Increased Nitric Oxide Production Research Articles

Immunostimulatory activity of stem bark of Kalopanax pictus in RAW 264.7 macrophage

The purpose of this study was to examine the potential immune-enhancing effects of hot water extracts from Kalopanax pictus (KPW) stem barks on mouse macrophage RAW 264.7 cells. Cytotoxicity and nitric oxide (NO) production were measured after KPW treatment via MTT and Griess assays, respectively. The levels of immune-related proteins such as iNOS and COX-2 were determined by Western blot analysis, and cytokines related to immune responses and phagocytic activity were examined by ELISA. KPW significantly increased NO production in a concentration-dependent manner without any cytotoxicity. In addition, the levels of certain enzymes (iNOS and COX-2) and cytokines (TNF-α, IL-1β, IL-6, and IL-12) were also significantly upregulated. Furthermore, the phagocytic activity of RAW 264.7 cells was significantly enhanced by KPW treatment. Taken together, these results suggest that KPW have a potential as an immunostimulatory agent.

Nitric oxide is induced and required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human malignancies. KSHV lytic replication promotes the spread of infection and progression of KSHV-associated malignancies; however, the mechanism regulating KSHV lytic replication remains unclear. In this study, we investigated the role of nitric oxide (NO) in KSHV lytic replication. In the TREx BCBL1-RTA KSHV lytic replication cell system, induction of KSHV lytic replication increased intracellular and extracellular NO. Chemical inhibition of NO production resulted in a lower level of KSHV lytic replication as shown by a reduced level of infectious virions, and decreased levels of viral lytic transcripts and proteins. In a second KSHV lytic replication system of iSLK-RGB-BAC16 cells, we confirmed that KSHV lytic replication increased NO production. Chemical inhibition of NO production resulted in reduced numbers of cells expressing enhanced green fluorescent protein and blue fluorescent protein, two reporters that closely track the expression of KSHV early and late genes, respectively. Consistent with these results, inhibition of NO production resulted in reduced levels of infectious virions, and viral lytic transcripts and proteins. Importantly, exogenous addition of a NO donor was sufficient to enhance the full KSHV lytic replication program. These results demonstrate that NO is required for efficient KSHV lytic replication, and NO playsa crucial role in the KSHV life cycle and KSHV-induced malignancies.

Crambescin C1 Acts as A Possible Substrate of iNOS and eNOS Increasing Nitric Oxide Production and Inducing In Vivo Hypotensive Effect.

Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.

The vascular response to acute hyperglycaemia: What is the role of exercise capacity?

The vascular response to acute hyperglycaemia: What is the role of exercise capacity?

The Release of Nitric Oxide Is Involved in the β-Arrestin1-Induced Antihypertensive Effect in the Rostral Ventrolateral Medulla.

β-Arrestin1 is a multifunctional scaffold protein with the ability to interact with diverse signaling molecules independent of G protein-coupled receptors. We previously reported that overexpression of β-arrestin1 in the rostral ventrolateral medulla (RVLM) decreased blood pressure (BP) and renal sympathetic nerve activity (RSNA) in spontaneously hypertensive rats (SHRs). Nitric oxide (NO) is widely reported to be involved in central cardiovascular regulation. The goal of this study was to investigate whether NO signaling contributes to the β-arrestin1-mediated antihypertensive effect in the RVLM. It was found that bilateral injection of adeno-associated virus containing Arrb1 gene (AAV-Arrb1) into the RVLM of SHRs significantly increased NO production and NO synthase (NOS) activity. Microinjection of the non-selective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME; 10 nmol) into the RVLM prevented the β-arrestin1-induced cardiovascular inhibitory effect. Furthermore, β-arrestin1 overexpression in the RVLM significantly upregulated the expression of phosphorylated neuronal NOS (nNOS) by 3.8-fold and extracellular regulated kinase 1/2 (ERK1/2) by 5.6-fold in SHRs. The β-arrestin1-induced decrease in BP and RSNA was significantly abolished by treatment with ERK1/2 small interfering RNA (ERK1/2 siRNA). Moreover, ERK1/2 siRNA attenuated the β-arrestin1-induced NO production, NOS activity, and nNOS phosphorylation in the RVLM. Taken together, these data demonstrate that the antihypertensive effect of β-arrestin1 in the RVLM is mediated by nNOS-derived NO release, which is associated with ERK1/2 activation.

Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells.

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

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.

Metformin has no impact on nitric oxide production in patients with pre-diabetes

Aims/IntroductionThe authors evaluated the impact of different dose of metformin on NO (nitric oxide) production in subjects with pre-diabetes. Materials and MethodsThe metformin-naïve patients from one Diabetic Center with newly diagnosed pre-diabetes, without cardio-vascular diseases, were randomized (based on the identification number, individual for each inhabitant in the country) for treatment with different doses of metformin (group A 3 × 500 mg, group B 3 × 1000 mg) for 12 weeks. Then, the subjects from group B were switched to dose 3 × 500 for the last 3 weeks. The wide panel of L-arginine/NO pathway metabolites concentrations was assessed using the liquid chromatography-mass spectrometry technique. ResultsBetween October 2017 and December 2018, 36 individuals were initially randomized to intervention groups. The study was completed with 25 subjects: 14 patients in group A, 11 in group B; also 11 healthy volunteers were recruited. There was no difference between participants with pre-diabetes and healthy volunteers as regards the baseline characteristics except for fasting glucose and fatty liver. The decrease of L-citrulline concentration only was reported for treatment groups during the intervention period, with no change for the other NO-production related substances. ConclusionIt was the first study on the in vivo release of NO in humans with different metformin doses in patients with pre-diabetes. Metformin did not seem to increase NO production measured by the citrulline plasma levels, irrespective of the dose. The citrulline concentration change might indicate the drug impact on the condition of the enterocytes.

Bio- and Hemo-Compatible Silk Fibroin PEGylated Nanocarriers for 5-Fluorouracil Chemotherapy in Colorectal Cancer: In Vitro Studies.

5-fluorouracil (5-FU) remains the gold standard of treatment for colorectal cancer, but its poor bioavailability and high systemic toxicity highlight the urgent need for the development of novel delivery strategies to increase the efficacy of 5-FU treatment. The present study is aimed to design and validate a PEGylated Silk Fibroin Nanocarrier (SF/PEG nanoparticles (NPs)) as an efficient 5-FU delivery system for potential intravenous administration. Using the human adenocarcinoma HT–29 cell line as an in vitro model for colorectal cancer, the cytotoxicity screening of the SF/PEG NPs showed that pristine nanocarriers were highly biocompatible, while the addition of 5-FU triggers a dramatic reduction in tumor cell viability, proliferation potential and mitochondrial integrity as well as a significant increase in nitric oxide production. Despite their high in vitro cytotoxicity, the 5-FU SF/PEG NPs were found hemocompatible as no impact on red blood cells hemolysis or the phagocytic activity of the granulocytes was observed. Exposure of HT–29 tumor cells and blood samples to 5-FU SF/PEG NPs augmented the tumor necrosis factor-α levels. Moreover, 5-FU SF/PEG NPs showed an impact on tumor cell migration and invasive potential as both of these processes were inhibited by the NP treatment.

Sauchinone Blocks Ethanol Withdrawal-Induced Anxiety but Spares Locomotor Sensitization: Involvement of Nitric Oxide in the Bed Nucleus of the Stria Terminalis.

