Total Nitric Oxide Synthase Activity Research Articles

Dl-trans-3,4-Dihydroxy-1-selenolane (DHSred) heals indomethacin-mediated gastric ulcer in mice by modulating arginine metabolism

BackgroundThe importance of the arginine metabolism in gastric ulcer-healing is given relatively less attention. Hence the role of controlling this pathway by dl-trans-3,4-dihydroxy-1-selenolane (DHSred) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated. MethodsSwiss albino mice were ulcerated with indomethacin followed by treatment with the test samples, and the activities of myeloperoxidase (MPO), total nitric oxide synthase (NOS) and arginase, the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and the pro-/anti-inflammatory cytokine levels were assayed. NOS-inhibitors were also used to establish the biochemical mechanism. ResultsIndomethacin induced maximum ulceration in mice on the 3rd day, associated with reduced arginase activity, eNOS expression, along with increased MPO and total NOS activities, nitric oxide (NO) generation, iNOS expression, and pro-/anti-inflammatory (Th1/Th2) cytokine ratio. Treatment with DHSred (2.5mgkg−1×3days) restored the cytokine balance to shift the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity and eNOS expression, and reduced iNOS expression, total NOS activity and NO level. ConclusionsThe ulcer-healing property of DHSred, but not of omeprazole was due to a favorable pro-/anti-inflammatory cytokine ratio that shifted the arginine metabolism to the polyamine pathway and increased the eNOS/iNOS ratio. The healing action of omeprazole was not significantly associated with these parameters. General significanceThe shift in the ariginine-metabolism from the iNOS/NO axis to the arginase/polyamine axis is guided by Th1/Th2 cytokines ratio and plays an important role in gastric ulcer-healing. The favourable effects of the non-toxic and water-soluble compound, DHSred on these pathways and other COX-dependent and antioxidative parameters suggested it to be a promising anti-ulcer formulation for further studies.

Growth inhibition and coordinated physiological regulation of zebrafish (Danio rerio) embryos upon sublethal exposure to antidepressant amitriptyline

Amitriptyline is a tricyclic antidepressant used for decades. It is present at low detectable concentrations in the aquatic environment, but relative few studies have focused on its ecotoxicological effects on non-target aquatic animals. The present study conducted an acute toxicity test of waterborne amitriptyline exposure using zebrafish (Danio rerio) embryos 4 to 124h-post-fertilization. Time-dependent lethal concentrations were firstly determined and at mg/L levels. Effects of amitriptyline on zebrafish embryos were then evaluated under amitriptyline exposure at sublethal concentrations of 1, 10, 100ng/L, 1, 10, 100μg/L and 1mg/L. Our results showed that amitriptyline significantly reduced the hatching time and body length of embryos after exposure in a concentration-dependent manner. Our study also revealed that the exposure evoked a coordinated modulation of physiological and biochemical parameters in exposed zebrafish embryos, including alterations of adrenocorticotropic hormone (ACTH) level, oxidative stress and antioxidant parameters, as well as nitric oxide (NO) production and total nitric oxide synthase (TNOS) activity. A U-shaped concentration-dependent response curve was observed in ACTH level in response to amitriptyline exposure. However, both U-shaped and inversed U-shaped curves were indicated in the responses of antioxidant parameters, including total antioxidant capacity, antioxidant enzyme activities (catalase, superoxide dismutase and peroxidase), glutathione content and glutathione reductase activity. Correspondingly, hydroxyl radical formation and lipid peroxidation indices changed in similar U-shaped concentration-dependent patterns, which together the results of antioxidant parameters suggested induction of oxidative stress in embryos exposed to amitriptyline at high concentrations. Moreover, NO production and TNOS activity were both significantly affected by amitriptyline exposure. Notably, significant correlations between these measured parameters were revealed, which suggested a dynamic adaptation process and coordinated regulation of multiple physiological systems in fish embryos to amitriptyline treatment. Furthermore, our study demonstrated that the effective concentrations of amitriptyline for measured parameters in zebrafish embryos were as low as 10ng/L, and thus revealed the potential risk of amitriptyline and other antidepressants to aquatic life.

