Reduced Nitric Oxide Production Research Articles

Anti-Inflammatory and Anti-Oxidant Effects of Epilobium amurense subsp. cephalostigma via Activation of Nrf2/HO-1 and Inhibition of NF-κB/p38 MAPK Signaling in LPS-Stimulated Macrophages

The genus Epilobium consists of approximately 200 species that are distributed worldwide. Some of these herbs have been used for the treatment of diarrhea, infection, irritation, and other disorders associated with inflammation. Unlike that of other Epilobium species, there is little scientific understanding of the pharmacological effect of Epilobium amurense subsp. cephalostigma (Hausskn.) C. J. Chen, Hoch & P. H. Raven. In this study, we demonstrated the anti-inflammatory and antioxidative properties of an E. amurense 95% ethanol extract (EACEE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and observed the underlying mechanism of this effect. We measured the productions of nitric oxide (NO) and reactive oxygen species, and examined the actions of EACEE on transcription factors in the macrophages. EACEE reduced NO production and inducible nitric oxide synthase protein levels via the inhibition of the nuclear factor (NF)-κB pathway. Additionally, EACEE suppressed redundant reactive oxygen species production and regulated nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling. Furthermore, EACEE significantly inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK). Overall, these results indicate that EACEE exerts anti-inflammatory and antioxidant effects via the activation of Nrf2/HO-1 and inhibition of NF-κB/p38 MAPK signaling.

Auraptene, a Monoterpene Coumarin, Inhibits LTA-Induced Inflammatory Mediators via Modulating NF-κB/MAPKs Signaling Pathways.

Objective Oxidative stress-mediated inflammatory events involve in the progress of several diseases such as asthma, cancers, and multiple sclerosis. Auraptene (AU), a natural prenyloxycoumarin, possesses numerous pharmacological activities. Here, the anti-inflammatory effects of AU were investigated in lipoteichoic acid- (LTA-) induced macrophage cells (RAW 264.7). Methods The expression of cyclooxygenase (COX-2), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, c-Jun N-terminal kinase (JNK), heme oxygenase (HO-1), p65, and IκBα were all identified by western blotting assay. The level of nitric oxide (NO) was measured by spectrometer analysis. The nuclear translocation of p65 nuclear factor kappa B (NF-κB) was assessed by the confocal microscopic staining method. Native polyacrylamide gel electrophoresis was performed to perceive the activity of antioxidant enzyme catalase (CAT). Results AU expressively reduced NO production and COX-2, TNF-α, IL-1 β, and iNOS expression in LTA-stimulated cells. AU at higher concentration (10 µM) inhibited ERK and JNK, but not p38 phosphorylation induced by LTA. Moreover, AU blocked IκB and p65 phosphorylation, and p65 nuclear translocation. However, AU pretreatment was not effective on antioxidant HO-1 expression, CAT activity, and reduced glutathione (GSH, a nonenzymatic antioxidant), in LTA-induced RAW 264.7 cells. Conclusion The findings of this study advocate that AU shows anti-inflammatory effects via reducing NF-κB/MAPKs signaling pathways.

Glucose availability limits microglial nitric oxide production.

Metabolic intermediates influence inflammation not only through signaling effects, but also by fueling the production of pro-inflammatory molecules. Microglial production of nitric oxide (NO) requires the consumption of NADPH. NADPH consumed in this process is regenerated from NADP+ primarily through the hexose monophosphate shunt, which can utilize only glucose as a substrate. These factors predict that glucose availability can be rate-limiting for glial NO production. To test this prediction, cultured astrocytes and microglia were incubated with lipopolysaccharide and interferon-γ to promote expression of inducible nitric oxide synthase, and the rate of NO production was assessed at defined glucose concentrations. Increased NO production was detected only in cultures containing microglia. The NO production was markedly slowed at glucose concentrations below 0.5mM, and comparably reduced by inhibition of the hexose monophosphate shunt with 6-aminonicotinamide. Reduced NO production caused by glucose deprivation was partly reversed by malate, which fuels NADPH production by malate dehydrogenase, and by NADPH itself. These findings highlight the role of the hexose monophosphate shunt in fueling NO synthesis and suggest that microglial NO production in the brain may be limited at sites of low glucose availability, such as abscesses or other compartmentalized infections.

