Lowered Nitric Oxide Levels Research Articles

The effects of HIV-1-proteins and antiretroviral therapy on aortic endothelial cells (AECs). A mechanistic in vitro approach

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation Introduction Endothelial dysfunction (ED) is an early precursor of cardiovascular disease characterized by decreased nitric oxide (NO) and increased oxidative stress, creating a pro-inflammatory environment. Increased ED and cardiovascular risk have been observed in HIV-1 infection and antiretroviral therapy (ART). Purpose The United Nations AIDS update indicates that in 2015 17.0 million people worldwide were living with HIV and receiving antiretroviral therapy. Considering the extent of this global pandemic and its comorbidity with CVD, zooming in on HIV’s intersection with antiretroviral therapy and cardiovascular health is of paramount importance. Yet, many of the mechanisms involved are still poorly understood. This knowledge can ultimately assist with the careful selection of antiretroviral-agents in patients with underlying cardiovascular risk factors. Objectives Establishing a simulated model of in vitro HIV-1-infection in aortic endothelial cells and determining the effects of non/nucleoside reverse transcriptase inhibitors (NRTI/NNRTIs) and protease inhibitors (PIs) on markers of endothelial function and the expression/activation of vascular signalling proteins. Methods A simulated HIV-1-model was established by adding recombinant HIV-1 proteins to growth medium in a single cocktail containing Tat, Gp160 and Nef. ART1 (NRTI/NNRTIs) were administered as a single cocktail to growth medium, containing efavirenz (EFV), emtricitabine (FTC) and tenofovir (TDF). ART2 (PIs) were administered as a single cocktail to growth medium, containing lopinavir (LPV) and ritonavir (RTV). All the aforementioned treatments were subject to dose-response investigations and treatment lasted 24 hours. Cell viability, NO-production and oxidative stress were measured using fluorescent probes. Selected dosages from above studies were further used and important vascular signalling proteins in HIV-1 exposed AECs were investigated by western blotting. Results HIV-1 (100ng/ml, 24h) resulted in ↓NO-levels (DAF-fluorescence intensity: 100±1.25% vs. 76.00±3.67%; p<0.05) with no oxidative stress. HIV-1+PI treatment lowered NO levels (DAF-fluorescence: 100±0.72% vs. 94.47±1.91%; p<0.05). HIV-1+ NRTI/NNRTI treatment had no effects. HIV-1+PI treatment ↓IκBα expression (inhibitory signalling protein in pro-inflammatory NF-κB pathway) (1.0 ± 0.15 vs 0.37 ± 0.03; p<0.05) and ↓eNOS expression (1.0 ± 0.21 vs. 0.28 ± 0.04; p<0.05). The same was observed for PKB/Akt (1.0 ± 0.03 vs. 0.68 ± 0.02; p<0.05). No effects on cell viability and oxidative stress were observed. Conclusion Exposure of AECs to combined HIV-1-protein and PI treatment lowered NO-production, which may be linked to underlying PKB/Akt and eNOS downregulation. HIV-1+PI treatment was associated with increased pro-inflammatory NF-κB signalling. Overall, our results suggest that a dysfunctional state was induced in AECs exposed to HIV-1 proteins and PI treatment.

Comparison of Total Phenolic Contents, Antioxidant and Anti-Inflammation Activity in Sweetpotato Leaves Obtained from 24 Different Cultivars

