Induced Nitric Oxide Production Research Articles

Effect of Fractions from Lycopus lucidus Turcz. Leaves on Genomic DNA Oxidation and Matrix Metalloproteinase Activity.

We investigated the inhibitory effects of fractions from Lycopus lucidus Turcz. leaves on genomic DNA oxidation, Nitric Oxide (NO) production, and Matrix Metalloproteinase (MMP) activity. Oxidative damage of genomic DNA was detected after Fenton reaction with H2O2 using DNA electrophoresis. Western blotting was performed to compare the expression levels of MMP-2 in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 cells. Lipopolysacchride (LPS)-induced NO production in RAW 264.7 cells was measured using Griess reagent. All fractions (n-Hexane, 85% aq. MeOH, n-BuOH, and water fractions) from the leaves of L. lucidus Turcz. significantly inhibited intracellular production of reactive oxygen species (ROS) (p<0.05). Particularly, 85% aq. MeOH and n-BuOH fractions showed higher ROS inhibitory activity than the other fractions. n-Hexane, 85% aq. MeOH, n-BuOH and water (0.05 mg/mL) fractions significantly inhibited oxidative DNA damage by 57.97%, 68.48%, 58.97%, and 68.39%, respectively (p <0.05). Treatment of RAW 264.7 cells with each fraction reduced LPS-induced NO production in a dose-dependent manner (p<0.05). n-Hexane and 85% aq. MeOH fractions notably reduced MMP-2 secretion levels in the culture supernatants from HT-1080 cells. Overall, these results indicated that L. lucidus Turcz. leaves can be exploited as plant based sources of antioxidants in the pharmaceutical, cosmetic, nutraceutical, and food industries.

Bacteriocin BacSp222 and Its Succinylated Forms Exhibit Proinflammatory Activities Toward Innate Immune Cells .

PurposeThe zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules.MethodsThe study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides.ResultsBacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide.ConclusionAll naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.

Sex Difference in Renal Artery Contractility in a Novel CRISPR/Cas9‐Generated P2X7 Knockout Rat

Purinergic signaling regulates numerous physiological processes, including renal hemodynamics. The P2X7 receptor (P2X7) is a non‐selective cation channel activated by extra‐cellular adenosine triphosphate (ATP) which plays an important role in immune cell response and inflammation. P2X7 is also expressed in endothelial cells. In the kidney, acute pharmacological blockade of P2X7 increases renal perfusion in healthy rats (Kidney Int. 88(5): 1079–1087, 2015). Here, we generated a novel P2X7 knockout (KO) rat and examined ex vivo contractility of the renal artery in male and female rats.A P2rx7 global KO rat was generated on a F344 IcoCrl background using CRISPR/Cas9 technology. KO was achieved via a two‐adenine insertion predicted to introduce a premature stop codon in exon 2. RNA and protein were extracted from KO and wild‐type (WT) kidneys to detect P2X7 by RTqPCR (n=4‐5/group) and western blotting (n=2/group). Bone marrow‐derived macrophages (BMDM) from KO and WT rats were used to assess P2X7 function by measuring interleukin (IL)‐1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5‐6/group). RNA from BMDM was extracted after LPS to detect P2rx7, Il1b, Tnf and Il6 mRNA by RTqPCR (n=3/group), and Griess reaction was used to assess BMDM nitric oxide (NO) production (n=5‐6/group). One main renal artery from 2.5‐to‐4.5‐month‐old male and female KO and WT rats (n=8‐11/group) was mounted on a wire myograph to assess the response to 125 mM potassium physiological solution (KPSS) and to cumulative doses (1 nM‐30 μM) of phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP).The P2rx7 mutation in exon 2 was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was decreased by ~60% in kidney and ~53% in BMDM from KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. P2X7 KO did not alter BMDM polarization after LPS stimulation. The induction of NO production was not different between genotypes (2.14±0.26 vs 2.31±0.43 μM, NS) and mRNA expression of M1 markers Il1b, Tnf and Il6 was similarly increased. In BMDM from male WT rats, ATP stimulation induced IL‐1β release; this response was markedly suppressed in KO BMDM (2577±70 vs 413±19 pg/mL, p&lt;0.0001). In the renal artery, the constriction induced by KPSS depolarization or by PE was not different between genotypes for either sex. In male rats, the maximum vasodilation induced by ACh was significantly decreased in KO compared to WT (IC50=0.25 vs 0.20 μM, NS; %Imax= 24±3 vs 10±2%, p=0.0015); this was not observed in females (IC50=0.11 vs 8.28 μM, NS; %Imax= 19±4 vs 12±4%, NS). The dilation induced by the direct NO donor, SNP was not affected by either genotype or sex.Global genetic deletion of P2X7 impaired endothelial‐dependent vasodilation in the renal artery of male rats that may reflect diminished NO production in response to ACh. Thus, the increased renal perfusion induced by P2X7 inhibitor may arise from off‐target effects. The potential sexual dimorphism in vascular expression and/or function of P2X7 requires further investigation.