Cytotoxicity and Anti-Inflammatory Effects of Polyherbal Formulations, Joint Pain Spl and Rumalaya Forte on Lipopolysaccharide Induced Inflammation in IC-21 Macrophages.

To test the effectiveness of marketed polyherbal formulations on lipopolysaccharide-induced inflammatory conditions in macrophages. Usage of herbal compounds among patients suffering from arthritis and cancer is increasing every year. Many anti-inflammatory herbal products available in the market should be screened thoroughly for their possible mechanism of action. Joint Pain Spl (JPS) is a polyherbal dietary food supplement composed of 13 herbal plants, and Rumalaya Forte (RF) is a polyherbal formulation comprising of 6 herbal plants. These were tested for their cytotoxicity, as well as antioxidant and anti-inflammatory activities in LPS treated IC-21 peritoneal macrophages. Commercially available JPS and RF powder was used to prepare the extract. The aqueous and methanol extracts were quantified for the presence of phenolic and flavonoid compounds and confirmed with HPLC. In vitro DPPH free scavenging activity was performed. Cytotoxicity was tested by MTT assay. Anti-inflammatory activity was tested using lipopolysaccharide-stimulated IC-21 peritoneal macrophage cells. The phytochemical screening showed the presence of phenolic and flavonoid compounds in JPS and RF. The aqueous and methanol extracts of JPS and RF possesses significant DPPH free radical scavenging activity. MTT assay revealed that 90.64% (aqueous extract) and 92.21% (methanol extract) of exposed macrophages are viable even after 24h exposure of maximal tested concentrations of herbal formulations. Pre-treatment of JPS and RF on LPS induced IC-21 macrophages showed a reduction in nitric oxide production (maximal 79.95%) and a high level of superoxide anion scavenging activity (maximal 82.5%) over control. The two tested polyherbal formulations, such as JPS and RF possess anti-inflammatory activity by modulating free radical generation in IC-21 macrophages. Thus the presence of the phenolic and flavonoid compounds may contribute to the antioxidant activity.

Substance P ameliorates TNF-α-mediated impairment of human aortic vascular cells in vitro.

Vascular diseases are caused by endothelial dysfunction due to inflammation. On endothelial injury, the expression of extracellular matrix (ECM) is enhanced and nitric oxide (NO) bioavailability becomes deficient. This condition affects endothelial metabolism and leads to vascular destruction. The aim of this investigation was to determine whether substance P (SP) is able to protect the endothelium against inflammatory stress. To this end, aortic endothelial cells were pre-treated with SP, followed by tumour necrosis factor α (TNF-α), and cellular responses were evaluated using a combination of cell biology and quantification assays, as well as western blot analyses. Our results show that TNF-α enhanced ECM expression and reduced NO production within 4hours, promoting immune cell adhesion to the endothelium and monocyte chemoattractant protein-1 (MCP-1) secretion from aortic smooth muscle cells. However, SP treatment ameliorated TNF-α-induced endothelial impairment by maintaining low ECM levels. Our data suggest that this protective effect is mediated by Akt activation and NO-enriched conditions. The inhibition of aortic endothelial cell injury by SP also reduced MCP-1 production in aortic smooth muscle cells. Together, our data indicate that SP can protect aortic endothelial and smooth muscle cells from inflammatory injury, which suggests that SP may prevent cardiovascular disease.

Aging Additively Influences Insulin- and Insulin-Like Growth Factor-1-Mediated Endothelial Dysfunction and Antioxidant Deficiency in Spontaneously Hypertensive Rats.