Abstract Objectives Sweetpotato is one of the most important crops in the world. However, most of the sweetpotato leaves (SPLs) are discarded despite their high nutritional values. To evaluate the potential utility of SPLs as a functional food, the objectives were to measure the total phenolic contents and their antioxidant activities and to determine the anti-inflammatory effects of SPL phenolic extracts from 24 different cultivars in Arkansas. Methods Sweetpotato leaves were analyzed for total phenolics (TP) and antioxidant activities using Folin-Ciocalteu's assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, respectively. To assess cell viability and nitric oxide (NO) inhibition activities, RAW 264.7 cells were pretreated with SPL phenolic extracts at the concentrations of 50–500 μg/mL for 1 h and stimulated with lipopolysaccharide (LPS, 100 ng/mL). Cell viability and NO production were measured using the Cell Proliferation assay and Griess reaction system, respectively. All statistical analyses were performed using the ANOVA procedure (SAS 9.4) with significance at P < 0.05 level. Results Among 24 SPL extracts, SPL#1, 3, 9, 38, and 11 exhibited the strong DPPH scavenging activity with 247, 137, 117, 111 and 106 mM Trolox equivalent/g, respectively. Extracts of SPL#1, 9, 3, 7, and 10 showed a high content of phenolics with containing 17.6, 14.8, 14.7, 14.3 and 14.2 mg gallic acid equivalent/g. When the RAW 264.7 cells were treated with 50–500 μg/mL of SPLs for 24 h, SPL extracts up to 250 μg/mL did not cause any significant cytotoxicity. After inducing inflammation with LPS, 50–100 μg/mL of 24 SPL treatments lowered NO levels (11–44%) in a dose-dependent manner compared to the positive control (P < 0.05). Also, SPL#1, 3, 9, 38, and 11, indicating the highest DPPH radical scavenging activity, showed significant NO reduction (15–49%) at the concentrations of 50–200 μg/mL (P < 0.05). Conclusions The findings showed that sweetpotato leaves are a good source of phenolics to exert significant antioxidant activity and NO inhibitory effects in a cellular system, suggesting that sweetpotato leaves may be used as a functional food for improving immune response. Funding Sources The work was supported by USDA-NIFA.

Jasonia glutinosa (L.) DC., a traditional herbal medicine, reduces inflammation, oxidative stress and protects the intestinal barrier in a murine model of colitis.

Jasonia glutinosa (L.) DC., known as rock tea (RT), is traditionally used in Spain as a digestive due to its beneficial properties in bowel disorders. The pharmacological nature of these properties has not been established yet. The aim of this work was to evaluate the therapeutic utility of RT in experimental colitis and to identify chemical constituents with anti-inflammatory and/or anti-oxidative properties. RT extract was prepared with ethanol in a Soxhlet apparatus and analysed by HPLC-DAD. Superoxide radical scavenging properties, xanthine oxidase and lipoxygenase (5-LOX) inhibitory activity, and capability to lower nitric oxide (NO) and tumor necrosis factor α (TNF-α) levels were measured in cell-free and cell-based assays. In the 2.5%-dextran-sodium sulphate (DSS) injury-repair model of ulcerative colitis (UC), mice were daily treated with sulfasalazine (SSZ, as reference drug, 100mg/kg bw), RT (5, 25 and 50mg/kg bw, p.o.), or vehicle over 20 days. Colitis was scored daily. Colon samples were examined macroscopically and histopathologically. Protein levels of myeloperoxidase (MPO), interleukins 6, and 10 (IL-6, IL-10), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied as markers of oxidative stress and inflammatory activity. The integrity of the apical epithelial layer was assessed by immunofluorescence staining of zonula ocludens-1 (ZO-1). Finally, intestinal contractility was also evaluated by isometric myography. Fifteen phenolic compounds and three pigments were identified and quantified, of which caffeoylquinic acids, and the flavonoid, quercetin-3-O-galactoside, were the most abundant. RT extract significantly scavenged superoxide radicals, inhibited 5-LOX activity, and lowered NO and TNF-α levels. DSS-treated mice receiving RT scored clinically lower than controls during the first 3 days of DSS treatment and during the recovery period. SSZ was less effective than RT. Anatomical and histological examination of colon samples revealed that RT significantly prevented colon shortening, increased colon thickness, and lowered the macroscopic damage score. RT also significantly prevented the increase of MPO activity, IL-6 levels, iNOS and COX-2 expression, the loss of ZO-1 apical expression, and normalized contractility disturbances. In conclusion, daily administration of RT showed therapeutic properties in the DSS-model of UC. The benefits of RT can likely be attributed to its anti-inflammatory and antioxidant phenolic and flavonoid constituents.

