The emerging role of nitric oxide in the synaptic dysfunction of vascular dementia

With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.

Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

L-arginine, the substrate of nitric oxide synthase,inhibits fertility of male rats

Aim: To examine the effect of L-arginine, the substrate of nitric oxide (NO) synthase, on reproductive function ofmale rats. Methods: Male rats were gavaged with either L-arginine (100 or 200 mg·kg^(-1)·d^(-1)), D-arginine (200mg·kg^(-1)·d^(-1)) or vehicle (0.9% NaCl) for seven consecutive days. Their sexual behaviour and fertility were evaluat-ed using receptive females. Results: L-arginine (200 mg/kg) had no significant effect on sexual competence (interms of sexual arousal, libido, sexual vigour and sexual performance). In mating experiments, the higher dose of L-arginine effectively and reversibly inhibited fertility, whilst the lower dose and the inactive stereoisomer D-arginine hadno significant effect. The antifertility effect caused by L-arginine was due to a profound elevation in the preimplantationloss mediated possibly by impairment in epididymal sperm maturation, hyperactivated sperm motility and sperm capaci-tation. Conclusion: Elevated NO production may be detrimental to male fertility.

Effects of L-arginine on serum nitric oxide, nitric oxide synthase and mucosal Na^+-K^+-ATPase and nitric oxide synthase activity in segmental small-bowel autotransplantation model

AIM:To explore a simple method to create intestinal autotransplantation in rats and growing pigs and to investigate the effect of L-arginine supplementation on serum nitric oxide (NO), nitric oxide synthase (NOS) and intestinal mucosal NOS and Na+-K+-ATPase activity during cold ischemia-reperfusion (IR) in growing pigs. METHODS: In adult Wistar rat models of small bowel autotransplantation, a fine tube was inserted into mesenteric artery via the abdominal aorta. The superior mesenteric artery and vein were occluded. Isolated terminal ileum segment was irrigated with Ringer's solution at 4℃ and preserved in the same solution at 0-4℃ for 60 min. Then, the tube was removed and reperfusion was established. In growing pig models, a terminal ileum segment, 50 cm in length, was isolated and its mesenteric artery was irrigated via a needle with lactated Ringer's solution at 4℃. The method and period of cold preservation and reperfusion were described above. Ten white outbred pigs were randomly divided into control group and experimental group. L-arginine (150 mg/kg) was continuously infused for 15 min before reperfusion and for 30 min after reperfusion in the experimental group. One, 24, 48, and 72 h after reperfusion, peripheral vein blood was respectively collected for NO and NOS determination. At the same time point, intestinal mucosae were also obtained for NOS and Na+-K+-ATPase activity measurement. RESULTS: In adult rat models, 16 of 20 rats sustained the procedure, three died of hemorrhage shock and one of deep anesthesia. In growing pig models, the viability of small bowel graft remained for 72 h after cold IR in eight of 10 pigs. In experimental group, serum NO level at 1 and 24 h after reperfusion increased significantly when compared with control group at the same time point (152.2±61.4μmol/L /s60.8±31.6μmol/L, t=2.802, P=0.02<0.05; 82.2±24.0μmol/L vs 54.0±24.3μmol/L, t=2.490, P=0.04<0.05). Serum NO level increased significantly at 1 h post-reperfusion when compared with the same group before cold IR, 24 and 48 h post-reperfusion (152.2±61.4μmol/L vs 75.6±16.2μmol/L,t=2.820, P=0.02<0.05,82.2±24.0μmol/L,t=2.760, P= 0.03<0.05, 74.2±21.9μmol/L, t=2.822, P= 0.02<0.05). Serum NOS activity at each time point had no significant difference between two groups. In experimental group, intestinal mucosal NOS activity at 1 h post-reperfusion reduced significantly when compared with pre-cold IR (0.79±0.04 U/mg vs 0.46±0.12 U/mg, t = 3.460, P= 0.009<0.01). Mucosal NOS activity at 24, 48, and 72 h post-reperfusion also reduced significantly when compared with pre-cold IR (0.79±0.04 U/mg vs 0.57±0.14 U/mg, t= 2.380, P=0.04 <0.05, 0.61±0.11 U/mg, t= 2.309, P = 0.04<0.05, 0.63±0.12U/mg, t = 2.307, P= 0.04<0.05). In control group, mucosal NOS activity at 1 and 24 h post-reperfusion was significantly lower than that in pre-cold IR (0.72±0.12 U/mg vs 0.60±0.07 U/mg, t= 2.320, P= 0.04<0.05, 0.58±0.18 U/mg, t=2.310, P= 0.04<0.05). When compared to the normal value, Na+-K+-ATPase activity increased significantly at 48 and 72 h post-reperfusion in experimental group (2.48±0.59μmol/mg vs 3.89±1.43μmol/mg, t=3.202, P= 0.04<0.05, 3.96±0.86μmol/mg, t=3.401, P= 0.009 <0.01) and control group (2.48±0.59μmol/mg vs 3.58±0.76 μmol/mg, t=2.489, P= 0.04<0.05, 3.67±0.81μmol/mg, t= 2.542, P= 0.03<0.05). CONCLUSION: This novel technique for intestinal autotransplantation provides a potentially consistent and practical model for experimental studies of graft cold preservation. L-arginine supplementation during cold IR may act as a useful adjunct to preserve the grafted intestine.

