. pylori infection and cultured for 6 hours (for determination of the levels of p-GSK3β, GSK3β, and β-catenin) and 24 hours (for determination of cell viability and protein levels of c-myc and cyclin E). Cell viability was determined by the MTT assay and protein levels were determined via western blot-based analysis.Resultsβ-Carotene inhibited H. pylori-induced increases in the percentage of viable cells, phosphorylated GSK3β (p-GSK3β), and the levels of β-catenin, c-myc and cyclin E.Conclusionsβ-Carotene inhibits H. pylori-induced hyper-proliferation of gastric epithelial cells by suppressing β-catenin signaling and oncogene expression.

P-Chloro-diphenyl diselenide attenuates plasma lipid profile changes and hepatotoxicity induced by nonionic surfactant tyloxapol in rats

Tyloxapol is a nonionic surfactant oligomer inductor of dyslipidemia, which in turn is a risk factor for liver damage. Selenium-based compounds have emerged as promising therapeutic candidates for treating different experimental disorders. This study investigated the effects of p-chloro-diphenyl diselenide (p-ClPhSe)2 on toxicity induced by Tyloxapol in rats. Plasma lipid profile, hepatic functionality and oxidative stress parameters were evaluated in adult male Wistar rats treated with (p-ClPhSe)2 (10 mg/kg; oral administration by gavage) for seven days and exposed to a single Tyloxapol injection (400 mg/kg; intraperitoneal route) 30 min after the last (p-ClPhSe)2 treatment. Tyloxapol exposure increased the plasma levels of total cholesterol, triacylglycerol, non-HDL-cholesterol and the calculated cardiac risk index (CRI). The plasma activities of alanine and aspartate aminotransferase (ALT and AST, liver function markers) were increased in rats exposed to Tyloxapol, which demonstrates a hepatic lipotoxicity. In the liver, reactive oxygen species (ROS) content was enhanced and the non-protein sulfhydryl (NPSH) levels were decreased by Tyloxapol. The data revealed that repeated treatment with (p-ClPhSe)2 reduced plasma lipid alterations and hepatotoxicity induced by Tyloxapol. Although (p-ClPhSe)2 did not reduce ROS levels increased by Tyloxapol, it increased NPSH content in the liver. Pearson’s correlation coefficient revealed a positive relationship between the levels of hepatic NPSH and plasma HDL. HDL is known by eliciting antioxidant activity; therefore, the improvement in HDL function could be suggested as a therapeutic target. In conclusion, the results demonstrate the protective effects of (p-ClPhSe)2 on the hepatic lipotoxicity induced by Tyloxapol in rats.

Melatonin protects against defects induced by malathion during porcine oocyte maturation.

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.

Glycyrrhizin protects IGFBP-3 knockout mice from retinal damage

We previously reported that insulin-like growth factor binding protein 3 (IGFBP-3) knockout (KO) mice have neuronal and vascular damage to the retina. We also reported that glycyrrhizin, a high mobility growth factor binding protein 1 (HMGB1) inhibitor, is protective to the diabetic retina. In this study, we investigated whether glycyrrhizin could reduce neuronal and vascular damage in the IGFBP-3 KO mouse retina. We used measurements of retinal thickness, cell number in the ganglion cell layer, degenerate capillaries, reactive oxygen species (ROS) and protein levels of HMGB1, tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1β) and sirtuin 1 (SIRT1) to determine whether glycyrrhizin could protect the retina. Data show that glycyrrhizin in the drinking water was effective in reducing neuronal damage at 2 months and vascular damage at 6 months. Glycyrrhizin reduced ROS levels at 6 months, and reduced levels of HMGB1, TNFα, and IL-1β at both 2 and 6 months. Taken together, the data suggest that glycyrrhizin is protective to the retina of IGFBP-3 KO mice through anti-inflammatory mechanisms.

Overexpression of microRNA‑155 alleviates palmitate‑induced vascular endothelial cell injury in human umbilical vein endothelial cells by negatively regulating the Wnt signaling pathway.