Inhibitory effects of cranberry polyphenol and volatile extracts on nitric oxide production in LPS activated RAW 264.7 macrophages.

Cranberry volatiles have received little attention for health-promoting properties. In this study, we compared the inhibitory effects of cranberry polyphenol and volatile extracts and volatile standards on nitric oxide (NO) production in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Polyphenols were analyzed by HPLC/HPLC-MS and volatiles were analyzed by GC/GC-MS. The inhibition of NO production of the fresh cranberry polyphenol and volatile extracts and α-terpineol, linalool, linalool oxide, and eucalyptol standards at 2, 4, and 8-fold dilutions of their original concentrations in fresh cranberries was evaluated by treating these extracts/standards for 1 h before or after LPS application for 24 h. After inducing inflammation with LPS, the polyphenol treatments (317.8 and 635.7 μg g-1) and 1.8 μg g-1 volatile treatment lowered NO levels 46-62% compared to the positive control (P < 0.05). When the cells were treated with polyphenol and volatile extracts before inducing inflammation, the 635.7 μg g-1 and 317.8 μg g-1 polyphenol treatments and 1.8 μg g-1 and 0.9 μg g-1 volatile treatments lowered NO levels (13-52%) compared to the positive control (P < 0.05). Polyphenol and volatile extracts from cranberry were effective in reducing NO production whether applied before or after the application of LPS. α-Terpineol at a concentration found in fresh cranberries (1.16 μg mL-1) was also found to be effective in reducing NO production whether cells were treated before or after application of LPS. Future studies are needed to reveal the mechanisms by which volatile compounds, especially α-terpineol act to mitigate inflammation and to determine the bioavailability of terpenes.

The role of nitric oxide in the interaction of Arabidopsis thaliana with the biotrophic fungi, Golovinomyces orontii and Erysiphe pisi

Powdery mildews are a diverse group of pathogenic fungi that can infect a large number of plant species, including many economically important crops. However, basic and applied research on these devastating diseases has been hampered by the obligate biotrophic lifestyle of the pathogens, which require living host cells for growth and reproduction, and lacking genetic and molecular tools for important host plants. The establishment of Arabidopsis thaliana as a host of different powdery mildew species allowed pursuing new strategies to study the molecular mechanisms governing these complex plant–pathogen interactions. Nitric oxide (NO) has emerged as an important signaling molecule in plants, which is produced upon infection and involved in activation of plant immune responses. However, the source and pathway of NO production and its precise function in the regulatory network of reactions leading to resistance is still unknown. We studied the response of Arabidopsis thaliana to infection with the adapted powdery mildew, Golovinomyces orontii (compatible interaction) and the non-adapted, Erysiphe pisi (incompatible interaction). We observed that NO accumulated rapidly and transiently at infection sites and we established a correlation between the resistance phenotype and the amount and timing of NO production. Arabidopsis mutants with defective immune response accumulated lower NO levels compared to wild type. Conversely, increased NO levels, generated by treatment with chemicals or expression of a NO-synthesizing enzyme, resulted in enhanced resistance, but only sustained NO production prevented excessive leaf colonization by the fungus, which was not achieved by a short NO burst although this reduced the initial penetration success. By contrast, lowered NO levels did not impair the ultimate resistance phenotype. Although our results suggest a function of NO in mediating plant immune responses, a direct impact on pathogen growth and development cannot be excluded.