This study aimed to investigate the aging-related endothelial dysfunction mediated by insulin and insulin-like growth factor-1 (IGF-1) and antioxidant deficiency in hypertension. Male spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar–Kyoto rats (WKYs) were randomly divided into 24-week-old (younger) and 48-week-old (older) groups, respectively. The endothelial function was evaluated by the insulin- and IGF-1-mediated vasorelaxation of aortic rings via the organ bath system. Serum levels of nitric oxide (NO), malondialdehyde (MDA), catalase, and total antioxidant capacity (TAC) were examined. The insulin- and IGF-1-mediated vasorelaxation was significantly impaired in both 24- and 48-week-old SHRs compared with age-matched WKYs and was significantly worse in the 48-week-old SHR than the 24-week-old SHR. After pretreatments of phosphoinositide 3-kinase (PI3K) or NO synthase (NOS) inhibitors, the insulin- and IGF-1-mediated vasorelaxation became similar among four groups. The serum level of MDA was significantly increased, while the NO, catalase, and TAC were significantly reduced in the 48-week-old SHR compared with the 24-week-old SHR. This study demonstrated that the process of aging additively affected insulin- and IGF-1-mediated endothelial dysfunction in SHRs, which could be partly attributed to the reduced NO production and antioxidant deficiency.

Hypericum Perforatum Extract Modifies Serum and Tumoral Redox Status and Nitric Oxide Metabolites in Mice

Plant extracts with antioxidant efficacy and/or suppressive activity on nitric oxide (NO) production are highly being used as supplements by cancer patients. Despite very high concentrations of NO can kill malignant cells; NO can also propagates cancer growth even at supraphysiological levels. Hence, discovery of safe compounds diminishing NO production may be beneficial in reducing cancer progression. Hypericum extracts are shown to exert redox-active efficacies and to reduce NO production in non-malignant conditions; but it is not studied in terms of modifying systemic and tumoral redox status, nitrite and nitrate concentrations. In this study, we showed that Hypericum perforatum extracts significantly reduces intratumoral levels of nitrite and nitrate in solid tumors formed by Ehrlich ascites carcinoma cells. These results may suggest that Hypericum perforatum extracts may act as efficient adjuvants for malignancies which growth are highly influenced and propagated with NO. Keywords: Hypericum perforatum; total oxidant status; total antioxidant status; nitric oxide; nitrite; nitrate DOI: 10.7176/JSTR/7-05-05

Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by increasing tetrahydrobiopterin via regulation of GTP-cyclohydrolase 1 and reducing uncoupled endothelial nitric oxide synthase activity

Background and aimsThe apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. MethodsLow-density lipoprotein (LDL) receptor null (LDLr−/−) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. ResultsUnexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. ConclusionsHypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

The role of Physalis angulata as potential anti-type 2 diabetic agent

Background: Medicinal plants play important roles in the management of several diseases including diabetes. Objective: The aim of this study is to investigate the mechanism of action of Physalis angulata (PA) for its anti-diabetic activity using an in vitro model. Materials and Methods: Alpha-amylase inhibition was investigated through dinitrosalicylic acid assay. Glucose uptake was determined using LO-2 cell model. Radical scavenging activity was performed through 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) assay. Nitric oxide (NO) production was measured using the Griess reaction. Cell viability was examined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Results: The result showed that PA extract was able to inhibit enzyme alpha-amylase activity up to (56.6% ± 4.7%) at the concentration of 200 μg/ml. Moreover, PA possessed glucose adsorption and glucose uptake capacity up to (2.2 ± 0.18) mM glucose/g extract and (156% ± 10.1%), respectively. In addition, PA extract scavenged (52.6% ± 3.5%) DPPH and (59.7% ± 2.6%) ABTS + radicals and reduced NO production to (34.2% ± 3.8%) from RAW264.7 cells without any cytotoxic effects. Conclusion: PA could be suggested as pharmaceutical ingredient for the development of anti-diabetic products.

Seasonal Variations of the Nutritive Value and Phytotherapeutic Potential of Cladium mariscus L. (Pohl.) Targeting Ruminant's Production.

