Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia.

BACKGROUND: The urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. METHODS: The effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model. Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. RESULTS: We initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1ß, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. CONCLUSION: Crebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

Renal protective effects of astragalus root in rat models of chronic kidney disease.

BACKGROUND: Astragalus root is a commonly used herb in traditional Chinese medicine. Although renoprotective effects have been reported in some clinical and experimental studies, the details remain unknown. METHODS: We used 5/6 nephrectomized rats as chronic kidney disease (CKD) models. At 10 weeks, they were divided into four groups, namely, CKD, low-dose astragalus (AR400), high-dose astragalus (AR800), and sham groups. At 14 weeks, they were sacrificed for the evaluation of blood, urine, mRNA expression in the kidney, and renal histopathology. RESULTS: Kidney dysfunction was significantly improved following astragalus administration (creatinine clearance: sham group; 3.8 ± 0.3 mL/min, CKD group; 1.5 ± 0.1 mL/min, AR400 group; 2.5 ± 0.3 mL/min, AR800 group; 2.7 ± 0.1 mL/min). Blood pressure, urinary albumin, and urinary NGAL levels were significantly lower in the astragalus-treated groups than those in the CKD group. Excretion of urinary 8-OHdG, an oxidative stress marker, and intrarenal oxidative stress were lower in the astragalus-treated groups than those in the CKD group. Furthermore, the mRNA expression of NADPH p22 phox, NADPH p47 phox, Nox4, renin, angiotensin II type 1 receptor, and angiotensinogen in the kidney was lower in the astragalus-treated groups compared with the CKD group. CONCLUSION: This study suggests that astragalus root slowed CKD progression, possibly through the suppression of oxidative stress and the renin-angiotensin system.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

Combined ß-sitosterol and trimetazidine mitigate potassium dichromate-induced cardiotoxicity in rats through the interplay between NF-κB/AMPK/mTOR/TLR4 and HO-1/NADPH signaling pathways.

Hexavalent chromium salt, like potassium dichromate (PD), is chromium's most precarious valence state in industrial wastes. Recently, there has been increasing interest in ß-sitosterol (BSS), a bioactive phytosterol, as a dietary supplement. BSS is recommended in treating cardiovascular disorders due to its antioxidant effect. Trimetazidine (TMZ) was used traditionally for cardioprotection. Through the administration of BSS and TMZ, the cardiotoxic effects of PD were to be countered in this study, in addition to examining the precise mechanism of PD-induced cardiotoxicity. Thirty male albino rats were divided into five groups; the control group: administered normal saline daily (3 mL/kg); the PD group: administered normal saline daily (3 mL/kg); BSS group: administered BSS daily (20 mg/kg); TMZ group: administered TMZ daily (15 mg/kg); and the BSS + TMZ group: administered both BSS (20 mg/kg) and TMZ (15 mg/kg) daily. All experimental groups, except the control, received on the 19th day a single dose of PD (30 mg/kg/day, S.C.). Normal saline, BSS, and TMZ were received daily for 21 consecutive days p.o. The exposure to PD promoted different oxidative stresses, pro-inflammatory, and cardiotoxicity biomarkers. BSS or TMZ succeeded solely in reducing these deleterious effects; however, their combination notably returned measured biomarkers close to normal values. The histopathological investigations have supported the biochemical findings. The combination of BSS and TMZ protects against PD cardiotoxicity in rats by reducing oxidative stress and apoptotic and inflammatory biomarkers. It may be promising for alleviating and protecting against PD-induced cardiotoxicity in people at an early stage; however, these findings need further clinical studies to be confirmed. HIGHLIGHTS: • Potassium dichromate induces cardiotoxicity in rats through the upregulation of oxidative stress, proinflammatory, and apoptotic pathways biomarkers. • ß-Sitosterol possesses a possible cardioprotective effect by modulating several signaling pathways. • Trimetazidine, the antianginal agent, has a potential cardioprotective impact on PD-intoxicated rat model. • The combination of ß-Sitosterol and trimetazidine was the best in modulating different pathways involved in PD cardiotoxicity in rats via the interplay between NF-κB/AMPK/mTOR/TLR4 and HO-1/NADPH signaling pathways.