Antinociceptive Effects of Radon Inhalation on Formalin-Induced Inflammatory Pain in Mice

Radon therapy is clinically useful for the treatment of inflammatory diseases. The mechanisms of pain relief remain to be fully elucidated. In this study, we investigated the antinociceptive effects of radon inhalation in a mouse model of formalin-induced inflammatory pain. Immediately, after radon inhalation at a concentration of background level (ca. 19Bq/m(3)), 1,000 or 2,000Bq/m(3) for 24h, 1.35% formalin (0.5% formaldehyde in saline, 20μl) was subcutaneously injected into the hind paw of mice, and we measured licking response time. Radon inhalation inhibited the second phase of response in formalin test. Formalin administration induced nociception and increased tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) levels in serum and leukocyte migration in paws. Concurrently, formalin injection decreased antioxidative functions. Radon inhalation produced antinociceptive effects, i.e., lowered serum TNF-α and NO levels, and restored antioxidative functions. The results showed that radon inhalation inhibited formalin-induced inflammatory pain.

Preventive and curative effects of radon inhalation on chronic constriction injury‐induced neuropathic pain in mice

Radon therapy is clinically useful for the treatment of pain-related diseases. However, there have been no studies regarding the effects of radon inhalation on neuropathic pain. In this study, we aimed to determine whether radon inhalation actually induced a remission of neuropathic pain and improved the quality of life. First, we investigated the antinociceptive effects of radon inhalation in the chronic constriction injury (CCI) model of neuropathic pain. We evaluated pain behaviour in mice before and after CCI surgery, using von Frey test. Pretreated mice received CCI surgery immediately after 24-h inhalation of radon at background (BG) concentration (c. 19 Bq/m(3) ), or at a concentration of 1000 or 2000 Bq/m(3) , and post-treated mice inhaled similar levels of radon 2 days after CCI surgery. CCI surgery induced mechanical allodynia and hyperalgesia on a plantar surface of mice, as assessed using von Frey test, and 2000 Bq/m(3) radon inhalation alleviated hyperalgesic conditions 22-37% compared to BG level concentration. Concurrently, CCI surgery increased norepinephrine (NE), tumour necrosis factor-alpha (TNF-α) and nitric oxide (NO) concentrations in plasma, and leukocyte migration in paws. Furthermore, CCI-induced neuropathy reduced superoxide dismutase (SOD) activity. Treatment with radon inhalation, specifically at a concentration of 2000 Bq/m(3) , produced antinociceptive effects, i.e., lowered plasma TNF-α, NE and NO levels and restored SOD activity, as well as pain-related behaviour. This study showed that inhalation of 2000 Bq/m(3) radon prevented and alleviated CCI-induced neuropathic pain in mice.

Selenium has a protective effect on ischemia/reperfusion injury in a rat ovary model: biochemical and histopathologic evaluation

Background/PurposeThe aim of the study was to evaluate the effects of selenium (Se) on ischemia/reperfusion (I/R) injury in rat ovaries. MethodsThirty-five female Sprague-Dawley rats were randomly divided into 5 groups (n = 7): sham (S), I/R1, I/R2, Se1, and Se2. In the I/R1 and Se1 groups, 4 hours of ischemia was followed by 6 hours of reperfusion, and in the I/R2 and Se2 groups, 4 hours of ischemia was followed by 12 hours of reperfusion. In the Se groups, 30 minutes before reperfusion, a single dose of 0.2 mg/kg Se was administered intraperitoneally. The ovarian tissue levels of malondialdehyde (MDA) and nitric oxide (NO), and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured biochemically. Tissue damage to ovarian tissue was scored by histopathologic examination. ResultsThe I/R groups had significantly higher MDA levels and lower CAT, SOD, and GPx activities than the sham group (P < .05). Although NO levels were significantly higher in the I/R1 group than in the sham group (P < .05), the NO levels in the I/R2 and sham groups were similar. Selenium pretreatment significantly lowered tissue MDA and NO levels and increased tissue SOD and GPx activities in the Se groups, compared with those in the I/R groups (P < .05). Catalase activities were significantly higher in the Se2 group than in the I/R2 group (P < .05). Catalase activities were higher in the Se1 group than in the I/R1 group, but the difference was not statistically significant. Treatment with Se significantly decreased the ovarian tissue damage scores in the Se2 group compared with those in the I/R2 group (P < .05). ConclusionSelenium is effective in preventing tissue damage induced by I/R in rat ovaries.