The present study aimed to investigate the effect of microRNA155 (miR-155) on palmitate-induced vascular endothelial cell injury in human umbilical vein endothelial cells (HUVECs) via the regulation of the Wnt signaling pathway. HUVECs were treated with 0.1 mM palmitate. After transfection with mimic, antagomir or the Wnt pathway inhibitor XAV939, HUVECs were divided into six treatment groups: Control, palmitate, mimic + palmitate, mimic + palmitate + XAV939, antagomir + palmitate, antagomir + palmitate + XAV939. miR-155 expression was detected using reverse transcription-quantitative PCR. The expression levels of the Wnt signaling pathway-related factors β-catenin and Cyclin D, and the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were detected using western blot analysis. MTT and Transwell assays were used to detect the proliferation and migration of cells, respectively. Apoptosis and reactive oxygen species (ROS) levels were determined using flow cytometry. The localization of β-catenin in cells was determined by immunofluorescence. Palmitate reduced the expression level of miR-155 in HUVECs. In palmitate-induced HUVECs, overexpression of miR-155 promoted cell proliferation, reduced the levels of apoptosis, downregulated IL-6 and TNF-α expression, and reduced ROS levels. Inhibition of the Wnt signaling pathway enhanced the anti-endothelial cell injury effect caused by the overexpression of miR-155 in palmitate-induced HUVECs, thereby promoting proliferation, reducing apoptosis, downregulating the levels of inflammatory factors and reducing ROS levels. In summary, overexpression of miR-155 inhibited palmitate-induced apoptosis, ROS production and levels of inflammatory factors, and promoted the proliferation of HUVECs by negatively regulating the Wnt signaling pathway. This present study provides a theoretical basis for the prevention and treatment of cardiovascular diseases associated with endothelial cell injury.

CDNA Library for Mining Functional Genes in Sedum alfredii Hance Related to Cadmium Tolerance and Characterization of the Roles of a Novel SaCTP2 Gene in Enhancing Cadmium Hyperaccumulation.

Heavy metal contamination presents serious threats to living organisms. Functional genes related to cadmium (Cd) hypertolerance or hyperaccumulation must be explored to enhance phytoremediation. Sedum alfredii Hance is a Zn/Cd cohyperaccumulator exhibiting abundant genes associated with Cd hypertolerance. Here, we developed a method for screening genes related to Cd tolerance by expressing a cDNA-library for S. alfredii Hance. Yeast functional complementation validated 42 of 48 full-length genes involved in Cd tolerance, and the majority of them were strongly induced in roots and exhibited diverse expression profiles across tissues. Coexpression network analysis suggested that 15 hub genes were connected with genes involved in metabolic processes, response to stimuli, and metal transporter and antioxidant activity. The functions of a novel SaCTP2 gene were validated by heterologous expression in Arabidopsis, responsible for retarding chlorophyll content decrease, maintaining membrane integrity, promoting reactive oxygen species (ROS) scavenger activities, and reducing ROS levels. Our findings suggest a highly complex network of genes related to Cd hypertolerance in S. alfredii Hance, accomplished via the antioxidant system, defense genes induction, and the calcium signaling pathway. The proposed cDNA-library method is an effective approach for mining candidate genes associated with Cd hypertolerance to develop genetically engineered plants for use in phytoremediation.

Tomato Root Growth Inhibition by Salinity and Cadmium Is Mediated By S-Nitrosative Modifications of ROS Metabolic Enzymes Controlled by S-Nitrosoglutathione Reductase.

S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanum habrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.

Antioxidant Defenses: A Context-Specific Vulnerability of Cancer Cells.

Reactive oxygen species (ROS) are well known for their capacity to cause DNA damage, augment mutagenesis, and thereby promote oncogenic transformation. Similarly, agents that reduce ROS levels (antioxidants) are frequently thought to have anti-cancer properties given their propensity to minimize DNA damage and mutagenesis. However, numerous clinical studies focused on antioxidants suggest that this is a facile premise and that antioxidant capacity can be important for cancer cells in a similar fashion to normal cells. As a consequence of this realization, numerous laboratories have been motivated to investigate the biological underpinnings explaining how and when antioxidant activity can potentially be beneficial to cancer cells. Relatedly, it has become clear that the reliance of cancer cells on antioxidant activity in certain contexts represents a potential vulnerability that could be exploited for therapeutic gain. Here, we review some of the recent, exciting findings documenting how cancer cells utilized antioxidant activity and under what circumstances this activity could represent an opportunity for selective elimination of cancer cells.