The effect of ischemic preconditioning on nitric oxide synthase activity during myocardial ischemia and reperfusion in anesthetized dogs

obJeCtives: To examine whether changes in nitric oxide (NO) bioavailability occurring during a 25 min occlusion of the left anterior descending coronary artery and following ischemic preconditioning (PC) in anesthetized dogs (chloralose and urethane, intravenous) are associated with changes in the activation of NO synthase (NOS) enzymes. Methods: In 12 control and 16 preconditioned dogs, total NOS activity (measured using radioimmunoassay), superoxide (measured using confocal microscopy) and nitrotyrosine (NT; measured using Western blot) production were assessed in tissue samples obtained from the left ventricular wall both after the PC (two 5 min occlusions) procedure and at two time points (5 min and 25 min) during prolonged ischemia. Plasma nitrate/nitrite concentrations in the blood of the coronary sinus were also determined. ResuLts: Compared with the sham control group that was not subjected to ischemia, occlusion of the left anterior descending coronary artery resulted in an initial increase, followed by a significant decrease in NOS activity and nitrate/nitrite levels. There were parallel increases in superoxide and NT production by the end of the ischemic period. PC itself enhanced NOS activity and the concentration of NO metabolites, which were maintained during the prolonged occlusion. Furthermore, PC prevented prolonged ischemia-induced superoxide and NT formation. ConCLusions: The present study demonstrated that changes in NO bioavailability during ischemia are closely associated with changes in NOS activation. PC stimulates and maintains the activation of NOS, thereby providing better NO bioavailability and suppressed free radical formation during sustained ischemia. All of these effects may have importance in the previously observed marked antiarrhythmic effect of PC.

Relationships between maternal plasma levels of homocysteine, nitric oxide synthase, inducible nitric oxide synthase and fetal neural tube defects

Objective To investigate the relationships between fetal neural tube defects (NTD) and maternal plasma homocysteine (Hcy) level,total nitric oxide synthase (NOS) and inducible NOS (iNOS) activity.Methods A total of 28 pregnant women with clinically or prenatal ultrasound diagnosed NTD offspring in Shihezi City and its surrounding areas of the Xinjiang Uygur Autonomous Region from January 6,2008 to May 23,2011 were enrolled as NTD group.Another 41 normal pregnant women,matched in corresponding area and period,were enrolled as the control group.The plasma level of Hcy was determined by rate method,and the activities of total NOS and iNOS were detected by spectrophotometry.Linear correlation analysis and t-test were used as statistical method.Results The plasma level of Hcy in NTD group was higher than that in the control group with statistically significant difference [(15.2±7.9) μmol/L vs (8.4±3.7) μmol/L,t=4.269,P＜0.01].The activity of total NOS was lower in the NTD group than that in the control group [(20.0±9.0) U/ml vs (26.0±4.1) U/ml,t=3.258],so was the activity of iNOS [(7.5±2.9) U/ml vs (12.0±4.5) U/ml,t=4.670],the differences were statistically significant (both P＜0.01).Linear correlation analysis showed a negative correlation between the level of Hcy and the activity of total NOS (r=0.325,P＜0.01),but no correlation was found with iNOS (r=0.205,P＞0.05).Conclusions High level of Hcy and low activity of NOS and iNOS might be correlated with fetal NTD. Key words: Neural tube defects; Nitric oxide synthase; Homocysteine

Mitochondrial Modulation of Apoptosis Induced by Low-dose Radiation in Mouse Testicular Cells