Pharmacodynamics assessment of β-carboline from the roots of Psammosilene tunicoides as analgesic compound

Ethnopharmacological relevanceThe root of Psammosilene tunicoides (W. C. Wu et C. Y. Wu) is a well-known medicinal herb for the treatment of pain, hemostasia and rheumatoid arthritis among Chinese people. Aim of the studyThe present study aimed to investigate the antinociceptive activity and mechanism of β-carboline alkaloids 1–4 which were extracted from the roots of P. tunicoides. Materials and methodsThe analgesic effects were evaluated using peripheral and central pain mouse models of nociception, including the formalin test and the tail flick test. The levels of glutamic acid (Glu) and nitric oxide (NO) in cerebellar cortexes and spinal cords (L4-6) were determined. The anti-inflammatory of all compounds were then assessed on RAW264.7 cells. ResultsOur results showed that compounds 1–4 had significant analgesic effects on both phases of formalin test of mice. Furthermore, all compounds showed suppressive effects on Glu in the brain and NO levels in the brain cortex and the spinal cord. Except for compound 1, the others could extend the pain threshold of hot water tail-flick in mice. In addition, compounds 2 and 3 (60 μmol/kg) could decrease GABAAα1 protein levels in spinal cord. All compounds exhibited anti-inflammatory effects by inhibiting lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells with half-maximal inhibitory concentration (IC50) 1.1–34.9 μM. Conclusionβ-carboline alkaloids from the roots of P. tunicoides had significant analgesic activity by both central and peripheral mechanisms. Our findings suggested that regulating the release of NO or Glu or GABAα1 are some of the mechanisms of analgesic activity of β-carboline alkaloids.

Anagliptin prevents lipopolysaccharide (LPS)- induced inflammation and activation of macrophages

Sepsis is a multiple organ dysfunction syndrome (MODS) induced by infection, which significantly threatens public health. The overactivation of inflammatory reactions and oxidative stress participate in the pathogenesis of sepsis. Anagliptin, a novel anti-diabetic agent widely applied for the treatment of type II diabetes, has been recently claimed to possess anti-inflammatory properties. Here, the protective effects of anagliptin on lipopolysaccharide (LPS)- stimulated macrophages will be checked to explore the possible pharmacological property of anagliptin on sepsis. The state of oxidative stress was dramatically activated by LPS, accompanied by the upregulation of toll-like receptor 4 (TLR4) and high mobility group box-1 (HMGB-1), as well as the elevated expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). After treatment with anagliptin, the state of oxidative stress in macrophages was alleviated, with the downregulation of TLR4, HMGB-1, iNOS, and the declined release of NO. The excessive secretion of inflammatory factors, activation of the NF-κB pathway, and promoted expression level of receptor-interacting protein 1 (RIP1) were observed in LPS- stimulated macrophages, all of which were greatly reversed by the introduction of anagliptin. Lastly, the protective properties of anagliptin on LPS- treated macrophages, including the inhibitory effects on inflammation and the NF-κB pathway, were dramatically abolished by the overexpression of RIP1 in macrophages. Collectively, anagliptin prevented LPS-induced inflammation and activation of P338D1 macrophages by repressing the expression level of RIP1.

Direct evidence of nitric oxide production induced by lactoferrin and its enhancement by magnesium ions in cultured endothelial cells.

Bovine lactoferrin (BLF) reportedly lowers blood pressure and induces vasorelaxation, but its effect on nitric oxide (NO) production has not been established. Accordingly, we aimed to determine whether BLF induces NO production in bovine aortic endothelial cells, and the effects of extracellular free magnesium (Mg) ion concentrations on this NO production. BLF induced NO production time-dependently. NO production was markedly inhibited by the NO synthase inhibitor, NG-nitro-L-arginine methyl ester, in an effect abolished by L-arginine, but not D-arginine. NO production was suppressed at low concentrations, and enhanced at high concentrations, of Mg ions in culture medium. These results suggest that BLF has an important role in hypotensive effects. Mg ions may affect BLF-induced NO production.