In our endeavor to identify salt-tolerant plants with potential veterinary uses in ruminants’ production strategies, we focused on Cladium mariscus L. Pohl (sawgrass), due to its high total phenolic and tannin content, anti-radical properties, and ethnomedicinal uses. Aerial parts were collected along the year in Southern Portugal and evaluated for the nutritional profile and in vitro organic matter digestibility (IVOMD), aiming for its use as feed. Acetone extracts were appraised for total contents in phenolics (TPC), flavonoids (TFC), and tannins (CTC), as well as the chemical composition by HPLC-DAD and in vitro antioxidant and anti-inflammatory properties, targeting its exploitation as phytotherapeutic products. Sawgrass biomass has a limited nutritive value, due to its high neutral detergent fiber (NDF; 596–690 g kg−1 dry matter (DM)) and acid detergent fiber (ADF; 330–418 g kg−1 DM) contents, low crude protein (51.8–87.3 g kg−1 DM) and IVOMD (172–317 g kg−1 organic matter (OM)). Despite differences among seasons, the mineral profile was adequate. The extracts were rich in TPC (88–112 mg g−1), CTC (115–169 mg g−1), and TFC (18.5–20.2 mg g−1), and displayed significant antioxidant capacity, particularly in summer and autumn, whilst no seasonal influence was detected for anti-inflammatory properties (30% reduction of nitric oxide production). Eleven phenolics were quantified: chlorogenic, ferulic, and syringic acids were the most abundant, especially in the autumn sample. Overall, despite the low nutritional interest, sawgrass extracts hold the potential as a source of antioxidant and anti-inflammatory phenolic compounds.

Evaluation of the efficacy of ethanol leaf extract of Helichrysum petiolare Hilliard and B.L. Burtt against skin aging

Purpose: To determine the efficacy of Helichrysum petiolare ethanol leaf extract against skin aging.Methods: The cytotoxic potential of the plant extract towards human dermal fibroblast (MRHF) cells was determined by Hoechst 33342/Propidium iodide (PI) staining. Effect of H. petiolare extract on reactive oxygen species (ROS) levels in MRHF cells and NO (nitric oxide) production in RAW 246.7 cells activated by LPS (lipopolysaccharides) was investigated. The inhibitory effect of the extract againstcollagenase, elastase, tyrosinase and protein glycation was also evaluated.Results: The extract did not display cytotoxicity towards MRHF cells at the tested concentrations when compared to the trend seen with the untreated control (p < 0.05). The extract caused a significant decrease (p < 0.05) in ROS levels in MRHF cells in a concentration-dependent manner and also demonstrated a reduction in NO production in RAW cells with no toxicity. Furthermore, the extract produced a weak inhibition of collagenase, elastase and tyrosinase activities when compared to the corresponding positive controls, but effectively inhibited protein glycation at the tested concentrations.Conclusion: The findings suggest that the ethanol leaf extract from H. petiolare has the potential to mitigate skin aging and therefore needs to be further investigated for possible clinical applications.
 Keywords: Cytotoxicity, Efficacy, Helichrysum petiolare, MRHF cells, Oxidative stress, Protein glycation; skin aging