One-time ozone treatment improves the postharvest quality and antioxidant activity of Actinidia arguta fruit.

The major aim of this study was to check the effect of one-time ozonation on selected quality parameters and antioxidant status of Actinidia arguta fruit. For this purpose, A. arguta fruit was ozonated with gas at a concentration of 10 and 100 ppm, which was carried out successively for 5, 15 and 30 min. Next, the selected quality attributes, antioxidants level as well as NADPH and mitochondrial energy metabolism in mini-kiwi fruit after ozonation were analysed. Our research has shown that ozonation reduced the level of yeast and mould without affecting the content of soluble solids or acidity. In turn, ozonation clearly influenced the antioxidant activity and the redox status of the fruit. The ozonated fruit was characterised by a lower level of ROS due to the higher level of low molecular weight antioxidants, as well as the higher activity of superoxide dismutase and catalase. In addition, improved quality and antioxidant activity of the fruit were indirectly due to improved energy metabolism and NADPH level. The ozonated fruit showed a higher level of ATP, due to both higher activity of succinate dehydrogenase and higher availability of NADH. Moreover, the increased level of NAD+ and the activity of NAD+ kinase and glucose-6-phosphate dehydrogenase contributed to higher levels of NADPH in the fruit.

Pleurotus albidus (Agaricomycetes) Antioxidant Action Induces Vasodilation in Aorta Arteries: The Influence of the NADPH/NOS Balance.

The main objective of this work was to evaluate whether Pleurotus albidus extract exerts influences on aorta artery tone by its antioxidant properties. The hearts and aortic arteries of male Wistar rats were removed for use in biochemical analysis and vascular reactivity. Both tissues were exposed to P. albidus extract at different concentrations for 30 min and were then exposed to a free radical generation system for 30 min. The extract reduced lipid peroxidation levels and increased catalase and glutathione peroxidase activity in cardiac tissue. In the aorta, P. albidus extract demonstrated a direct vasodilatory effect, which was associated with a reduction in nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and an increase in sulfhydryl levels and nitric oxide synthase (NOS) activity. Our findings suggest that P. albidus extract has regulatory potential on aorta arteries, regulating the balance of NOX/NOS enzymes and then influencing vessel tone. Further studies are needed to determine the protective mechanisms of the extract.

NADPH levels affect cellular epigenetic state by inhibiting HDAC3-Ncor complex.

NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3-Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells.

The Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca2+ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca2+ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca2+ release (but not cADPR- or IP3-evoked Ca2+ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen 'hits' exhibiting >80% inhibition of NAADP-evoked Ca2+ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca2+ release without depleting acidic Ca2+ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca2+ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP3-dependent Ca2+ signaling with potential therapeutic value.

Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice.

BACKGROUND & AIMS: Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. METHODS: Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. RESULTS: Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity. CONCLUSIONS: NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

Glycine restores glutathione and protects against oxidative stress in vascular tissue from sucrose-fed rats.

The attenuation of oxidative stress could be an important mechanism whereby the incidence of vascular complications in the MS (metabolic syndrome) may be diminished. The present study was undertaken to investigate the mechanism by which glycine, supplemented to the diet of SF (sucrose-fed) rats, modulates glutathione biosynthesis and protects against oxidative stress and altered endothelium-dependent relaxation in isolated aorta. Glycine reduced O2•- (superoxide anion radical) release in the presence of NADPH, and decreased protein carbonyl and lipid peroxidation. This effect of glycine could be because of the increased amount of glutathione synthetase, which may be responsible for increased glutathione (GSH) content in vascular tissue from SF rats. Moreover, glycine increased the amount of Cu,Zn-SOD (copper/zinc superoxide dismutase) and eNOS (endothelial NO synthase) in aorta from SF animals. Finally, it improved the relaxation response to ACh (acetylcholine) found impaired in aortic rings from SF rats. In the presence of NAC (N-acetylcysteine), a precursor of GSH, an improved ACh-mediated aortic relaxation of aortic rings from SF rats was observed, whereas BSO (buthionine sulfoximine), an inhibitor of glutathione biosynthesis, inhibited the relaxing effect of NAC in aortas from both control and SF rats. This experiment emphasizes the role of GSH in endothelial function in SF rats. The present data suggest that glycine rectifies vascular reactivity by increasing the biosynthesis of glutathione. Glutathione protects vascular tissue against oxidative stress, and enhances the availability of NO, which exerts its relaxing effect, thus contributing to the reduction of high BP (blood pressure) in the SF rats.