5-aminolevolinic acid enhances sunflower resistance to Orobanche cumana (Broomrape)

The root holoparasitic weed sunflower broomrape (Orobanche cumana Wallr.) specifically infects sunflowers (Helianthus annuus L.) and causes significant loss of their quality and production. This experiment was designed to evaluate the ameliorative effects of 5-aminolevolinic acid (ALA) on susceptible sunflower (cv. TK0409) under O. cumana stress. Sunflower seeds were pretreated at five concentrations (0, 5, 10, 15, or 20 mg L−1) of ALA, and then the germinated seeds were grown in O. cumana infested and non-infested soil. Results revealed that malondialdehyde (MDA) and reactive oxygen species (ROS) were significantly increased in both roots and leaves along with O. cumana penetration, suggesting oxidative bursts in sunflower, whereas exogenously applied ALA significantly reduced the levels of MDA and ROS by inducing anti- and non-antioxidant enzyme activities under O. cumana stress. Besides, ALA obviously recovered the ultrastructural damages of broomrape-invaded leaf mesophyll and root tip cells in sunflower. Moreover, application of ALA significantly decreased the expressions of xyloglucan endotransglucosylase/hydrolase related genes (XTH6 and XTH9) in sunflower under O. cumana stress compared with the O. cumana infected plants alone, indicating that the increased resistance of sunflower might be related to hypersensitive reactions which were activated by ALA. All the applied dosages of ALA had the effects of enhancing the resistance of sunflower against O. cumana, and the most effective mitigation was observed at 10 mg L−1 ALA pretreatment. In conclusion, ALA enhanced sunflower resistance to broomrape by promoting antioxidant defence system, reducing ROS levels, decreasing cellular damage and regulating the expressions of stress related genes.

Glutathione peroxidase 3 (GPX3) suppresses the growth of melanoma cells through reactive oxygen species (ROS)-dependent stabilization of hypoxia-inducible factor 1-α and 2-α.

In this study, we aimed to explore the mechanism of glutathione peroxidase 3 (GPX3) in the growth of malignant melanoma (MM) cells by hypoxia-inducible factor-1α (HIF1-α) and HIF2-α regulating the metabolism through reactive oxygen species (ROS). The messenger RNA and protein expression of GPX3, HIF1-α, HIF2-α in tissues, and cell lines were measured by reverse transcription-quantitative PCR and Western blot analysis. A375 cells were transfected with GPX3 overexpression plasmid, small interfering RNA (siRNA) targeting GPX3, or siRNA targeting HIF1-α/HIF2-α to upregulate or downregulate the expression of GPX3 or HIF1-α/HIF2-α. The effects of H2 O2 and N-acetylcysteine (NAC) on the levels of HIF1-α and HIF2-α after overexpression of GPX3 were studied. The cell viability was detected by Cell Counting Kit-8. The levels of ROS, glucose uptake and lactic acid production, oxidative phosphorylation, and glycolysis of cells were measured for assessment of cellular metabolism. The expression of GPX3 decreased, while ROS, HIF1-α, and HIF2-α increased in MM tissues and cells. Overexpression of GPX3 inhibited the viability of MM cells and the growth of melanoma xenografts. The overexpression of GPX3 reduced the glucose uptake, extracellular lactic acid content, and extracellular acidification rate and increased the oxygen consumption rate level. Overexpression of GPX3 could reduce the levels of HIF1-α and HIF2-α, which could regulate metabolic levels. GPX3 reduced ROS level in MM to inhibit HIF1-α and HIF2-α. The addition of H2 O2 increased while NAC reduced the protein levels of HIF1-α and HIF2-α in the cells overexpressing GPX3. Our study demonstrates that GPX3 inhibits the growth of MM cells through its inhibitory effect on cell metabolic disorder by inhibiting HIF1-α via regulating ROS.