Both the positive (manifested by locomotor sensitization) and negative (withdrawal symptoms) reinforcing effects of ethanol (EtOH) involve central nitric oxide (NO) signaling. Sauchinone (a bioactive lignan in Saururus chinensis) has been shown to improve methamphetamine-induced behavioral and neurochemical changes via the NO signaling pathway. Thus, this study evaluated the effects of sauchinone on locomotor sensitization and anxiety during EtOH withdrawal (EtOHW). Male adult Sprague-Dawley rats were treated with 1.5 g/kg/day of EtOH (20%, vol/vol) via intraperitoneal injection for 28 days, followed by a 3-day withdrawal. During withdrawal, the rats were given intragastric sauchinone (2.5, 7.5, or 25 mg/kg/day) once a day. EtOH locomotor sensitization was determined by challenging EtOHW rats with 0.75 g/kg EtOH, while EtOHW-induced anxiety was assessed using the elevated plus maze (EPM). None of the three doses of sauchinone affected EtOH locomotor sensitization. However, in the EPM, treatment of EtOHW rats with sauchinone at 7.5 or 25 mg/kg/day increased both the number of entries into and the time spent in the open arms. Moreover, the two doses of sauchinone inhibited the oversecretion of plasma corticosterone during EtOHW. In the bed nucleus of the stria terminalis (BNST), EtOHW increased NO production, enhanced gene and protein expression of both inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS), and also elevated protein levels of corticotropin-releasing factor, which were all inhibited by 25 mg/kg/day sauchinone. In an in vitro experiment, sauchinone (3, 10, and 30 μM) inhibited H2O2-stimulated nNOS protein expression in neuronal PC12 cells. Finally, intra-BNST infusion of sodium nitroprusside, a NO donor, after sauchinone (25 mg/kg/day) administration, abolished its expected anxiolytic effect. Taken together, these results indicate that sauchinone attenuates anxiety-like behavior in rats during EtOHW but spares EtOH locomotor sensitization, and the anxiolytic effect is mediated via the NO signaling pathway in the BNST.

Regulation of NOS3 by Novel Acetylation Sites

Nitric oxide (NO) is important for cardiovascular and renal health. NO is primarily produced via the enzymatically catalyzed reaction of L-arginine and oxygen to produce NO and L-citrulline by nitric oxide synthase (NOS) isoforms, NOS1, NOS2, and NOS3. NOS3 was first identified in the endothelium but is also present in the kidney epithelium. Endothelial NOS3 maintains cardiovascular health by preventing platelet aggregation, smooth muscle proliferation, and leukocyte adhesion. Epithelial NOS3 controls water and electrolyte homeostasis by regulating ion transporter activity along the nephron. NOS3 activity is tightly controlled by posttranslational modifications. While phosphorylation sites have been well characterized, less is known about regulation by acetylation. Lysine acetylation is a dynamic posttranslational modification observed in both the nucleus and cytosol to regulate intracellular signaling. Our lab and others have shown NOS3 to be regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites on NOS3 which modulate its activity. Based on posttranslational modification prediction software we chose seven lysines to investigate: K486, K497, K504, K607, K610, K727, and K731. We hypothesized that deacetylation of any or all of these lysine sites increases NOS3-mediated NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation in the media over 48 hours. Constructs containing either K486R, K497R, or K504R did not show a significant increase in NO production compared to WT (WT: 1703±499.3 pmol NO2/mg protein; K486R: 1984 ± 1003 pmol NO2/mg protein; K497: 1528 ± 1032 pmol NO2/mg protein; K504: 4337±576.7 pmol NO2/mg protein; p < 0.05 vs WT). However, mutations of four lysines, K607R, K610R, K727R, and K731R (K607/610/727/731R), dramatically increased NO production (21971 ± 1941 pmol NO2/mg protein; WT: 1703±499.3 pmol NO2/mg protein; p < 0.0001 vs WT). To further identify which of the four lysines are responsible for the observed phenotype, we made two new constructs containing either K607/610R or K727/731R. K607/610R was similar to WT (WT: 2271 ± 729.3 pmol NO2/mg protein; K607/610R: 3523 ± 805.8 pmol NO2/mg protein: p > 0.05), yet mutations of K727/731R demonstrated a large increase in NO production (9431 ± 1772 pmol NO2/mg protein; WT: 2271 ± 729.3 pmol NO2/mg protein; p<0.05). These studies suggest K727 and/or K731 is basally acetylated and deacetylation increases NO production uncovering novel posttranslational modifications which regulate NOS3 activity. Future studies will identify whether K727, K731, or both sites are responsible for the increase in NO production.