ObjectiveTo investigate whether apoptosis induced by low-dose radiation (LDR) is regulated by mitochondrial pathways in testicular cells. MethodsMale mice were exposed to whole-body LDR, and changes in mitochondrial function and in expression of apoptotic factors were analyzed in the testicular cells as follows. Total nitric-oxide synthase (T-NOS) and Na+/K+ ATPase activities were biochemically assayed. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were determined by flow cytometry using fluorescent probes. Levels of mRNAs encoding cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) were quantified by real-time reverse-transcription PCR (RT-PCR). Expression of Cyt c, AIF, caspase-9, and caspase-3 at the protein level was assessed by western blotting and immunohistochemistry. ResultsLDR induced an increase in T-NOS activity and ROS levels, and a decrease in Na+/K+ ATPase activity and mitochondrial Δψm, in the testicular cells. The intensity of these effects increased with time after irradiation and with dose. The cells showed remarkable swelling and vacuolization of mitochondria, and displayed a time- and dose-dependent increase in the expression of Cyt c, AIF, procaspase-9, and procaspase-3. Activation of the two procaspases was confirmed by detection of the cleaved caspases. The changes in expression of the four apoptotic factors were mostly limited to spermatogonia and spermatocytes. ConclusionLDR can induce testicular cell apoptosis through mitochondrial signaling pathways.

Regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord

Accumulating evidence suggests that the metabolism of l-arginine, a metabolically versatile amino acid, is critically involved in the aging process. The present study compared the activity and protein expression of nitric oxide synthase (NOS) and arginase, and the levels of l-arginine and its eight down-stream metabolites in the brain stem (pons and medulla) and the cervical spinal cord in 3- (young) and 22- (aged) month-old male Sprague–Dawley rats. Total NOS activity was significantly reduced with age in the spinal cord (but not brain stem), and there were no age-related changes in arginase activity in both regions. Western blot revealed decreased protein expression of endothelial NOS, but not neuronal NOS, with age in both regions. Furthermore, there were significantly decreased l-arginine, glutamate, GABA and spermine levels and increased putrescine and spermidine levels with age in both regions. Although the absolute concentrations of l-arginine and six metabolites were significantly different between the brain stem and spinal cord in both age groups, there were similar clusters between l-arginine and its three main metabolites (l-citrulline, l-ornithine and agmatine) in both regions, which changed as a function of age. These findings, for the first time, demonstrate the regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord. Future research is required to understand the functional significance of these changes and the underlying mechanisms.

Protective Effect of the Nitric Oxide Pathway in L-Citrulline Renal Ichaemia-Reperfusion Injury in Rats

To observe the protective effects of L-citrulline on the renal I/R injury and elucidate the mechanisms involved, 48 rats were randomized into eight groups: Group 1: sham operated; Group 2: I/R (45 min renal ischaemia and 24 h reperfusion); Group 3: I/R + L-citrulline (300 mg/kg, i.g.); Group 4: I/R + L-citrulline (600 mg/kg, i.g.); Group 5: I/R + L-citrulline (900 mg/kg, i.g.); Group 6: I/R + normal saline (NS, i.g.); Group 7: I/R + N sup ω nitro-L-arginine ester (L-NAME, 20 mg/kg, i.p.); Group 8: I/R + L-citrulline (900 mg/kg, i.g.) + L-NAME (20 mg/ kg, i.p.). At the end of the reperfusion period, serum was collected and the kidneys underwent histological and biochemical examinations. Our results showed that pre-treatment with L-citrulline (300, 600, and 900 mg/kg) significantly ameliorated the renal injury caused by I/R. Moreover, L-citrulline prevented induction of lipid peroxidation and increased the activity of superoxide dismutase and the levels of glutathione and nitric oxide. The I/R-induced decreases in total nitric oxide synthase activity, inducible nitric oxide activity, constitutive nitric oxide activity and endothelial nitric oxide protein expression in the renal cortex were significantly prevented. However, the L-citrulline-mediated protection was significantly antagonized by co-administration of L-NAME. These results suggested that L-citrulline administration exhibited significant protection against renal I/R injury. This protective effect, at least in part, via up-regulation of the endothelial nitric oxide protein expression and constitutive nitric oxide synthase activity, maintained production of nitric oxide at the basal level.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot−1, 0.40 ± 0.06 U mgprot−1, P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot−1, 93.0 ± 6.3 nmol mgprot−1, P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