Protective Effects of Trimetazidine in Retarding Selenite-Induced Lens Opacification

ABSTRACTPurpose: Cataracts are the leading cause of vision loss worldwide, and the over-production of reactive oxygen species (ROS) is the foremost underlying cause of cataracts. Reducing ROS levels can efficiently prevent lens opacification, as evidenced by many studies. Here, we inhibited ROS overproduction with trimetazidine (TMZ), which is an antioxidant, to explore the therapeutic effects of TMZ and the mechanism of lens opacification.Materials and methods: Sodium selenite-induced cataract formation resulted in a significant loss of lens transparency. This effect could be efficiently rescued by TMZ, which was further found to be an inhibitor of ROS production, as determined by assaying oxidative stress-related parameters (SOD activity, MDA, ·OH and H2O2 levels) during cataract formation. The experimental protocols involving animal research were approved by the Animal Care and Ethics Committee of Wenzhou Medical University and conducted according to the Association for Research in Vision and Ophthalmology under the guidelines of the Animal Welfare Act (SYXK 2015–0009).Results: Our study found that TMZ can retard the onset and progression of lens opacification in vivo in experiments using Sprague-Dawley (SD) suckling rats and can rescue the morphology of HLEB3 cells in vitro. The flow cytometry and DNA fragmentation assays showed that TMZ could prevent sodium selenite-induced apoptosis. The western blot analysing showed that the levels of apoptosis-associated Bcl-2 and Nrf2 were dramatically decreased following the sodium selenite treatment. In addition, the bisulfate DNA sequencing revealed that the demethylation of CpGs in the promoter region of Keap1 was stimulated, and that this demethylation could be inhibited by TMZ by rescuing the Nrf2 expression level.Conclusions: Our findings indicate that the antioxidant TMZ strongly reduces ROS production, which ultimately delays the progression of cataract formation, suggesting that treatment with TMZ represents a novel, promising antioxidant protection to retard cataract formation.

Paeoniflorin protects spiral ganglion neurons from cisplatin-induced ototoxicity: Possible relation to PINK1/BAD pathway.

The objective of this study was to elucidate whether paeoniflorin (PF) exerted an effect on cisplatin‐induced spiral ganglion neuron (SGN) damage, with special attention given to the role of PINK1/BAD pathway in this process. Middle cochlear turn culture and C57BL/6 mice were utilized to identify the character of PF in vitro and in vivo. We found that cisplatin treatment led to SGN damage, in which reactive oxygen species (ROS) generation increased, PINK1 expression decreased, BAD accumulation on mitochondria raised and mitochondrial apoptotic pathway activated. Conversely, we demonstrated that PF pre‐treatment obviously mitigated cisplatin‐induced SGN damage. Mechanistic studies showed that PF could reduce ROS levels, increase PINK1 expression, decrease the BAD accumulation on mitochondria and, thus, alleviate the activated mitochondrial apoptosis in SGNs caused by cisplatin. Overall, the findings from this work reveal the important role of PF and provide another strategy against cisplatin‐induced ototoxicity.