Ragweed pollen extract intensifies lipopolysaccharide-induced priming of NLRP3 inflammasome in human macrophages.

Ragweed pollen extract (RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species (ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1ß (IL-1ß), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1ß production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1ß production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1ß production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1ß production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro-IL-1ß and key components of the inflammasome via a ROS-dependent mechanism.

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide.

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 µM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 µM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

The effects of Urtica dioica L. leaf extract on aniline 4-hydroxylase in mice.

The effects of hydroalcoholic (80% ethanol-20% water) extract of Urtica dioica L. on microsomal aniline 4-hydroxylase (A4H) were investigated in the liver of Swiss albino mice (8- 10-weeks-old) treated with two doses (50 and 100 mg/kg body weight, given orally for 14 days ). The activities of A4H showed a significant increase in the liver at both dose levels of extract treatment. The hydroalcoholic extract of Urtica dioica induced the activities of A4H that had been increased by treatment of metal ions (Mg2+ and Ca2+) and the mixture of cofactors (NADH and NADPH). At saturated concentration of cofactor, microsomal A4H exhibited significantly even higher activities in the presence of the mixture of cofactors than NADPH and NADH. Mg2+ and Ca2+ ions acted as stimulants in vitro. The present results suggest that the hydroalcoholic extract of Urtica dioica may have modalatory effect on aniline hydroxylase at least in part and enhance the activity of A4H adding metals ions and cofactors.

Comparative mutagenic effects of structurally similar flavonoids quercetin and taxifolin on tester strains Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA.

Quercetin (QT) and Taxifolin (TF) are structurally similar plant polyphenols. Both have been reported to have therapeutic potential as anti-cancer drugs and antioxidants. Mutagenic effects of QT and TF were evaluated using Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA tester strains. Either in the presence or absence of S9 mix, QT was mutagenic to TA102 and WP2 uvrA. However, the mutagenicity of QT was significantly enhanced in the presence of S9 mix. Likewise, in the presence of Iron (Fe2+) and NADPH generating system (NGS) and absence of S9 mix, QT induced significantly high mutations in both TA102 and WP-2 uvrA. Mutagenicity of QT decreased in both strains in the presence of Iron (Fe2+) or NGS alone. TF was not mutagenic in the presence or absence of S9 mix in both TA102 and WP-2 uvrA 2, regardless of the presence of iron or NGS. Incorporation of antioxidants (ascorbate, superoxide dismutase (SOD), catalase (CAT)) and/or iron chelators (desferroxamine (DF) and ethylenediamine-tetraacetate (EDTA)) in the test systems markedly decreased QT-induced mutations in both tester strains. These results suggest that QT but not TF, could induce mutations in the presence or absence of rat liver S9 or Iron (Fe2+) and NGS in both tester strains by redox cycling and Fenton reactions to produce oxygen free radicals. Our results indicate that a minor structural variation between the two plant polyphenols could elicit a marked difference in their genotoxicities. These results provide a basis for further study into the potential use of QT in combination with iron supplements.

Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases.

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.

Inhibition on human liver cytochrome P450 3A4 by constituents of fennel (Foeniculum vulgare): identification and characterization of a mechanism-based inactivator.

Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).

NO-Dependent mechanisms of amygdalofugal modulation of hypothalamic autonomic neurons.

Neurophysiological and histochemical experiments on rats were performed to study the effects of the central nucleus of the amygdala on the activity of cells in various areas of the hypothalamus. Electrical stimulation of the medial part of the nucleus evoked marked excitatory reactions in neurons in the medial part of the paraventricular nucleus of the hypothalamus and the rostral part of the lateral hypothalamic area. Intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) led to increases in evoked neuron responses. A series of histochemical studies following activation of the central nucleus demonstrated increases in the quantity and optical densities of NADP diaphorase (NADP-d)-positive neurons in the parvocellular zone of the paraventricular nucleus of the hypothalamus and the medial part of the lateral hypothalamic area. The activity of nitroergic cells in the ventrolateral part of the lateral hypothalamic area was suppressed in these conditions. These mechanisms may underlie the amygdalofugal modulation of the autonomic functions of the hypothalamus.