Inhibitory Effect of Ethanol Extract from Folium et Ramulus Securinegae on TNF‐α induced vascular inflammation in Human Umbilical Vein Endothelial Cells

Folium et Ramulus Securinegae (FRS) is known as a drug that protects and strengthen kidney and spleen by improving the blood circulation and relaxing muscles and tendons. This study investigated whether FRS has protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. Results showed that FRS suppressed TNF-α (Tumor necrosis factor–α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with FRS decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, FRS inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. FRS also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. FRS increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, FRS increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. Overall, the present data suggest that FRS has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

Blue laser-induced selective vasorelaxation by the activation of NOSs

Phototherapy has been tried for treating cardiovascular diseases. In particular, ultraviolet and blue visible lights were suggested to be useful due to their nitric oxide (NO)-production ability in the skin. However, the effects of blue light on the arterial contractility are controversial. Here, we hypothesized that appropriate protocol of blue laser can induce selective vasorelaxation by activating vasodilating signaling molecules in arteries. Using organ chamber arterial mechanics, NO assay, Matrigel assay, and microarray, we showed that a 200-Hz, 300-μs, 445-nm pulsed-laser (total energy of 600 mJ; spot size 4 mm) induced selective vasorelaxation, without vasocontraction in rat mesenteric arteries. The laser stimulation increased NO production in the cord blood-endothelial progenitor cells (CB-EPCs). Both the laser-induced vasorelaxation and NO production were inhibited by a non-selective, pan-NO synthase inhibitor, L-NG-Nitro arginine methyl ester. Microarray study in CB-EPCs suggested up-regulation of cryptochrome (CRY)2 as well as NO synthase (NOS)1 and NOSTRIN (NOS trafficking) by the laser. In conclusion, this study suggests that the 445-nm blue puled-laser can induce vasorelaxation possibly via the CRY photoreceptors and NOSs activation. The blue laser-therapy would be useful for treating systemic hypertension as well as improving local blood flow depending on the area of irradiation.

Characterizing the immune response of chickens to Campylobacter jejuni (Strain A74C).

Campylobacter is one of the major foodborne pathogens causing bacterial gastroenteritis worldwide. The immune response of broiler chickens to C. jejuni is under-researched. This study aimed to characterize the immune response of chickens to Campylobacter jejuni colonization. Birds were challenged orally with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or with 0.5 mL of 0.85% saline. Campylobacter jejuni persisted in the ceca of challenged birds with cecal colonization reaching 4.9 log10 CFU/g on 21 dpi. Campylobacter was disseminated to the spleen and liver on 7 dpi and was cleared on 21 dpi from both internal organs. Challenged birds had a significant increase in anti-Campylobacter serum IgY (14&21 dpi) and bile IgA (14 dpi). At 3 dpi, there was a significant suppression in T-lymphocytes derived from the cecal tonsils of birds in the challenge treatment when compared to the control treatment after 72 h of ex vivo stimulation with Con A or C. jejuni. The T-cell suppression on 3 dpi was accompanied by a significant decrease in LITAF, K60, CLAU-2, IL-1β, iNOS, and IL-6 mRNA levels in the ceca and an increase in nitric oxide production from adherent splenocytes of challenged birds. In addition, on 3 dpi, there was a significant increase in CD4+ and CD8+ T lymphocytes in the challenge treatment. On 14 dpi, both pro and anti-inflammatory cytokines were upregulated in the spleen, and a significant increase in CD8+ T lymphocytes in Campylobacter-challenged birds’ ceca was observed. The persistence of C. jejuni in the ceca of challenged birds on 21 dpi was accompanied by an increase in IL-10 and LITAF mRNA levels, an increase in MNC proliferation when stimulated ex-vivo with the diluted C. jejuni, an increase in serum specific IgY antibodies, an increase in both CD4+ and CD8+ cells, and a decrease in CD4+:CD8+ cell ratio. The balanced Th1 and Th2 immune responses against C. jejuni might explain the ceca’s bacterial colonization and the absence of pathology in Campylobacter-challenged birds. Future studies on T lymphocyte subpopulations should elucidate a pivotal role in the persistence of Campylobacter in the ceca.