S857 THE NUCLEOTIDE KINASE NADK CONSTITUTES A METABOLIC VULNERABILITY OF NOTCH1‐DRIVEN T‐ALL

Background:Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer with a cumulative risk of ∼1 in 2,000 children by the age of 15 years and an increasing incidence over the last 30 years.Activating mutations in the NOTCH1gene are found in more than 60% of cases of T‐cell Acute Lymphoblastic leukaemia (T‐ALL) and are being targeted therapeutically with compounds such as γ‐secretase inhibitors (GSIs) and Notch inhibiting antibodies (mAbs).Aims:To identify novel therapeutic vulnerabilities of Notch1 driven T‐ALL.Methods:A CRISPR‐Cas9 drop‐out screen was used to identify genetic vulnerabilities of NOTCH1‐driven T‐ALL. Amongst these was the nucleotide kinase NADK, a novel therapeutic vulnerability of T‐ALL that we chose to investigate in downstream studies. These included in‐vitro and in‐vivo validation of NADK as a therapeutic target using CRISPR/gRNA and a compound inhibitor, and investigation of its function and relationship NOTCH1 activation using diverse approaches.Results:A genome wide CRISPR‐Cas9 screen was performed in T‐ALL cell lines driven by NOTCH1 overexpression (Jurkat, CEM‐CCRF), or mutation (PEER) and in a line not dependent on NOTCH1 (Loucy) (Figure 1A). We identified 69 NOTCH‐specific essential genes, including well‐known players in NOTCH signaling such as NOTCH1, RBPJ, BCL11B, GATA3 and CTPB1. We also identified a separate group of genes associated to cellular pathways involved in the reduction of reactive oxygen species (ROS). One of these was NADK, the gene for nicotinamide adenine dinucleotide kinase, which we investigate further. NADK drives the conversion of NAD+to NADP+, which in its reduced form (NADPH) is used in the reduction of ROS levels through the production of reduced Glutathione. Exposure of Loucy (NOTCH1‐WT) to H2O2 showed that it was not able to reduce intracellular ROS levels as efficiently as NOTCH1‐driven lines, suggesting increased NADK activity in the latter. In keeping with this, intracellular NADP+/NADPH levels that were significantly higher in NOTCH1‐driven cell lines than in Loucy (Figure 1C). In addition, pharmacological or genetic inhibition of NADK led to a significant increase in ROS levels in the NOTCH1‐driven Jurkat cells, while Loucy cells were not affected (Figure 1B). It was previously reported that, in both human and mouse T‐ALL cells, the intracellular domain of Notch1 (NiCD) suppresses production of ROS levels. This was confirmed by demonstrating that the intracellular levels of NADP+/NADPH increased by more than two‐fold when the human NiCD was overexpressed in Loucy (Figure 1F). In‐vivo experiments demonstrated that genetic or pharmacological inhibition of NADK reduced leukaemia proliferation and significantly prolonged the survival of mice xenografted with Jurkat or PEER, but not Loucy (Figure 1D/1E). We hypothesize that NOTCH1 drives ROS detoxification through NADK activation, a function required to support its proliferative effect. This frames NADK as a therapeutic vulnerability in NOTCH1‐driven T‐ALL (Figure 1G).Summary/Conclusion:Our findings propose that the intracellular domain of NOTCH1 activates NADK to enhance the production NADP+/NADPH, a function required to support it proliferative effects. This frames NADK as a putative novel therapeutic vulnerability of NOTCH1‐driven T‐ALL and potentially other NOTCH1‐driven cancers.image

Oxygen-sensitive interneurons exhibit increased activity and GABA release during ROS scavenging in the cerebral cortex of the western painted turtle.

The western painted turtle (Chrysemys picta bellii) has the unique ability of surviving several months in the absence of oxygen, which is termed anoxia. One major protective strategy that the turtle employs during anoxia is a reduction in neuronal electrical activity, which may result from a natural reduction in reactive oxygen species (ROS). We previously linked a reduction in ROS levels to an increase in γ-amino butyric acid (GABA) receptor currents. The purpose of this study is to understand how fast-spiking, GABA-releasing neurons respond to reductions in ROS and how this affects GABA release. Using a fluorescence-coupled enzymatic microplate assay for GABA, we found that anoxia, the ROS scavenger N-(2-mercaptopriopionyl)glycine (MPG), or the mitochondria-specific ROS scavenger MitoTEMPO resulted in a 2.5-, 2.0-, and 2.5-fold increase in extracellular GABA concentration, respectively. This phenomenon could be blocked with TTX, indicating that it is activity dependent. Using whole cell patch-clamping techniques, we found that fast-spiking, burst-firing GABAergic turtle neurons increase the duration and number of action potentials per burst by 26% and 42%, respectively, in response to ROS scavenging via MPG. These results suggest that the reduction in mitochondrially produced ROS that occurs during anoxia leads to increased GABA release, which promotes postsynaptic inhibitory activity through activation of GABA receptors.NEW & NOTEWORTHY This is a novel study examining the response of cerebral cortical stellate interneurons to anoxia and mitochondrial reactive oxygen species (ROS) scavenging with MitoTEMPO. Under both conditions burst firing increases in these cells, and we show that extracellular GABA release increases in the presence of the ROS scavenger. We conclude that in the anoxia-tolerant painted turtle brain, a decrease in ROS levels is an important low oxygen signal.