Effects of curcumin on arginase enzyme activity, ornithine and nitric oxide levels in experimental breast cancer model

Breast cancer accounts for almost 30% of all cancer types, making it the most common type of tumor among women in the world. Arginase, an essential enzyme of the urea cycle, leads to the formation of urea and ornithine from L-arginine using the same substrate as nitric oxide synthase (NOS). Arginase has been reported to be higher in cancer patients and can be used as a useful biomarker. In this study, we aimed to investigate the effects of curcumin, an anticarcinogenic agent, on arginase enzyme activity, ornithine, and nitric oxide (NO) levels in experimental breast cancer model in mice. 43 male Balb/c mice were used, and Erhlich acid tumor model was created in the study. Mice were divided into five groups as healthy control group, curcumin treatment before tumor formation, curcumin treatment after tumor formation and cancer control groups. 100 mg/kg curcumin were given orally. Serum and tissue arginase enzyme activities, NO levels and tissue ornithine levels were determined spectrophotometrically. Increased serum arginase activity decreased with curcumin treatment, but this difference was not statistically significant. On the other hand, decreased NO levels were increased with curcumin treatment. In tumor tissue, arginase activity and ornithine levels decreased significantly with curcumin treatment and tissue NO levels increased significantly with curcumin treatment. In our study, we show that curcumin may have a protective effect on the development of breast cancer by inhibiting arginase enzyme activity and ornithine levels, which are the precursors of polyamines, as well as inducing NO production via NOS. As a promising anticancer agent, the net effects of curcumin in this mechanism should be supported by more advanced studies and new parameters.

Microbial transformation and inhibitory effect assessment of uvaol derivates against LPS and HMGB1 induced NO production in RAW264.7 macrophages

For the discovery of new pentacyclic triterpenes as a potential anti-inflammatory agent, microbial transformation of uvaol by Penicilium griseofulvum CICC 40293 and Streptomyces griseus ATCC 13273 was investigated. Stereoselective hydroxylation and epoxidation reactions were observed in the biotransformation. Moreover, six new metabolites were isolated and structurally elucidated by HR-ESI-MS and NMR spectrum. All the compounds were evaluated upon the inhibitory effects of nitric oxide (NO) release in RAW 264.7 cells induced by lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1). Among them, compound 3 (13, 28-epoxy-3β, 7β, 21β-trihydroxy-urs-11-ene) with the unique epoxy structure and compound 5 (3β, 21β, 24, 28-tetrahydroxy-urs-12-en-30-oic acid), exhibited a considerable inhibitory effect on both models while compound 2 (urs-12-ene-3β, 7β, 21β, 28-tetraol) showed a significant bias in the LPS-induced inflammatory response with IC50 value of 2.22 μM. Therefore, this study could provide some insights on the discovery of the pentacyclic triterpene leads for the treatment of either DAMPs or PAMPs triggered inflammation.

Incorporation of Zinc into Binary SiO2-CaO Mesoporous Bioactive Glass Nanoparticles Enhances Anti-Inflammatory and Osteogenic Activities.

During the healing and repair of bone defects, uncontrolled inflammatory responses can compromise bone regeneration. Biomaterials with anti-inflammatory activity are favorable for bone tissue regeneration processes. In this work, multifunctional Zn-containing mesoporous bioactive glass nanoparticles (Zn-MBGs) exhibiting favorable osteogenic and anti-inflammatory activities were produced employing a sol-gel method. Zn-MBGs exhibited a mesoporous spherical shape and nanoscale particle size (100 ± 20 nm). They were degradable in cell culture medium, and could release Si, Ca, and Zn in a sustained manner. Zn-MBGs also exhibited a concentration-dependent cellular response. The extract of Zn-MBGs obtained by incubation at 0.1 mg/mL (in culture medium) for 24 h could enhance in vitro mineralization, alkaline phosphatase activity, the expression of osteogenesis-related genes, and the production of intracellular protein osteocalcin of rat bone marrow stromal cells (BMSCs). Moreover, the extract of Zn-MBGs at 0.1 mg/mL could significantly downregulate the expression of inflammatory genes and the production of inducible nitric oxide in RAW 264.7 cells, particularly under stimulation of inflammatory signals interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Zn-MBGs also inhibited the pro-inflammatory M1 polarization of RAW264.7 cells induced by LPS and IFN-γ. In summary, we successfully synthesized Zn-MBGs with concentration-dependent osteogenic and anti-inflammatory activities. Zn-MBGs show their great potential in immunomodulation strategies for bone regeneration, representing a multifunctional biomaterial that can be applied to regenerate bone defects under inflammatory conditions.

Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation.

Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia. Effects that go beyond lipid-lowering actions have been suggested to contribute to their beneficial pharmacological properties. Whether and how statins act on macrophages has been a matter of debate. In the present study, we aimed at characterizing the impact of statins on macrophage polarization and comparing these to the effects of bempedoic acid, a recently registered drug for the treatment of hypercholesterolemia, which has been suggested to have a similar beneficial profile but fewer side effects. Treatment of primary murine macrophages with two different statins, i.e., simvastatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tumor necrosis factor (TNF), interleukin (IL) 1β, and IL6. In contrast, no differences were observed under long-term inflammatory (M1) or anti-inflammatory (M2) conditions, and neither inducible NO synthase (iNOS) expression nor nitric oxide production was altered. Statin treatment led to extracellular-signal regulated kinase (ERK) activation, and the pro-inflammatory statin effects were abolished by ERK inhibition. Bempedoic acid only had a negligible impact on macrophage responses when compared with statins. Taken together, our data point toward an immunomodulatory effect of statins on macrophage polarization, which is absent upon bempedoic acid treatment.

Characterization of an Immune-Enhancing Polysaccharide Fraction Isolated from Heat-Processed Ginseng Derived from Panax ginseng C.A. Meyer

Panax ginseng C.A. Meyer (ginseng) has shown immune-enhancing activity in many studies. The purpose of the present study was to analyze the chemical properties of a polysaccharide fraction (SGP) purified from heat- processed ginseng and to evaluate its immune-enhancing activity using RAW264.7 macrophages. The results showed that SGP increased inducible nitric oxide synthase expression and nitric oxide production in RAW264.7 macrophages. In addition, SGP increased mRNA expression and secretion of interleukin 6 and tumor necrosis factor alpha. Immunoblotting results showed that SGP increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and NF-κB subunit p65 at 500 μg/mL and 1000 μg/mL. Taken together, SGP can activate macrophages through the MAPK and NF-κB signaling pathways, and it may help maintain homeostasis during viral and bacterial infections.

Small-molecule Akt-activation in airway cells induces NO production and reduces IL-8 transcription through Nrf-2

BackgroundThe non-cancerous functions of Akt in the airway are understudied. In some tissues, Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) that has anti-inflammatory effects. NO production has antibacterial and antiviral effects in the airway, and increasing NO may be a useful anti-pathogen strategy. Akt also stimulates the nuclear factor erythroid 2–related factor 2 (Nrf-2) transcription factor, which transcribes antioxidant genes. Therefore, we hypothesized that activation of the Akt/eNOS pathway, which also activates Nrf-2, may have protective effects in human airway cells against injury.MethodsTo directly test the effects of Akt signaling in the airway, we treated A549 and 16HBE cells as well as primary bronchial, nasal, and type II alveolar epithelial cells with small molecule Akt activator SC79. We examined the effects of SC79 on eNOS activation, NO production, Nrf-2 target levels, and interleukin-8 (IL-8) transcription during exposure to TNF-α or Pseudomonas flagellin (TLR5 agonist). Additionally, air–liquid interface bronchial cultures were treated with cadmium, an oxidative stressor that causes airway barrier breakdown.ResultsSC79 induced a ~ twofold induction of p-eNOS and Nrf-2 protein levels blocked by PI3K inhibitor LY294002. Live cell imaging revealed SC79 increased acute NO production. Quantitative RT-PCR showed a ~ twofold increase in Nrf-2 target gene transcription. TNF-α or flagellin-induced IL-8 levels were also significantly reduced with SC79 treatment. Moreover, the transepithelial electrical resistance decrease observed with cadmium was ameliorated by SC79, likely by an acute increase in tight junction protein ZO-1 levels.ConclusionsTogether, the data presented here demonstrate SC79 activation of Akt induces potentially anti-pathogenic NO production, antioxidant gene transcription, reduces IL-8 transcription, and may protect against oxidative barrier dysfunction in a wide range of airway epithelial cells.