Effects of dietary live yeast Hanseniaspora opuntiae C21 on the immune and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine effects of Hanseniaspora opuntiae C21 on immune response and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Sea cucumbers were fed with either diets containing C21 at 104, 105 and 106 CFU g−1 feed or a control diet for 30–50 days, respectively. After feeding for 30 days and 45 days, five sea cucumbers from each tank were sampled for immunological analyses. Results indicated that C21 significantly improved the phagocytic activity in coelomocytes of sea cucumbers (P < 0.05). Moreover, C21 administration significantly enhanced lysozyme (LSZ), phenoloxidase activity (PO), total nitric oxide synthase (T-NOS), superoxide dismutase (SOD), alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in coelomic fluid, and LSZ, T-NOS, AKP and ACP activities in coelomocytes lysate supernatant (CLS) of sea cucumbers (P < 0.05). After feeding for 45 days, 10 sea cucumbers from each dose group were challenged with V. splendidus NB13. Cumulative incidence and mortality of sea cucumbers fed with C21 were found to be lower than those of control group. After feeding for 50 days, sea cucumbers in 104 CFU g−1 C21 treatment and control tanks were subjected to acute salinity changes (from 30 to 20) for 24 h in the laboratory, and the immunological parameters were measured to evaluate the immune capacities of the A. japonicus. Phagocytic, LAZ and T-NOS activities of C21-treated group were higher than those of control group, indicating that salinity stress tolerance of sea cucumber was enhanced by C21. The present results showed that a diet supplemented with C21 could stimulate the immune system of juvenile A. japonicus thus enhancing their resistance against V. splendidus.

THE EFFECT OF JIAWEI BUYANG HUANWU DECOCTION ON THE NO SYSTEM AFTER ILIAC ARTERY BALLOON INJURED IN RABBITS

ObjectivesTo investigate the effect of Jiawei Buyang Huanwu Decoction in preventing vascular restenosis and on the nitric oxide (NO) system after rabbit iliac artery balloon injury.Methods24 male New Zealand albino...

Immune responses and disease resistance of the juvenile sea cucumber Apostichopus japonicus induced by Metschnikowia sp. C14

Metschnikowia sp. C14 was isolated from the intestine of sea cucumber Apostichopus japonicus. It inhibited the growth of the pathogenic Shewanella marisflavi AP629 and Vibrio splendidus NB13 of A. japonicus in vitro. The yeast was added to basal diet at 104, 105 and 106CFUg−1 of feed to evaluate its effects on immune response and disease resistance against V. splendidus infection in juvenile sea cucumber A. japonicus. After feeding for 30days and 45days, five sea cucumbers from each tank were sampled for immunological analyses. Results indicated that C14 significantly improved the phagocytic activity in coelomocytes of sea cucumbers (P<0.05). Moreover, C14 administration significantly elevated lysozyme (LSZ), phenoloxidase activity (PO), total nitric oxide synthase (T-NOS), superoxide dismutase (SOD), alkaline phosphatase (AKP) in coelomic fluid, and LSZ, T-NOS, SOD and acid phosphatase (ACP) activities in CLS of sea cucumbers (P<0.05). After feeding for 45days, ten sea cucumbers from each dose group were challenged with V. splendidus NB13. Cumulative incidence and mortality of sea cucumbers fed with C14 were found to be lower than those of control group. The present results show that diet supplemented with C14 stimulates the immune system of juvenile A. japonicus thus enhancing their resistance against V. splendidus. C14 enhanced the immune responses and disease resistance of sea cucumbers.

Chronic inhibition of nitric oxide synthase activity by NG-nitro-l-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of NO synthase (NOS) in the CNS suggest a plastic change in NO synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N G -nitro- l -arginine (L-NNA) was examined on NO production, NO-sensitive cGMP levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5–200 mg/kg) to 21-day old rats reduced NOS activity and NO concentration dose dependently by 70–90% and the tissue cGMP level by 60–80%. By contrast, chronic application of L-NNA between postnatal days 4–21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10–50%. Chronic treatment of 10 mg/kg L-NNA induced nNOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50 mg/kg) of L-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced iNOS expression and activity in glial cells. Treatments did not affect eNOS expression. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced chronic inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.