The effects of the molecular weights of hyaluronic acid on the immune responses

BackgroundThe molecular weight of hyaluronic acid (HyA) depends on the type of organ in the body. When HyA of the desired molecular weight is implanted into the human body for regeneration of damaged tissue, it is degraded by hyaluronidase in associated with an inflammatory response. This study sought to evaluate the effects of HyA molecular weight and concentration on pro- and anti-inflammatory responses in murine macrophages.MethodsThe structures and molecular weights of HyAs (LMW-10, MMW-100, MMW-500, and HMW-1,500) were confirmed by 1 H NMR and gel permeation chromatography (GPC), respectively. After treatment of murine macrophages with a low (10 µg/mL) or high (100 µg/mL) concentration of each molecular weight HyA, cells were stimulated with lipopolysaccharide (LPS) and changes in immune response in both LPS-stimulated and untreated macrophages were evaluated by assessing nitric oxide (NO) production, and analyzing expression of pro- and anti-inflammatory genes including by RT-PCR.ResultsMolecular weights of LMW-10, MMW-100, MMW-500, and HMW-1,500 were 13,241 ± 161, 96,531 ± 1,167, 512,657 ± 8,545, and 1,249,500 ± 37,477 Da, respectively. NO production by LPS-stimulated macrophages was decreased by increasing concentrations and molecular weights of HyA. At a high concentration of 100 µg/mL, HMW-1,500 reduced NO production in LPS-stimulated macrophages to about 45 %. Using NanoString technology, we also found that the immune-related genes TNF-α, IL-6, IL-1β, TGF-β1, IL-10, IL-11, CCL2, and Arg1 were specifically over-expressed in LPS-stimulated macrophages treated with various molecular weights of HyA. An RT-PCR analysis of gene expression showed that HMW-1,500 decreased expression of classically activated (M1) macrophage genes, such as TNF‐α, IL-6, CCL2, and IL-1β, in LPS-stimulated macrophages, whereas medium molecular-weight HyA (MMW-100 and MMW-500) instead increased expression levels of these genes. HMW-1,500 at a high concentration (100 µg/mL) significantly decreased expression of pro-inflammatory genes in LPS-stimulated macrophages. Expression of genes associated with anti-inflammatory responses (M2 phenotype), such as TGF-β1, IL-10, IL-11, and Arg1, were increased by high concentrations of MMW-500 and HMW-1,500 in LPS-stimulated macrophages.ConclusionsHigh molecular-weight HyA (i.e., > 1,250 kDa) inhibits pro-inflammatory responses in LPS-stimulated macrophages and induces anti-inflammatory responses in a concentration dependent manner.

Serum ADMA levels were positively correlated with EDSS scores in patients with multiple sclerosis

Multiple sclerosis (MS) is an autoinflammatory, chronic central nervous system disease. In the pathogenesis of the disease increased nitric oxide (NO) levels play an important role. Nitric oxide (NO) has neuroprotective effects in physiological conditions, however, it is thought that excessive NO formation in MS may lead to demyelination and axonal damage. Derivatives of methylarginine including asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA), symmetric dimethyl arginine (SDMA) directly or indirectly reduce NO production. Our aim was to measure the levels of methylarginine derivatives and citrulline, ornithine, arginine, homoarginine levels, which are metabolites associated with NO metabolism, in MS subgroups.

Macrophage inflammatory state in Type 1 diabetes: triggered by NLRP3/iNOS pathway and attenuated by docosahexaenoic acid.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by insulin-producing pancreatic β-cell destruction and hyperglycemia. While monocytes and NOD-like receptor family-pyrin domain containing 3 (NLRP3) are associated with T1D onset and development, the specific receptors and factors involved in NLRP3 inflammasome activation remain unknown. Herein, we evaluated the inflammatory state of resident peritoneal macrophages (PMs) from genetically modified non-obese diabetic (NOD), NLRP3-KO, wild-type (WT) mice and in peripheral blood mononuclear cells (PBMCs) from human T1D patients. We also assessed the effect of docosahexaenoic acid (DHA) on the inflammatory status. Macrophages from STZ-induced T1D mice exhibited increased inflammatory cytokine/chemokine levels, nitric oxide (NO) secretion, NLRP3 and iNOS protein levels, and augmented glycolytic activity compared to control animals. In PMs from NOD and STZ-induced T1D mice, DHA reduced NO production and attenuated the inflammatory state. Furthermore, iNOS and IL-1β protein expression levels and NO production were lower in the PMs from diabetic NLRP3-KO mice than from WT mice. We also observed increased IL-1β secretion in PBMCs from T1D patients and immortalized murine macrophages treated with advanced glycation end products and palmitic acid. The present study demonstrated that the resident PMs are in a proinflammatory state characterized by increased NLRP3/iNOS pathway-mediated NO production, up-regulated proinflammatory cytokine/chemokine receptor expression and altered glycolytic activity. Notably, ex vivo treatment with DHA reverted the diabetes-induced changes and attenuated the macrophage inflammatory state. It is plausible that DHA supplementation could be employed as adjuvant therapy for treating individuals with T1D.