Wild radish (Raphanus sativus var. hortensis f. raphanistroides) root extract protects neuronal cells by inhibiting microglial activation

External stimulus-induced activation of microglia plays an important role in the protection of neurons in the central nervous system; however, over-activation of microglia could cause neuronal damage, and it is implicated in the pathogenesis of neurodegenerative diseases. The aim of the present study was to investigate the effects of wild radish (Raphanus sativus var. hortensis f. raphanistroides) root extract (WRE) on microglial over-activation. Mouse microglia BV-2 cells and rat primary microglia were stimulated with lipopolysaccharide (LPS), treated with WRE, and analyzed for nitric oxide (NO) production, pro-inflammatory cytokine secretion, inducible NO synthase (iNOS) expression, and p38 kinase phosphorylation. Human neuroblastoma SH-SY5Y cells were treated with microglia-conditioned medium and analyzed for cell viability. Stimulation with LPS increased NO production and iNOS expression in BV-2 cells and primary microglia, but the treatment with WRE decreased both. Furthermore, WRE downregulated the mRNA expression and secretion of inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), and inhibited the phosphorylation of p38 in LPS-activated microglia. Treatment with the conditioned medium of LPS-induced BV-2 cells decreased the viability of SH-SY5Y cells, but the damaging effect was significantly alleviated in cells treated with the conditioned medium of LPS plus WRE-cultured microglia. This indicated that the WRE treatment of microglia could protect neuronal cells from microglial activation-induced neurotoxicity. WRE may be a potential food product to attenuate neuroinflammation via the inhibition of microglial over-activation, which can slow down the neurodegenerative processes in the brain.

Nitric oxide inhibits interleukin-1-mediated protection against Escherichia coli K1-induced sepsis and meningitis in a neonatal murine model.

Neonatal meningitis-associated Escherichia coli (NMEC) is a leading cause of sepsis and meningitis in newborn infants. Neonates are known to have impaired inflammasome activation and interleukin (IL)-1 production. However, it is unknown what role this plays in the context of NMEC infection. Here we investigated the role of IL-1 signaling in the pathogenesis of NMEC infection. We found both IL-1β and IL-1α were secreted from macrophages and microglial cells in response to NMEC in a Toll-like receptor 4- and NLR family pyrin domain containing 3 (NPLR3)-dependent manner. Intracerebral infection of adult mice indicated a protective role of IL-1 signaling during NMEC infection. However, IL-1 receptor blockade in wild-type neonatal mice did not significantly alter bacterial loads in the blood or brain, and we, therefore, investigated whether protection conferred by IL-1 was age dependent. Neonates are known to have increased nitric oxide (NO) levels compared with adults, and we found NO inhibited the secretion of IL-1 by macrophages in response to NMEC. In contrast to our results in wild-type neonates, blockade of IL-1 receptor in neonates lacking inducible nitric oxide synthase (iNOS) led to significantly increased bacterial loads in the blood and brain. These data indicate IL-1 signaling is protective during NMEC infection in neonates only when iNOS is absent. Collectively, our findings suggest that increased NO production by neonates inhibits IL-1 production, and that this suppresses the protective role of IL-1 signaling in response to NMEC infection. This may indicate a general mechanism for increased susceptibility of neonates to infection and could lead to new therapeutic strategies in the future.

Nuclear localization of endothelial nitric oxide synthase and nitric oxide production attenuates aphidicolin-induced endothelial cell death

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 μM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.

Empagliflozin, alone or in combination with liraglutide, limits cell death in vitro: role of oxidative stress and nitric oxide