Agmatine selectively improves behavioural function in aged male Sprague–Dawley rats

L-arginine, a semi-essential amino acid, can be metabolized to form a number of bioactive molecules. Nitric oxide (NO), generated by NO synthase (NOS) from L-arginine, has been strongly implicated in the aging process. Agmatine, decarboxylated arginine, regulates the production of NO and other metabolites of L-arginine, modulates behavioural function, and has anti-inflammatory and neuroprotective effects. The present study investigated whether agmatine supplementation could improve behavioural function in aged male Sprague–Dawley rats, and could attenuate age-related changes in NOS activity and protein expression in memory-related structures. Aged rats treated with saline displayed significantly reduced exploratory activity and impaired spatial reference and working memory and object recognition memory. Agmatine (40mg/kg) administered intraperitoneally significantly improved spatial working memory and object recognition memory in aged rats, suppressed age-related elevation in total NOS activity, and restored endothelial NOS protein to the normal level. However, agmatine supplementation was unable to improve exploratory activity and spatial reference learning and memory in aged rats. These findings suggest that exogenous agmatine selectively improves behavioural function in aged rats under the present experimental condition, and merit future investigation of its therapeutic potential in cognitive decline during aging.

Protective effect of l-citrulline against ethanol-induced gastric ulcer in rats

We examined the protective effect of l-citrulline on ethanol-induced gastric ulcer in rats. Administration of l-citrulline at doses of 300, 600 and 900mg/kg body weight prior to ethanol ingestion protected the stomach from ulceration. The gastric lesions were significantly attenuated by all doses of l-citrulline as compared to the ethanol group. Pre-treatment with l-citrulline prevented the oxidative damage and the decrease of nitric oxide content as well as the increase of the myeloperoxidase activity. Consequently, significant changes observed included the attenuation in the elevation in total nitric oxide synthase activity and inducible nitric oxide synthase activity as well as the decrease in constitutive nitric oxide synthase activity in the gastric mucosa induced by ethanol. Analysis of serum cytokines of ethanol-induced rats showed a moderate decrease in interleukin-10 with considerable increase of interleukin-6 while l-citrulline inhibited the acute alteration of cytokines. These results suggested the gastroprotective effect of l-citrulline.

Cardiomyocyte overexpression of the α1a-adrenergic receptor in the rat phenocopies second but not first window preconditioning

We examined α(1A)-adrenergic receptor (AR) mediation of preconditioning in a novel α(1A)-AR cardiac transgenic (TG) rat model (α(1A)-TG). Compared with nontransgenic littermates (NTLs), in conscious α(1A)-TG rats, heart rate was reduced, contractility [left ventricle (LV) +dP/dt, ejection fraction, end-systolic elastance] was significantly enhanced, and triple product (LV systolic wall stress × LV +dP/dt × heart rate) was unchanged. However, infarct size (IS)/area at risk (AAR) in response to ischemia-reperfusion (30 min coronary occlusion/3 h reperfusion) was reduced to 35 ± 4.6% in α(1A)-TGs vs. 52 ± 2.2% in NTLs (P < 0.05). Second window preconditioning reduced IS/AAR in NTLs to 29 ± 2.7% but did not afford further protection in α(1A)-TGs. In contrast, with first window preconditioning, IS/AAR was reduced to similar levels in both α(1A)-TGs (12 ± 1.4%) and NTLs (10 ± 1.1%). In untreated α(1A)-TGs, cardioprotection was associated with enhanced myocardial phosphorylated (p)-mitogen/extracellular signal-regulated kinase (MEK), p-extracellular signal-regulated kinase (ERK), and inducible nitric oxide synthase (iNOS) at the protein level, along with a 1.3-fold increase in total nitric oxide synthase activity like in second window preconditioning. Affymetrix microarrays revealed that few genes (4.6% of 3,172 upregulated; 8.8% of 3,498 downregulated) showed directionally similar changes in α(1A)-TGs vs. NTLs subjected to second window preconditioning. Thus, second, but not first, window cardioprotection is evident in α(1A)-TGs in the absence of ischemic preconditioning and is mediated by iNOS activation associated with MEK/ERK phosphorylation. Transcriptionally, however, second window preconditioning is considerably more complex than α(1A)-TG preconditioning, with the alteration of thousands of additional genes affording no further protection than that already available in α(1A)-TG rats.