Anti-inflammatory potential of Portuguese thermal waters

In light of Medical Hydrology, thermal waters (TW) are all-natural mineral waters that emerge inside a thermal resort and have therapeutic applications. Their beneficial effect has been empirically recognized for centuries, being indicated for symptom alleviation and/or treatment of several diseases, almost all associated with inflammation. Indeed, an anti-inflammatory effect has been attributed to many different Portuguese TW but there is no scientific validation supporting this empiric knowledge. In the present study, we aimed to investigate the anti-inflammatory properties of 14 TW pertaining to thermal centers located in the Central Region of Portugal, and grouped according to their ionic profile. Mouse macrophage cells stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 agonist, were exposed to culture medium prepared in TW. Metabolism, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression levels and the scavenging capacity of TW, were investigated in vitro. 11 out of 14 TW reduced NO production and/or iNOS expression, and/or scavenging activity, in macrophages exposed to LPS. The sulphated/calcic TW did not show any effect on at least one of the inflammatory parameters evaluated. Two sulphurous/bicarbonate/sodic TW and the sulphurous/chlorinated/sodic TW promoted an increase in NO production and/or iNOS expression. Our results validate, for the first time, the anti-inflammatory properties of Portuguese TW, supporting their therapeutic use in the treatment of inflammation-related diseases and promoting their putative application in cosmetic products and medical devices.

The impact of glucose exposure on bioenergetics and function in a cultured endothelial cell model and the implications for cardiovascular health in diabetes

Cardiovascular disease is the primary driver of morbidity and mortality associated with diabetes. Hyperglycaemia is implicated in driving endothelial dysfunction that might underpin the link between diabetes and cardiovascular disease. This study was designed to determine the impact of chronic preconditioning of cells to hyperglycaemia and transient switching of cultured endothelial cells between hyper- and normo-glycaemic conditions on bioenergetic and functional parameters. Immortalised EA.hy926 endothelial cells were cultured through multiple passages under normoglycaemic (5.5 mM) or hyperglycaemic (25 mM) conditions. Cells were subsequently subjected (48 h) to continued normo- or hyperglycaemic exposure, or were switched to the alternative glycaemic condition, or to an intermediate glucose concentration (12.5 mM) and metabolic activity, together with key markers of function were measured. Cells habituated to hyperglycaemia were energetically quiescent. Functional activity, characterised by the measurement of nitric oxide, endothelin-1, tissue plasminogen activator and plasminogen activator inhibitor-1, was depressed by exposure to high glucose, with the reduction in nitric oxide production being the most notable. Function was more responsive to acute changes in extracellular glucose than were bioenergetic changes. We conclude that glucose is a key determinant of endothelial function. The study highlights the importance of chronic glucose exposure on cell phenotype and emphasises the need to pay close attention to glucose preconditioning in interpreting results under culture conditions.

Effect of Bacillus clausii-fermented spent coffee ground extract on Salmonella-infected macrophages

The coffee beverage market produces large amounts of waste as spent coffee grounds (SCGs). This by-product contains a high concentration of bioactive compounds that could be more accessible after bioprocessing. In this study, we identified the compounds present in Bacillus clausii-fermented SCG extract and determined the phenolic content and antioxidant activity of the extract. Additionally, in a macrophage model, we measured nitric oxide (NO) production and bactericidal activity against Salmonella enterica Typhimurium. The fermented SCG extract exhibited increased phenolic and flavonoid contents and antioxidant activity as compared to non-fermented SCG extract. The main compounds detected in fermented SCG extracts were cryptochlorogenic, caffeic, and chlorogenic acids. Macrophages RAW264.7 treated with 10, 50, or 100 μg/mL of fermented SCG exhibited significantly (P < 0.001) reduced NO production and showed no cytotoxic effects. Salmonella-infected macrophages treated with 50, 100, or 300 μg/mL of fermented SCG extracts exhibited 100% bactericidal efficacy after the first hour of infection up to 24 h. These results suggest that B. clausii-fermented SCG extract has a high antioxidant activity and may favor and enhance activation of macrophages against bacterial infections such as Salmonella enterica Typhimurium.