Garlic provides protection to mice heart against isoproterenol-induced oxidative damage: Role of nitric oxide

Garlic has been widely recognized as a cardioprotective agent. However, the molecular mechanism of its cardioprotective effects is not well established. Here we hypothesized that aqueous garlic homogenate may mediate cardioprotection via nitric oxide (NO). Mice were fed with saline and aqueous garlic homogenate (250 and 500mgkg−1day−1 orally) for 30days. In another set of experiment, mice were pre-treated with saline, aqueous garlic homogenate (AGH) (250mgkg−1day−1 for 30days), and AGH (30days) along with L-NAME (20mgkg−1day−1 i.p. for last 7days) before inducing acute myocardial infarction by isoproterenol (s.c. injection of isoproterenol 150mgkg−1day−1 for 2days) and sacrificed after 48h. Dose dependent increase in serum NO level was observed after garlic 250 and 500mgkg−1 dose feeding. While no change in serum SGPT and SGOT level, a significant decrease in serum LDH level was observed after garlic feeding. Garlic-induced NO formation was further confirmed in human aortic endothelial cells (HAEC). Administration of isoproterenol caused a significant decrease in endogenous antioxidants i.e., myocardial catalase, GSH and GPx activity, and mitochondrial enzyme activities like citrate synthase and β hydroxyacyl CoA dehydrogenase. All those deleterious cardiac changes induced by isoproterenol were significantly attenuated by garlic homogenate. However this beneficial effect of garlic was blunted when garlic was administered with L-NAME, a nonspecific inhibitor of nitric oxide synthase (NOS). Further, a significant increase in myocardial TBARS and decrease in total antioxidant activity was observed in L-NAME treated group compared to isoproterenol treated group. Administration of L-NAME in mice from control group lowered serum and cardiac NO levels without any change of oxidative stress parameters. In conclusion, our study provides novel evidence that garlic homogenate is protective in myocardial infarction via NO-signaling pathway in mice.

Inhibitory effects of palm α-, γ- and δ-tocotrienol on lipopolysaccharide-induced nitric oxide production in BV2 microglia

Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24 h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment ( p < .05). None of the tocotrienol doses tested affected microglia viability.

AQUEOUS EXTRACT OF HIBISCUS SABDARIFFA ALONE OR IN COMBINATION WITH VALSARTAN ATTENUATES RENOVASCULAR HYPERTENSION IN RATS

The present study was designed to investigate the possible modulative effects of aqueous extract of Hibiscus sabdariffa (HS) either alone or in combination with valsartan on hypertensive male albino rats. Renovascular bypertension was induced by ligation of left renal artery for 4 weeks. HS in a dose of 250 mg/kg, valsartan in a dose of 5 mg/kg and their combination were given daily to hypertensive rats for 45 days as single oral daily dose. HS either alone or in combination with valsartan reduced blood pressure (BP) and heart rate of hypertensive rats. It also increased aortic glutathione content (GSH) and super oxide dismutase (SOD) activity. It improved lipid profile, decreased nitric oxide (NO) levels and improved the histopathological changes of aorta. Valsartan alone decreased BP more than HS, increased aortic GSH content, aortic SOD activity and NO serum level but failed to correct lipid profile. Based on this study, it can be concluded that HS alone and in combination with valsartan showed potent antihypertensive effects. This effect may be mediated via antioxidant, hypolipidemic effects and correction of lowered serum NO levels. Valsartan has more potent antihypertensive effect than HS but it was not able to correct dyslipidemia and histopathological changes associated with hypertension.