Enhanced immune response by vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 macrophages.

Vacuoles are membrane vesicles in eukaryotic cells, the digestive system of cells that break down substances absorbed outside the cell and digest the useless components of the cell itself. Researches on anticancer and intractable diseases using vacuoles are being actively conducted. The practical application of the present study to animals requires the determination of the biocompatibility of vacuole. In the present study, we evaluated the effects of vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 cells. This showed a significant increase in the production of nitric oxide (NO) produced by macrophage activity. Using Reactive Oxygen Species (ROS) assay, we identified that ROS is increased in a manner dependent on vacuole concentration. Western blot analysis showed that vacuole concentration-dependently increased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2). Therefore, iNOS expression was stimulated to induce NO production. In addition, pro-inflammatory cytokines levels promoted, such as interleukin (IL) 6 (IL-6) and tumor necrosis factor (TNF) α (TNF-α). In summary, vacuoles activate the immune response of macrophages by promoting the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines.

Water extract of medicinal ink (WEMI) attenuates lipopolysaccharide-induced NO production of Raw264.7 cells via downregulating JAK2/STAT3-mediated iNOS expression

Ethnopharmacological relevanceMedicinal ink is used as a traditional topical medicine for treating inflammatory diseases via detoxification, relieving pain, hemostasis, and reducing swelling. However, the effect of medicinal ink on the inhibition of inflammatory responses and the underlying molecular mechanism remain unclear. Aim of the studyThe present study aimed to investigate the anti-inflammatory function of water extract of medical ink (WEMI) and elucidate its active mechanisms. Materials and methodsCell viability was assessed using crystal violet staining assay. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. Nitric oxide (NO) production was measured by Griess assay. The activation of inflammatory signaling molecules stimulated by lipopolysaccharide (LPS) was evaluated by assessing levels of inducible nitric oxide synthase (iNOS), phosphorylated Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) using Western blot assay. ResultsWater extract of medical ink (WEMI) did not present cytotoxic effect on murine macrophage Raw264.7 cells. High dosage of WEMI slightly rescued LPS-suppressed cell viability of Raw264.7 cells. WEMI did not induce NO production or IL-6 secretion, though WEMI significantly induced secretion of TNF-α on Raw264.7 cells not stimulated with LPS. On the other hand, LPS effectively stimulated inflammation on Raw264.7 cells; however, WEMI dramatically reduced LPS-induced NO production. WEMI alleviated LPS-stimulated IL-6 secretion but did not affect the content of TNF-α. In addition, WEMI effectively reduced expression of iNOS by abolishing LPS-mediated phosphorylation of JAK2 and STAT3 but not TLR4-mediated NF-κB and MAPK molecules. ConclusionsOur findings suggest that WEMI targets of the JAK2/STAT3-mediated iNOS expression play a key role in alleviating LPS-induced inflammatory responses in RAW264.7 macrophages. Therefore, medicinal ink may be a potential topical agent for treating fasciitis or synovitis via regulating the immune system.

Impact of a long-term high-glucose environment on pro-inflammatory responses in macrophages stimulated with lipopolysaccharide.

Cumulative evidence has established that macrophages orchestrate inflammatory responses that crucially contribute to the pathogenesis of insulin-resistant obesity and type 2 diabetes. In the present study, we examined the impact of hyperglycemia on macrophage pro-inflammatory responses under an inflammatory stimulus. To conduct this study, RAW264.7 macrophages were cultured under normal- (5.5mM) or high-glucose (22 or 40mM) conditions for 7days and stimulated with lipopolysaccharide (LPS). Long-term exposure to high glucose significantly enhanced the increase in the production of pro-inflammatory cytokines, including tumor necrosis-α, interleukin (IL)-1β, and IL-6, when macrophages were stimulated with LPS. The LPS-induced increases in inducible nitric oxide (NO) synthase (iNOS) expression and NO production were also significantly enhanced by long-term exposure of macrophages to high glucose. Treatment with N-acetyl-L-cysteine, a widely used thiol-containing antioxidant, blunted the enhancement of the LPS-induced upregulation of pro-inflammatory cytokine production, iNOS expression, and NO production in macrophages. When intracellular reactive oxygen species (ROS) were visualized using the fluorescence dye 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate, acetyl ester, a significant increase in ROS generation was found after stimulation of macrophages with LPS, and this increased ROS generation was exacerbated under long-term high-glucose conditions. LPS-induced translocation of phosphorylated nuclear factor-κB (NF-κB), a transcription factor regulating many pro-inflammatory genes, into the nucleus was promoted under long-term high-glucose conditions. Altogether, the present results indicate that a long-term high-glucose environment can enhance activation of NF-κB in LPS-stimulated macrophages possibly due to excessive ROS production, thereby leading to increased macrophage pro-inflammatory responses.