167: Intrauterine growth restriction increases nitric oxide synthase activity in the lungs of female but not male rats at birth

Intrauterine growth restriction (IUGR) increases the incidence of pulmonary hypertension (PH) in newborns and adults. Both forms of PH are associated with decreased nitric oxide synthase (NOS) activity. Normal vascular growth requires NOS activity at critical periods during development. Abnormal pulmonary vascular growth may predispose IUGR offspring to PH. We have shown that IUGR male rats develop right ventricular hypertrophy, a marker of elevated pulmonary vascular pressures, as adults. Whether IUGR alters age- and gender-specific levels of the endothelial (eNOS) and inducible (iNOS) NOS isoforms and total NOS activity in IUGR rat lungs is not known.

Effect of trichlorfon on oxidative stress and hepatocyte apoptosis of Carassius auratus gibelio in vivo

To investigate the effect of trichlorfon on oxidative stress and hepatocyte apoptosis of Carassius auratus gibelio in vivo, the fish were exposed to 0, 0.5, 1.0, 2.0 and 4.0mgl(-1) trichlorfon concentrations for 30days. At the end of the experiment, plasm antioxidant activities, hepatic total nitric oxide synthase (T-NOS) activity, xanthine oxidase (XOD) activity and hepatocyte apoptosis rate were analyzed. The results showed hepatic T-NOS activities, XOD activities and hepatocyte apoptosis rate were increased with trichlorfon concentration increase. The hepatic MDA content was increased significantly at 4.0mgl(-1) trichlorfon treatment. The disturbance of antioxidative balance was observed based on the monitoring of the plasm superoxide dismutase (SOD), catalase (CAT) activities and vitamin E levels. The current study revealed that trichlorfon influenced fish plasma antioxidative status, caused lipid peroxidation (LPO) then resulted in hepatocytes apoptosis.

Effects of dietary β-glucan, mannan oligosaccharide and their combinations on growth performance, immunity and resistance against Vibrio splendidus of sea cucumber, Apostichopus japonicus

A 4-week feeding trial was conducted to investigate the effects of dietary β-glucan, mannan oligosaccharide (MOS) and their combinations on growth performance, immunity and disease resistance of sea cucumber Apostichopus japonicus. Sea cucumbers (1215 individuals with initial weight of 3.8 ± 0.2 g) were fed nine practical diets according to a 3 × 3 factorial design: the basal diet as the control supplemented with three levels of β-glucan (0, 0.075, 0.15% w/w), crossed with 0, 0.1% (w/w) or 0.2% (w/w) MOS. Immune indices including total coelomocytes count (TCC), phagocytosis, superoxide anion production, superoxide dismutase (SOD) activity and total nitric oxide synthase activity (T-NOS) were measured at days 7, 11, 15, 18, 22, 25 and 29. At the end of the feeding trial, all the sea cucumbers left were weighted to monitor growth, and then were challenged by Vibrio splendidus. The results showed that dietary β-glucan, MOS and their combinations significantly increased TCC, phagocytosis, superoxide anion production and SOD activity of sea cucumbers ( P < 0.05). Only 0.15% β-glucan and the combinations of β-glucan and MOS significantly increased the T-NOS activity ( P < 0.05). A synergistic effect was found between dietary β-glucan and MOS. Moreover, combinations of β-glucan and MOS prolonged the high levels of immune indices compared with β-glucan or MOS supplementation alone. Except the 0.15% β-glucan group, all the other treatments showed significantly lower cumulative mortality compared with control ( P < 0.05). Furthermore, combination of 0.15% β-glucan and 0.1% MOS had the best effects on enhancing disease resistance of sea cucumber. All treatments showed significantly higher specific growth rate (SGR) compared with control ( P < 0.05), and the combination of 0.15% β-glucan and 0.1% MOS was significantly higher than other treatments ( P < 0.05). In conclusion, our results confirm the potential of β-glucan and MOS as dietary immunostimulants and the synergistic effects of β-glucan and MOS on A. japonicus.