Carnosine Protects Mouse Podocytes from High Glucose Induced Apoptosis through PI3K/AKT and Nrf2 Pathways

Diabetic nephropathy is the complication of diabetes mellitus that can lead to chronic renal failure. Reactive oxygen species (ROS) production plays an important role in its pathological process. Previous studies showed that carnosine may reduce diabetic nephropathy by antioxidant effect. However, the molecular mechanism of its antioxidant was not fully understood. In the current study, we developed high glucose containing different concentrations of carnosine to reduce ROS levels and podocytes apoptosis, and Cell Counting Kit-8 test was used to observe the cell viability. Carnosine (5-20mM) was found to protect mouse podocytes (MPC5) cells from HG-induced injury. Quantitative real-time PCR, Western blotting, and immunofluorescence staining revealed that high glucose induced ROS levels and podocytes apoptosis were downregulated by PI3K/AKT and Nrf2 signaling pathways. The current findings suggest that carnosine may reduce ROS levels and MPC5 cells apoptosis by PI3K/AKT and Nrf2 signaling pathways activation.

Polydatin and I-CBP112 protects early bovine embryo against nicotinamide-induced mitochondrial dysfunction

The mammalian Sirtuin family of seven enzymes, members of the NAD+-dependent histone deacetylase family that modify histones via direct deacetylation, is involved in the regulation of many antioxidant and oxidative stresses. In the present study, we explored the effects of nicotinamide (NAM)-induced oxidative stress on the in vitro development of bovine embryos, on the acetylation of histone H3 lysine 56 (H3K56ac) and on expression of apoptosis-related genes. Treatment with NAM (10, 20 or 40 mM for 24, 48 or 196 h) during IVC resulted in significantly decreased blastocyst formation (24 h: 38.8 vs. 33.1, 27.3 and 10.2%, with P > 0.05, P < 0.05 and P < 0.01, respectively; 48 h: 37.5 vs. 28.2, 13.4 and 0%, with P < 0.05 and P < 0.01, respectively; 196 h: 35.8 vs. 23.4, 0 and 0%, with P < 0.05, respectively). Treatment with NAM (20 and 40 mM for 24 h) resulted in increased intracellular reactive oxygen species (ROS) levels in 2-cell and blastocysts, and apoptotic cell numbers in blastocysts and decreased mitochondrial membrane potential (ΔΨ) in 2-cell embryos (P < 0.05). Polydatin (PD) and I-CBP112 rescued the 20 mM NAM-induced embryo developmental defects and reduced ROS levels and apoptotic cell numbers in blastocysts (P < 0.05). The gene expression of NF-κB, COX2 and p53 was significantly increased in the NAM-treated group. Immunofluorescence analysis confirmed that the protein levels of nuclear factor-kappa B (NF-κB) decreased significantly after PD and I-CBP112 treatment compared with the control (P < 0.05). High level of H3K56ac induced by NAM was decreased after PD and I-CBP112 treatment (P < 0.05). These findings suggest that NAM treatment induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced bovine developmental defects, which can be tolerated by PD and I-CBP112 treatment.

N-acetylcysteine reduces reactive oxygen species and pro-inflammatory cytokine production in HLA-B27 positive macrophages and shapes them towards a quiescent metabolic state

Abstract HLA-B27, which predisposes to spondyloarthritis (SpA), has a tendency to misfold, generate endoplasmic reticulum (ER) stress, and activate the unfolded protein response (UPR). The UPR may contribute to SpA pathogenesis by promoting the production of pro-inflammatory cytokines like IL-23 and TNF. During ER stress, cells produce more reactive oxygen species (ROS), which can further worsen the UPR and promote inflammation. Here, we examined whether reducing oxidative stress in HLA-B27+ macrophages affects pro-inflammatory cytokine production and UPR gene expression. Bone marrow-derived macrophages from HLA-B27-transgenic (Tg) treated with IFNg and LPS (stimulated) showed increased ROS production compared to HLA-B7-Tg and wild type (WT) control rats. This was accompanied by robust activation of the UPR only in HLA-B27-expressing cells. Treatment with N-acetylcysteine (NAC), a ROS scavenger, significantly reduced ROS levels in HLA-B27+ macrophages and strongly decreased the transcription of many pro-inflammatory cytokines including Il-23, Il-12, Tnf, Il-6 and Il-1b. The effect of NAC was further verified by ELISA assays available for rat IL-1b, IL-6, and TNFa. NAC treatment also partially reduced UPR markers including spliced Xbp1, and BiP, Chop, Manf and Derlin-3 in HLA-B27+ macrophages. Interestingly, NAC reduced metabolic activity in stimulated macrophages to levels similar to untreated control cells as measured by the Seahorse Assay. Our data demonstrate that reducing ROS levels with NAC ameliorates ER stress-related pro-inflammatory effects of HLA-B27 misfolding. Alteration of the oxidative stress pathway should be further explored for therapeutic intervention in SpA.