Raloxifene decreases serum malondialdehyde and nitric oxide levels in postmenopausal women with end‐stage renal disease under chronic hemodialysis therapy

We prospectively aimed to investigate the relationship between raloxifene administration and serum malondialdehyde (MDA), nitric oxide (NO), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride levels in postmenopausal women undergoing hemodialysis treatment for chronic renal failure. Twenty-four women scheduled for twice-weekly hemodialysis treatment were given 60 mg/day raloxifene for 3 months. Serum MDA, estimated NO, HDL, and LDL measurements were assessed initially and at month 3. We used the Mann-Whitney U and Wilcoxon signed-rank tests for comparisons. Five women discontinued the study. After controlling for baseline characteristics, mean serum MDA and estimated NO levels decreased (P = 0.0001 and P = 0.0001, respectively) from 2.01 +/- 0.40 micromol/L and 21.3 +/- 17.9 micromol/L at baseline to 1.27 +/- 0.23 micromol/L and 7.7 +/- 7.5 micromol/L at month 3, respectively. Mean serum LDL level declined (P = 0.004) from 120.0 +/- 24.3 mg/dL to 103.3 +/- 12.1 mg/dL, and mean HDL level increased (P = 0.024) from 52.2 +/- 7.8 mg/dL to 57.2 +/- 5.7 mg/dL with raloxifene administration. Oral raloxifene administration (60 mg/day) for 3 months lowered serum MDA and NO levels with favorable effects on serum lipid parameters in postmenopausal women, who were undergoing long-term hemodialysis treatment for chronic renal failure.

Selective Inhibitors of Inducible Nitric Oxide Synthase: Potential Agents for the Treatment of Inflammatory Diseases?

Nitric Oxide (NO) is widely recognized as an important messenger and effector molecule in a variety of biological systems. There is strong evidence from animal models that elevated or lowered NO levels are associated with a variety of pathological states. In nature, NO is synthesised from the amino acid l-arginine by a small family of closely related oxygenase enzymes: the nitric oxide synthases (NOS). A number of studies in animals have associated excessive NO production by one of these enzymes--the inducible NOS isoform (iNOS or NOS-II)--with acute and chronic inflammation in model systems and have also demonstrated that administration of NOS inhibitors can produce beneficial effects. Regrettably, however, the relatively poor potency, selectivity and pharmacokinetic (ADME) profiles of the available inhibitors have so far precluded a convincing demonstration of their efficacy in the clinic. This review will describe the current state of knowledge of the structure and function of NOS and the various approaches that are being followed in the search for truly selective NOS inhibitors as therapeutic agents for inflammatory diseases.

Gastrointestinal nitric oxide generation in germ-free and conventional rats.

Nitric oxide (NO) is a central mediator of various physiological events in the gastrointestinal tract. The influence of the intestinal microflora for NO production in the gut is unknown. Bacteria could contribute to this production either by stimulating the mucosa to produce NO, or they could generate NO themselves. Using germ-free and conventional rats, we measured gaseous NO directly in the gastrointestinal tract and from the luminal contents using a chemiluminescence technique. Mucosal NO production was studied by using an NO synthase (NOS) inhibitor, and to evaluate microbial contribution to the NO generation, nitrate was given to the animals. In conventional rats, luminal NO differed profoundly along the gastrointestinal tract with the greatest concentrations in the stomach [>4,000 parts per billion (ppb)] and cecum (approximately 200 ppb) and lower concentrations in the small intestine and colon (< or =20 ppb). Cecal NO correlated with the levels in incubated luminal contents. NOS inhibition lowered NO levels in the colon, without affecting NO in the stomach and in the cecum. Gastric NO increased greatly after a nitrate load, proving it to be a substrate for NO generation. In germ-free rats, NO was low (< or =30 ppb) throughout the gastrointestinal tract and absent in the incubated luminal contents. NO also remained low after a nitrate load. Our results demonstrate a pivotal role of the intestinal microflora in gastrointestinal NO generation. Distinctly compartmentalized qualitative and quantitative NO levels in conventional and germ-free rats reflect complex host microbial cross talks, possibly making NO a regulator of the intestinal eco system.

Glutathione depletion in CYP2E1-expressing liver cells induces toxicity due to the activation of p38 mitogen-activated protein kinase and reduction of nuclear factor-kappaB DNA binding activity.