Role of Nitric Oxide Synthase in Bladder Pathologic Remodeling and Dysfunction Resulting From Partial Outlet Obstruction

To investigate the relationship between nitric oxide synthase (NOS) and oxidative stress and pathologic remodeling in the partial obstructed bladder of a rat model. We surgically established partial bladder outlet obstruction (PBOO) in 2 groups of rats and allowed it to persist for 3-6 weeks. Normal and sham-operated rats served as the controls. Each group contained 6 rats for a total of 24 rats. Cystometry was used to evaluate the bladder function. The bladders were removed, and histopathologic measurements were performed to evaluate bladder hypertrophy and fibrosis and NOS immunolocalization. Biochemical measurements were used to evaluated NOS mRNA and activity and the oxidative stress level. The obstructed rats experienced significant increases in bladder weight, muscle hypertrophy, and deposits of collagen fibers compared with the normal and sham-operated groups. PBOO debilitated bladder contractibility, increased the residual urine volume and voiding interval, decreased the voiding volume, and caused poor bladder emptying, with an increased residual urine volume and decompensated bladder in the PBOO rats at 6 weeks. The elevation in malondialdehyde and reduction in superoxide dismutase activity in the PBOO rats suggested that oxidative stress injury occurred in the obstructed bladder. Lower inducible NOS and endothelial NOS (eNOS) mRNA expression was demonstrated through quantitative polymerase chain reaction. In particular, eNOS was significantly downregulated in the PBOO rats compared with the normal and sham-operated rats. The normal and PBOO bladder tissues did not express detectable levels of neuronal NOS mRNA or exhibit neuronal NOS immunoreactivity. The total NOS activity had decreased progressively in the PBOO groups in conjunction with the significantly decreased eNOS activity compared with that in the normal and sham-operated groups. These findings suggest that decreases in NOS activity and expression (mainly of eNOS) concomitant with increases in reactive oxygen species generation represent the underlying pathogenic mechanism of obstructed bladder remodeling and dysfunction.

Immune response of sea cucumber Apostichopus japonicus coelomocytes to several immunostimulants in vitro

The in vitro effects of β-glucan, mannan oligosaccharides (MOS), CpG oligodeoxynucleotide (CpG ODN), lactoferrin and vitamin C on the non-specific immune response of sea cucumber Apostichopus japonicus coelomocytes were studied. Coelomocytes were cultured in L-15 medium containing different concentrations of β-glucan (0, 5, 25 and 100 µg ml − 1), CpG ODN (0, 0.5, 2.5 and 5 µM), MOS (0, 40, 80 and 120 µg ml − 1), lactoferrin (0, 5, 25 and 100 µg ml − 1) and vitamin C (0, 25, 100 and 250 µg ml − 1). Coelomocytes were incubated with the above immunostimulants for 1 h, 3 h, 6 h, 12 h or 24 h except vitamin C for 3 h, 6 h, 12 h, 24 h or 48 h. Coelomocytes incubated in medium without immunostimulants were taken as controls at each incubation time. After exposure to these substances, coelomocytes were sampled and immune parameters viz., phagocytosis, superoxide anion production, superoxide dismutase (SOD) activity and total nitric oxide synthase (T-NOS) activity were determined. All immune parameters were significantly ( P < 0.05) enhanced by β-glucan, MOS and CpG ODN. Lactoferrin induced significant ( P < 0.05) increase in superoxide anion production and SOD activity of coelomocytes but did not affect phagocytosis and T-NOS activity. Vitamin C significantly ( P < 0.05) enhanced the SOD and T-NOS activities of coelomocytes while no significant effects were observed in phagocytosis and superoxide anion production. The results suggest that β-glucan, MOS, CpG ODN, lactoferrin and vitamin C can enhance the non-specific immune response of sea cucumber in vitro.