646 Activated-Grape Seed Extract (AGSE) inhibits oxidative stress and demethylation of PP2A in human skin

Environmental stimuli attack the skin daily resulting in oxidative stress and the generation of reactive oxygen species (ROS) and inflammation. One pathway that regulates oxidative stress in skin involves Protein phosphatase 2A (PP2A), a phosphatase that has been previously linked to Alzheimer’s Disease and aging. Oxidative stress bursts via hydrogen peroxide (H2O2) and Ultraviolet A (UVA) treatments have previously been shown to dramatically decrease PP2A methylation in human dermal fibroblasts (HDFs), moreover, in vivo studies demonstrate that oxidative stress induces PP2A inactivation. Thus, we hypothesize, agents that increase PP2A methylation and activity will promote skin health and combat aging. To discover novel inhibitors of PP2A demethylation activity, we screened a small library of ∼30 natural botanical extracts. We discovered Grape Seed Extract (GSE), which has previously been reported to have several benefits for skin to be one of the most potent PP2A demethylating extracts (IC50 = 0.96 μg/mL). Via several fractionation and extraction steps, we developed a novel grape seed extract called Activated-Grape Seed Extract (AGSE) which is enriched for PP2A-activating flavonoids resulting in a ∼5-fold increased potency (IC50 = 0.22 μg/mL) in preventing PP2A demethylation when compared to commercial GSE. We then investigated the antioxidant potential of AGSE and demonstrate it dose-dependently protects PP2A against oxidative stress-induced demethylation in HDFs by reducing ROS levels in cell-based and cell-free assays equal to or better than Vitamin C, E and Ferulic acid. Moreover, AGSE shows anti-inflammatory activity, dose-dependently inhibiting UVA induced IL-6 release in HDFs and TPA IL-8 release from NHEKs. These results demonstrate that AGSE is a novel, multi-functional extract that modulates methylation of PP2A with increased potency as compared to commercial GSE and supports the hypothesis of PP2A as a master regulator for oxidative stress signaling and aging in skin.