Potential Benefits of Berry Anthocyanins on Vascular Function.

Cardiovascular disease (CVD), such as hypertension and atherosclerosis, is the leading cause of global death. Endothelial dysfunction (ED) is a strong predictor for most CVD making it a therapeutic target for both drug and nutrition interventions. It has been previously shown that polyphenols from wine and grape extracts possess vasodilator activities, due to the increased expression and phosphorylation of the endothelial nitric oxide synthase (eNOS), and consequent vasodilator nitric oxide (NO) production. This is vital in the prevention of ED, as NO production contributes to the maintenance of endothelial homeostasis. Moreover, polyphenols have the ability to inhibit reactive oxygen species (ROS), which can cause oxidative stress, as well as suppress the upregulation of inflammatory markers within the endothelium. However, while the majority of the research has focused on red wine, this has overshadowed the potential of other nutritional components for targeting ED, such as the use of berries. Berries are high in anthocyanin flavonoids a subtype of polyphenols with studies suggesting improved vascular function as a result of inducing NO production and reducing oxidative stress and inflammation. This review focuses on the protective effects of berries within the vasculature.

The inhibition of inducible nitric oxide production in lipopolysaccharide-stimulated rat macrophages and in silico studies by flavonoids from Iris spuria L. rhizomes

Ethnopharmacological relevanceIris is the largest genus in the family Iridaceae. Iris plants are distributed in tropical regions of the world. They are used as ornamentals and traditionally used to treat a variety of ailments. AimThis study aimed to evaluate the anti-inflammatory effect of flavonoids isolated from Iris spuria L. Materials and methodsThe isolated flavonoids (1-4) were identified on the basis of different spectroscopic methods (1D- and 2D-NMR) and co-TLC with authentic samples. The anti-inflammatory effect was tested on lipopolysaccharide (LPS)-induced nitric oxide (NO) production from rat-isolated peritoneal macrophages. Modeling and docking simulations of the compounds were performed using Molecular Operating Environment software and the crystal structure of the murine inducible nitric oxide synthase (iNOS). ResultsFour flavonoids (1-4) had been isolated from the rhizomes of Iris spuria L. (Hocka Hoona) for the first time. They were characterized as 5,7,2'-trihydroxy-6-methoxyflavanone (1), tectorigenin 7-O-β-D-glucopyranoside (2), tectorigenin 4'-O-β-D-glucopyranoside (3), and tectorigenin 4'-O-[β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside] (4). The selective inducible NO synthase inhibitor; aminoguanidine was used as a positive control. The production of nitric oxide (NO) was inhibited in a dose-dependent manner of the isolated compounds along with isoflavonoids (5-9) previously isolated from Iris spuria L. (Calizona). A concentration of 60 μg/ml of all tested compounds showed a significant inhibitory effect compared to media with LPS. Molecular modeling experiments supported the obtained biological data. ConclusionOur results reveal that flavonoids isolated from I. spuria L. (Hocka Hoona) and I. spuria L. (Calizona) appear to have a potential anti-inflammatory effect via inhibition of iNOS.

Adlay hull extracts attenuate β-amyloid-induced neurotoxicity and oxidative stress in PC12 cells through antioxidative, anti-inflammatory, and antiapoptotic activities