Sympathoexcitation by oxidative stress in the brain mediates arterial pressure elevation in salt-sensitive hypertension.

Central sympathoexcitation is involved in the pathogenesis of salt-sensitive hypertension. We have suggested that oxidative stress in the brain modulates the sympathetic regulation of arterial pressure. Thus, we investigated whether oxidative stress could mediate central sympathoexcitation in salt-sensitive hypertension. Five- to 6-week-old male Dahl salt-sensitive rats and salt-resistant rats were fed with a normal (0.3%) or high- (8%) salt diet for 4 weeks. In urethane-anesthetized and artificially ventilated rats, arterial pressure, renal sympathetic nerve activity, and heart rate decreased in a dose-dependent fashion, when 20 or 40 micromol of tempol, a membrane-permeable superoxide dismutase mimetic, was infused into the lateral cerebral ventricle. The same degree of reduction was noted in salt-sensitive and salt-resistant rats without salt loading. Salt loading significantly increased central tempol-induced reductions in arterial pressure (-29.1+/-4.8% versus -10.6+/-3.3% at 40 micromol; P<0.01), sympathetic nerve activity (-18.7+/-2.0% versus -7.1+/-1.8%; P<0.01), and heart rate (-10.7+/-2.8% versus -2.0+/-0.7%; P<0.05) in salt-sensitive rats but not in salt-resistant rats. Intracerebroventricular diphenyleneiodonium, a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, also elicited significantly greater reduction in each parameter in salt-loaded salt-sensitive rats. Moreover, salt loading increased reduced nicotinamide-adenine dinucleotide phosphate-dependent superoxide production in the hypothalamus in salt-sensitive rats but not in salt-resistant rats. In addition, reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits p22(phox), p47(phox), and gp91(phox) mRNA expression significantly increased in the hypothalamus of salt-loaded salt-sensitive rats. In conclusion, in salt-sensitive hypertension, increased oxidative stress in the brain, possibly via activation of reduced nicotinamide-adenine dinucleotide phosphate oxidase, may elevate arterial pressure through central sympathoexcitation.

Free radical scavenging and antigenotoxic activities of natural phenolic compounds in dried flowers of Hibiscus sabdariffa L.

The antioxidant and free radical scavenging effects of two fractions of the ethanolic extract (HSCF, chloroform soluble fraction and HSEA, ethyl acetate soluble fraction) obtained from the dried flowers of Hibiscus sabdariffa L were investigated. The total antioxidant activity of the extracts was estimated to be 4.6 and 8.6 mM of vitamin C for HSCF and HSEA, respectively. Both HSCF and HSEA scavenged hydrogen peroxide (H(2)O(2)) (79-94%) at the dose of 500 microg. Similarly, the extracts showed inhibitory (70-80%) effects on superoxide anions radicals (O(2) (- *)) at a dose of 1000 microg. The concentrations required for a 50% scavenging of hydroxyl radical (OH) (IC(50)) were 380 and 200 microg for HSCF and HSEA, respectively. HSEA and HSCF were better scavengers of O(2) (- *), *OH and H(2)O(2) as compared to BHA, quercetin and alpha-tocopherol. At a concentration of 25 microg/mL HSCF and HSEA exhibited 32 and 38% inhibition on CCl(4)-NADPH-induced lipid peroxidation, respectively, while both extracts exhibited 80 and 89% inhibitory effects at 100 microg/mL. Pretreatment with H. sabdariffa extracts orally with 100 mg/kg and 250 mg/kg simultaneously with intraperitoneal injection FeCl(2)-ascorbic acid-ADP mixture reduced (p < 0.01) the formation of malondialdehyde content. Treatment of rats with HSCF, HSEA and vitamin C (standard antioxidant) significantly inhibited the induction of micronucleated polychromatic erythrocytes by sodium arsenite (2.5 mg/kg) (p < 0.001) after 24 h by 60, 70 and 50%, respectively. The results indicate that extracts of H. sabdariffa showed strong antimutagenic activity and free radical scavenging effects on active oxygen species.