Serotonin amide with anti‐inflammatory and wound healing properties

Some serotonin amides, as N‐behenoyl‐5‐hydroxytryptamine (C22:0‐5‐HT), are present in the surface wax of coffee beans and few studies show their possible biological activity. Thus, the aim of this work was to evaluate the anti‐inflammatory effect of C20:0‐5‐HT in the model of carrageenan‐induced inflammation into the subcutaneous air pouch (SAP), production of reactive oxygen species (ROS) by PMA‐stimulated leukocytes and MTT cell viability assay healing effect on the contraction model of the lesion in diabetic mice. C22:0‐5‐HT was synthesized by the group of Professor Claudia Rezende, Chemistry Institute, UFRJ. Female Webster mice (20–25g; n= 6–8) received oral administration of C22:0‐5‐HT (0.1, 1, 3 or 10 mg/kg) and were evaluated in model of SAP. Mice received carrageenan injection into the SAP and after 24 h were euthanized and the exsudate from SAP were collected for measurement of cell count. For the evaluation of ROS, leukocytes from the SAP were collected and incubated with C22:0‐5‐HT (0.1, 1 or 3 μM) for 1 h. Cells were incubated with 10 nM phorbol myristate acetate and 2 mM 2′‐7′‐dichlorodihidrofluoescein diacetate. The results were expressed as DCF‐DA fluorescence. Cell viability was measured by the MTT assay using RAW 264.7 mouse macrophages. For the analysis of healing activity female Webster mice (30–35g, n=10–12) received alloxan (65 mg/kg, iv) and after 7 days were anesthetized with ketamine and xylazine. The dorse was trichotomized and an area of 10 mm in diameter was exposed. The animals received topical administration (3 mg/animal) for 14 days and photos were taken on days 0, 3, 7, 10 and 14 to follow the retraction of the lesion. The image was processed in ImageJ program and the results are presented as the mean ± standard deviation of the wound area and expressed as arbitrary units (AU). Statistical significance between groups was determined by ANOVA followed by Bonferroni's test (*p&lt;0.05) and the number of the animal research ethical committee is DFBCICB015‐04/16. C22:0‐5‐HT showed anti‐inflammatory activity in the three higher doses tested with reduction on cell migration (vehicle= 60.8 ± 8 × 106 cells/mL versus 61.7 ± 7 × 106 cells/mL, 31.2 ± 6* × 106 cells/mL, 37.1 ± 7* × 106 cells/mL, and 30.4 ± 6* × 106 cells/mL), with the doses of 0.1, 1, 3, and 10 mg/kg, respectively. It was also observed reduction in levels of ROS by PMA‐stimulated leukocytes in all concentrations tested (vehicle= 197,237.4 ± 8 fluorescence versus 98,852.1 ± 9* fluorescence, 87,951.3 ± 8.* fluorescence, 77,932.1 ± 7* fluorescence) and did not reduce the cell viability. Was observed improvement in healing of the treated mice on days 10 and 14 after injury. Naïve: day 0 = 40.1 ± 3.1 AU; 3 = 29.2 ± 5.1 AU; 7 = 13.1 ± 3.4 AU; 10 = 6.1 ± 2.2 AU; 14 = 3.2 ± 1.1 AU; Diabetic mice: day 0 = 41.3 ± 5.1 AU; 3 = 30.2 ± 5.4 AU; 7 = 20.1 ± 4.4 AU; 10 = 13 ± 3.5 AU; 14 = 7.2 ± 2 AU; Diabetic mice C22:0‐5‐HT‐treated (3 mg/animal) day 0 = 37.4 ± 6 AU; 3 = 30.9 ± 4 AU; 7 = 14.1 ± 3 AU; 10 = 2.7 ± 0.9* AU; 14 = 0.3 ± 0.1* AU. The results demonstrate that C22:0‐5‐HT produces anti‐inflammatory and healing effect. The mechanism(s) still under investigation.Support or Funding InformationThis study was financed in part by the CNPq and FAPERJ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of plants and their root exudate on bacterial activities during rhizobacterium–plant remediation of phenol from water

We investigated remediation of phenol from water using microbe–plant partnerships. Co-introduction of maize seedlings, Pseudomonas fluorescens rifampicin-resistant P13 and P. stutzeri P7 carrying self-transmissible TOL-like plasmids reduced phenol content in water at lower phenol concentrations (25, 50, and 75 mg/L), similar to individual introduction of the bacteria. Co-introduction of plants and bacteria significantly reduced phenol content in water at higher phenol concentrations (100, 125, and 150 mg/L) compared to using individual introduction of the bacteria. Moreover, TOL-like plasmids were transferred from P7 to P13. Addition of plants promoted the growth of both strains, leading to increased plasmid transfer. At higher phenol concentrations, addition of plants resulted in increases of catechol 2, 3-dioxygenase (C23O) activity and reduction in level of reactive oxygen species (ROS) of bacteria in the degradation experiments. Increased plasmid transfer and C23O activity and reduction in ROS level might be the major reasons why plants promote bacterial degradation of phenol at higher phenol concentrations. Furthermore, root exudate of maize seedlings and artificial root exudate (ARE) constructed using major components of the root exudate had the same effects on bacterial activities. Unlike the ARE, deletion of glucose, arabinose, or fructose or all the monosaccharides from ARE resulted in no increase in numbers of both strains and in plasmid transfer. At the higher phenol concentrations, deletion of glutamic acid, aspartic acid, alanine, or glycine or all the amino acids did not stimulate bacterial C23O activity. Deletion of fumaric, oxaloacetic or citric acids still reduced bacterial ROS level as ARE did, but, deletion of all the organic acids or DIMBOA, a hydroxamic acid, did not reduce bacterial ROS level as ARE did. The data showed that each monosaccharide might be important for sufficient numbers of plant-associated bacteria and increased plasmid transfer while each amino acid might be important for maintaining bacterial C23O activity and that DIMBOA might be responsible for the decrease in ROS levels. These results are the basis for efficient remediation of phenol from water by microbe–plant partnerships and further studies on the mechanism of rhizobacterium-plant interaction.