Weissella cibaria CMU exerts an anti‑inflammatory effect by inhibiting Aggregatibacter actinomycetemcomitans‑induced NF‑κB activation in macrophages.

Periodontitis is a chronic inflammatory disease caused by various periodontal pathogens. Weissella cibaria CMU (oraCMU) is a probiotic that promotes oral health. However, its anti-inflammatory effects against periodontal pathogens have not yet been investigated. The present study evaluated the anti-inflammatory effects of live oraCMU against stimulation with the formalin-inactivated periodontal pathogen Aggregatibacter actinomycetemcomitans in RAW 264.7 macrophages. Cell viability was analyzed by the MTS assay in a dose-dependent manner (at multiplicities of infection of 0.1, 1, 10, 100 and 1,000). Nitric oxide (NO) was monitored using the Griess test. The mRNA expression of proinflammatory cytokines such as interleukin (IL)1β and IL6 was assessed by reverse transcription-quantitative PCR. Western blotting was used to examine the effects of oraCMU on the phosphorylation of NF-κB inhibitor α (IκBα) and IκBα kinase (IKK), the nuclear translocation of the NF-κB subunit p65 and the expression of inducible NO synthase (iNOS). Live oraCMU had no cytotoxic effects on RAW 264.7 macrophages. In A. actinomycetemcomitans-stimulated RAW 264.7 macrophages, oraCMU reduced NO production by suppressing iNOS expression and downregulating the mRNA expression of proinflammatory cytokines in a dose-dependent manner. IKK phosphorylation and IκBα degradation were dose-dependently inhibited by oraCMU and the nuclear translocation of p65 via the canonical NF-κB pathway was simultaneously reduced. The results indicated that oraCMU possessed anti-inflammatory activity associated with the inhibition of NF-κB signal activation in response to periodontal pathogens. This suggests that oraCMU is a beneficial anti-inflammatory probiotic that can aid in the maintenance of oral health.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.

Acrolein (ACR), a highly reactive α,β-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Diospyrin Modulates Inflammation in Poly I:C-Induced Macrophages via ER Stress-Induced Calcium-CHOP Pathway

Diospyrin, plant-derived bisnaphthoquinonoid, is known to have anticancer activity. However, pharmacological activity of diospyrin on viral infection is not well known. We investigated effects of diospyrin on macrophages induced by polyinosinic-polycytidylic acid (poly I:C), a mimic of double-stranded viral RNA. Various cytokines, intracellular calcium, nitric oxide (NO), phosphorylated p38 MAPK, and phosphorylated ERK1/2 as well as mRNA expressions of transcription factors were evaluated. Diospyrin significantly reduced NO production, granulocyte-macrophage colony-stimulating factor production, and intracellular calcium release in poly I:C-induced RAW 264.7. The phosphorylation of p38 MAPK and ERK1/2 was also significantly suppressed. Additionally, diospyrin inhibited mRNA levels of nitric oxide synthase 2, C/EBP homologous protein (CHOP), calcium/calmodulin dependent protein kinase II alpha, signal transducers and activators of transcription 1 (STAT1), STAT3, STAT4, Janus kinase 2, first apoptosis signal receptor, c-Jun, and c-Fos in poly I:C-induced RAW 264.7. Taken together, this study represents that diospyrin might have the inhibitory activity against viral inflammation such as excessive production of inflammatory mediators in poly I:C-induced RAW 264.7 via ER stress-induced calcium-CHOP pathway.