Alzheimer's disease (AD) is characterized by accumulation of β-amyloid (Aβ) in senile plaques, contributing to oxidative stress, mitochondrial diseases, and synaptic atrophy, consequently leading to the deterioration of brain function. Adlay (Coix lacryma-jobi L.) is an annual botanical. Here, a 95% ethanol extract of adlay hull (AHEE) was partitioned by ethyl acetate (AHEAE), n-butanol (AHBUE), and water (AHWE), and the effects of these extracts on lipopolysaccharide (LPS)-induced RAW264.7 cells and Aβ-induced PC12 cells, as experimental models of neurotoxicity, were evaluated. The expression of anti-inflammatory and antiapoptosis-related proteins was investigated and AHEE, AHEAE, and AHWE were found to exert anti-inflammatory effects. AHWE exhibited antiapoptotic effects and inhibited inducible nitric oxide synthase expression and nitric oxide production. We investigated the protective effects of AHWE against Aβ-induced neurotoxicity in dPC12 cells and explored the underlying mechanism. Pretreatment with AHWE significantly attenuated cell death and Aβ-mediated increase in B cell lymphoma (Bcl)-2/Bax ratio. AHWE significantly inhibited Aβ and enhanced protein kinase B (Akt) level in dPC12 cells, suggesting that its protective effect against Aβ-induced apoptosis in dPC12 cells was mediated through upregulation of the phosphoinositide 3-kinases (PI3K)/Akt signaling pathway. These extracts and its bioactive compound K36–21 may be potentially useful to treat neurodegenerative disorders.

Nitric oxide mediates disruption of human placental trophoblast invasion induced by perfluorobutane sulfonate

Perfluorobutane sulfonate (PFBS), an emerging pollutant, is associated with disruption of placental functions and adverse birth outcomes. However, the precise mechanism of this disruption remains unclear. Extravillous trophoblasts make up the majority of cells in the placenta, and have invasive abilities, which plays a critical role in a successful pregnancy. It has been reported that inducible nitric oxide (iNOS) and nitric oxide (NO) signaling is associated with trophoblast migration and invasion. In this study, PFBS exposure was found to enhance trophoblast invasion and increase matrix metalloproteinase 9 (MMP-9) levels. Additionally, PFBS upregulated iNOS levels and stimulated NO generation. iNOS inhibitor treatment attenuated the increased invasion of trophoblasts and MMP-9 expression induced by PFBS. Extracellular signal-regulated kinase (ERK) phosphorylation was also enhanced by PFBS exposure. In the presence of an ERK pathway inhibitor, however, the increases in trophoblast invasion, the induction of NO production, iNOS expression and MMP-9 expression induced by PFBS were attenuated. Taken together, these results suggest that iNOS/NO signaling is triggered by activation of the ERK signaling pathway, and that iNOS/NO signaling mediates PFBS-induced stimulation of trophoblast invasion.

THE KALLIKREIN-KININ SYSTEM IS FALLING INTO PIECES: BRADYKININ FRAGMENTS ARE BIOLOGICAL ACTIVE PEPTIDES

Objective: Bradykinin [BK-(1–9)] is an endogenous peptide involved in many physiological and pathological processes, such as cardiovascular homeostasis and inflammation. The central dogma of the kallikrein-kinin system is that BK-(1–9) fragments are biologically inactive. In this work, we proposed to test whether these fragments were indeed inactive. Design and method: Nitric oxide (NO) was quantified in human, mouse and rat cells loaded with DAF-FM after stimulation with BK-(1–9), BK-(1–7), BK-(1–5) and BK-(1–3). We used adult male rat aortic ring preparation to test vascular reactivity mediated by BK-(1–9) fragments. Changes in blood pressure and heart rate was measured in conscious adult male rats by intraarterial catheter method. Results: BK-(1–9) induced NO production in all cell types tested by B2 receptor activation. BK-(1–7), BK-(1–5) and BK-(1–3) also induced NO production in all tested cell types but this response was independent of the activation of B1 receptor and/or B2 receptor. BK-(1–7), BK-(1–5) or BK-(1–3) induced only vasorelaxant effect and in a concentration-dependent fashion. Vasorelaxant effects for BK-(1–7), BK-(1–5) or BK-(1–3) were independent of the kinin receptors. BK-(1–9) induced in conscious adult male Wistar rats a dose-dependent hypotension while BK-(1–7), BK-(1–5) and BK-(1–3) induced a dose-independent hypotension. Importantly, these observations diverged from the BK-(1–9) results, highlighting that indeed the BK-(1–9) fragments do not seem to act via the classical kinin receptors. Conclusions: In conclusion, BK-(1–7), BK-(1–5) and BK-(1–3) are biologically active components of the kallikrein-kinin system. Importantly, observed pathophysiological outcomes of these peptides are independent of B1R and/or B2R activation.