Depletion of glutathione (GSH) from CYP2E1-expressing cells by treatment with l-buthionine sulfoximine (BSO) causes decreased cell viability. The possible role of mitogen-activated protein kinases (MAPK) in this toxicity was evaluated. SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], an inhibitor of p38 MAPK decreased the BSO-dependent toxicity in HepG2 E47 cells, which express CYP2E1 and in hepatocytes from pyrazole-treated rats. Inhibitors of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and c-Jun amino-terminal kinase were not protective. SB203580 did not prevent the loss of GSH nor lower the increase in reactive oxygen production; hence, protection by SB203580 was downstream of the elevated oxidative stress. Treatment with BSO caused activation of p38 MAPK whereas activation of nuclear factor-kappaB (NF-kappaB) was decreased; these effects were prevented by SB203580. We speculated that the decrease in NF-kappaB activation prevented production of hepatoprotective factors. One such factor could be nitric oxide (NO); indeed a NO donor decreased the BSO plus CYP2E1-dependent toxicity, whereas inhibition of inducible NO synthase (iNOS) potentiated toxicity. BSO treatment down-regulated iNOS and lowered NO levels, reactions blocked by SB203580; however, protection by SB203580 was the same in the absence or presence of an iNOS inhibitor, indicating that recovery of iNOS and NO production was not the mechanism by which SB203580 afforded protection against the BSO plus CYP2E1-dependent toxicity. Presumably other protective factors besides nitric oxide may be produced from activated NF-kappaB when p38 MAPK is inhibited by SB203580. These results suggest that the activation of p38 MAPK by BSO treatment in CYP2E1-expressing liver cells cause a loss in NF-kappaB-dependent production of hepatoprotective factors. This loss, coupled to CYP2E1-generated oxidant stress, synergize to promote cell injury.

FR167653 attenuates murine immunological liver injury.

To study the effect of FR167653 on immunological liver injury (ILI) in mice. ILI was established by tail vein injection of 2.5 mg Bacillus Calmette-Guerin (BCG), and 10 d later with 10 mg lipopolysaccharide (LPS) in 0.2 mL saline (BCG plus LPS). Alanine aminotransferase (ALT), aspartate aminotransferase (AST) in sera and malondialdehyde (MDA), glutathione peroxidase (GSHpx) contents in liver homogenates were assayed by spectrophotometry. The levels of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) levels in sera were determined using ELISA. Interleukin-1 (IL-1) produced by peritoneal macrophages was determined by the method of (3)H-infiltrated cell proliferation. The nuclear factor-kappa B (NF-kappaB) p65 in liver tissue was analyzed with reverse transcription polymerase chain reaction (RT-PCR). Liver samples collected were stained with hematoxylin and eosin. FR167653 (50, 100, 150 mg/kg) could significantly decrease the serum transaminase (ALT, AST) activity and MDA content in liver homogenate, and improve reduced GSHpx level of liver homogenate. Liver histopathological examination showed FR167653 (100, 150 mg/kg) significantly reduced inflammatory cells infiltration and liver cells necrosis. FR167653 (50, 100, 150 mg/kg) significantly lowered TNF-alpha and NO levels in serum, and IL-1 produced by peritoneal macrophages. Moreover, expression of NF-kappaB mRNA in liver tissue of ILI induced by BCG plus LPS was significantly reduced by FR167653. All results showed that FR167653 had significant inhibitory action on ILI in mice.

Cardiac necrosis markers associated with low nitric oxide levels in the plasma of rabbits after treatment with vasopressin: protective effects of nitroglycerin administration