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitor empagliflozin and glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide are characterized as having cardiovascular benefits in patients with type 2 diabetes (T2D). Little is known regarding the underlying mechanisms nor the potential interactions between cardiovascular benefits of these two drugs when combined. We sought to investigate: (1) whether combination of empagliflozin and liraglutide has additive effect against diabetes-induced cytotoxicity, and (2) potential mechanisms involved in cardioprotective effect of empagliflozin and liraglutide in diabetes. Capacity of empagliflozin and liraglutide alone and in combination to reduce cardiac injury in diabetes was evaluated. HL-1 cells, a cardiac muscle cell line, were exposed to hyperglycemia/hyperinsulinemia and treated with/without empagliflozin, liraglutide or empagliflozin + liraglutide for 24h. At the end of treatments, cytotoxicity, oxidative stress, nitric oxide (NO) production, nitric oxide synthase (NOS) activity and phospho-eNOS (Thr495) expression were determined. We found that empagliflozin treatment alone and combined treatment decreased in vitro HL-1 cell death caused by hyperglycemia. Liraglutide treatment alone improved NOS activity followed by increased NO production, while empagliflozin had little effect. Furthermore, the effects of empagliflozin + liraglutide to decrease diabetes-induced cytotoxicity and oxidative stress were synergistic. While empagliflozin alone attenuated diabetes-induced cytotoxicity, combined treatment of liraglutide can synergistically ameliorates cell death and oxidative stress. This effect is potentially due to improved NOS activity and increased NO production induced by liraglutide.

Manool, a diterpene from Salvia officinalis, exerts preventive effects on chromosomal damage and preneoplastic lesions

The present study aimed to evaluate the effect of the manool diterpene on genomic integrity. For this purpose, we evaluated the influence of manool on genotoxicity induced by mutagens with different mechanisms of action, as well as on colon carcinogenesis. The results showed that manool (0.5 and 1.0 µg/ml) significantly reduced the frequency of micronuclei induced by doxorubicin (DXR) and hydrogen peroxide in V79 cells but did not influence genotoxicity induced by etoposide. Mice receiving manool (1.25 mg/kg) exhibited a significant reduction (79.5%) in DXR-induced chromosomal damage. The higher doses of manool (5.0 and 20 mg/kg) did not influence the genotoxicity induced by DXR. The anticarcinogenic effect of manool (0.3125, 1.25 and 5.0 mg/kg) was also observed against preneoplastic lesions chemically induced in rat colon. A gradual increase in manool doses did not cause a proportional reduction of preneoplastic lesions, thus demonstrating the absence of a dose-response relationship. The analysis of serum biochemical indicators revealed the absence of hepatotoxicity and nephrotoxicity of treatments. To explore the chemopreventive mechanisms of manool via anti-inflammatory pathways, we evaluated its effect on nitric oxide (NO) production and on the expression of the NF-kB gene. At the highest concentration tested (4 μg/ml), manool significantly increased NO production when compared to the negative control. On the other hand, in the prophylactic treatment model, manool (0.5 and 1.0 μg/ml) was able to significantly reduce NO levels produced by macrophages stimulated with lipopolysaccharide. Analysis of NF-kB in hepatic and renal tissues of mice treated with manool and DXR revealed that the mutagen was unable to stimulate expression of the gene. In conclusion, manool possesses antigenotoxic and anticarcinogenic effects and its anti-inflammatory potential might be related, at least in part, to its chemopreventive activity.

Resveratrol and grape juice: Effects on redox status and nitric oxide production of endothelial cells in in vitro preeclampsia model.

Preeclampsia (PE) is a hypertensive disorder of pregnancy and it is one of the main causes of maternal and fetal morbidity and mortality worldwide. It is known that oxidative stress plays a role in its pathophysiology, therefore we investigated the effects of trans-resveratrol, a potent antioxidant, on the Nrf2/ARE pathway, nitric oxide (NO) production, and reactive oxygen species (ROS) levels in an in vitro model of PE. Plasma from PE patients increased ARE activity in endothelial cells compared with plasma from healthy pregnant (HP), and the addition of resveratrol was able to potentiate this increase only in PE. Resveratrol also decreased ROS levels in the cells incubated with plasma from PE. Based on these results, we performed a pilot clinical study to compare the effects of serum from PE women before and 1h after ingestion of polyphenol-rich whole red grapefruit juice incubated on endothelial cells, since grapefruit contains large amounts of resveratrol. Serum from PE patients, obtained one hour after juice intake, decreased antioxidants markers in cells compared with the serum before juice intake, besides, it increased NO production. In conclusion, resveratrol and polyphenol-rich red grape juice have potentially beneficial effects on endothelial cells incubated with PE plasma/serum, which could aid in the management of PE.