It has been reported that the administration of vasopressin induces myocardial ischaemia in rats, which causes electrocardiographic ST segment alterations according to many authors. But rat electrocardiogram (ECG) lacks ST segment. Consequently it appears important to study the effects of vasopressin in rabbits, which show ST segment present in the ECG. Since cardiac necrosis markers are released in the plasma of humans with myocardial infarction, as well as in a variety of experimental models of myocardial necrosis, it is possible that the same may occur in rabbits with myocardial ischaemia induced by vasopressin. The main aim of this study was to investigate whether the administration of vasopressin causes the appearance of specific myocardial necrosis markers, such as cardiac troponin I, myoglobin or creatine kinase MB (CK MB) in rabbits in the presence or absence of modified ECG profile, and to also verify whether these markers are associated with the alterations in some coagulation parameters, that are known to be induced by vasopressin. As the effects of vasopressin are counteracted by nitric oxide (NO), another aim was to verify whether vasopressin also affects plasma NO levels and whether the administration of a NO donor can reverse these effects. Vasopressin was administered to rabbits and caused ischaemic alterations such as electrocardiographic changes, and significantly increased the levels of plasma cardiac necrosis markers (c-troponin I, myoglobin and CK MB). It also elevated diastolic blood pressure (BP), lowered heart rate (HR), increased procoagulation activity, and lowered plasma NO levels. The appearance of heart necrosis, demonstrated by plasma cardiac necrosis markers in the animals receiving vasopressin, was attributed to a drug-induced increase in vasoconstriction and coagulation activity. The intense vasoconstriction and thrombosis may lead to endothelium necrosis and a consequent drop in NO production. The administration of the NO donor nitroglycerin (NG) in the vasopressin treated animals restored NO values, and was capable of preventing the appearance of the plasma cardiac necrosis markers and altered coagulation values. The protective activity of NG was attributed to NO release, which lowers BP values and counteracts coagulation activity in vasopressin-treated animals. The described procedure may also be proposed for the study of early ischaemic myocardial lesions and the screening of NO donors preventing myocardial damage.

Inhibitory effects of melatonin on vascular reactivity: possible role of vasoactive mediators

Melatonin (MEL), the principal hormone of the vertebral pineal gland, elicits several neurobiological effects. However, the effects of MEL on vascular tissues are still vague. The first goal of this study was to investigate the effect of MEL on isolated rabbit aortic rings and its role in the vascular reactivity to contractile agents, noradrenaline (NA) and phenylephrine (PHE) and relaxant agents (acetylcholine and sodium nitroprusside). In addition, the levels of nitric oxide (NO), cGMP, total calcium, lipid peroxides, superoxide dismutase (SOD) and glutathione (GSH) were also investigated in tissue homogenates of rabbit aortic rings preincubated (20 min) in MEL with and without contractile agents. Our results revealed that MEL has an endothelium-dependent vaso-relaxant effect and potentiated significantly the vaso-relaxant effect of acetylcholine. Moreover, MEL (10 −4 M) had a significant inhibitory effect on the contractile responses of aortic rings to both NA and PHE. In comparison with control tissue rings, the levels of lipid peroxides were significantly increased while the levels of GSH, and SOD activities were significantly decreased in tissue homogenates of aortic rings pre-incubated (20 min) in NA or PHE. In addition, the levels of NO and cGMP were significantly lower in tissue rings pre-treated with NA and PHE, respectively. Also, the levels of total calcium were significantly increased only in tissue rings pre-treated with NA. The levels of lipid peroxides were significantly decreased, while the levels of GSH, NO and cGMP and SOD activities were significantly increased in tissue homogenates of aortic rings incubated (20 min) in MEL (10 −4 M) in comparison to ring tissues incubated in NA or PHE alone. In aortic rings incubated in MEL+PHE, the levels of lipid peroxides were significantly lower while the levels of GSH and cGMP and SOD activities were significantly higher than their levels in ring tissues incubated in PHE. In aortic rings incubated in MEL+NA, the levels of lipid peroxides and total calcium were significantly lower while the levels of NO were significantly higher than their levels in ring tissues incubated in NA alone. We conclude that MEL has an endothelium dependent vasorelaxant effect and potentiates the endothelium dependent vasorelaxation induced by acetylcholine. MEL inhibits the contractile responses of aortic rings to NA and PHE. These effects may be, in part, due to re-balancing the pro-oxidant/antioxidants system, lowered calcium content and elevated NO and cGMP levels in vascular tissue.