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine （L-NA） on the survival of retinal ganglion cells （RGCs） at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 （1.0, 3.0 and 4.5 mg/kg） and L-NA （1.5, 3.0, 4.5 and 6.0 mg/kg） increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.

Effect of nitric oxide synthase inhibitor N^G-nitro-L-arginine on the content of amino acid in ischemic brain tissues of rats

BACKGROUND： Nitric oxide synthase （NOS） inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids （EAA） during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine （L-NA） reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear. OBJECTIVE： By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid （GABA） in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism. DESIGN： A randomized and controlled experiment SETTING ： Department of Pharmacology, Hebei Academy of Medical Sciences MATERIALS： A total of 42 male healthy SD rats （grade Ⅱ, weighting 250-300 g） were provided by the Experimental Animal Center of Hebei Province （Certification： 04036）. Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride （TTC） were obtained from Sigma Chemicals Co, St Louis, MO, USA. HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC. METHODS： The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups： sham-operated group （n = 6）, ischemic group （n = 18）, L-NA group （n = 18）. The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared, but the filament was not inserted. Each group was further divided into 3 subgroups （n = 6 for each）： drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion （MCAO） respectively. L-NA （20 mg/kg, ip） was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain （IV%） in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett＇s test. MAIN OUTCOME MEASURES： （1） The volume of cerebral infarction; （2） The contents of aspartate, glutamate glycine and GABA in brain tissue after cerebral ischemia. RESULTS ： All 42 rats were involved in the final analysis. （1） Infarcted volume： Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was （20.13±3.59）% and （23.12±5.84）% in L-NA group, which was not similar to that in ischemic group [（22.10±3.98）%, （25.38± 5.37）%, P〉 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [（26.11±3.55）% and （37.15±3.58）%, P 〈 0.01]. Changes of amino acid content： At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group （ P〈 0.05-0.01）. However, contents in L-NA group were similar to those in ischemic group （P 〉 0.05）. At 12 hours after ischemia, the contents of aspartate [（0.21 ±0.06）, （0.36±0.05）, （0.29±0.12） mg/g] and glutamate [（0.55±0.06）, （0.78±0.10）, （0.52±0.10） mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [（0.49±0.17）, （0.63± 0.03）, （0.51±0.15） mg/g; （0.98±0.30）, （1.15±0.15）, （0.93±0.15） mg/g, P〈 0.05-0.01]. Glycine in hippocampus was （0.40±0.07） mg/g, which was higher than that in ischemic group [（0.21±0.07） mg/g, P 〈 0.05]. GABA in striatum, hippocampus and cortex was （0.93±0.10）, （0.62±0.12） and （0.81 ±0.10） mg/g, respectively, which was higher than that in ischemic group [（0.60±0.08）, （0.37±0.17）, （0.59±0.10） mg/g, P 〈 0.05-0.01]. CONCLUSION ： It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA.

Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity,which triggers cell death in various neuropathological diseases,including epilepsy.Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold,that is,has an anticonvulsant effect.However,the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear.In this study,we performed RNA sequencing,functional enrichment analysis,and weighted gene coexpression network analysis of the hippocampus of tremor rats,a rat model of genetic epilepsy.We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na+-K+-ATPase activity.In addition,we used a pilocarpine-induced N2a cell model to mimic epileptic injury.After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole,changes in malondialdehyde,lactate dehydrogenase and superoxide dismutase,which are associated with oxidative stress,were reversed,and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine.Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells.Furthermore,7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3,gasdermin-D,interleukin-1βand interleukin-18.This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death.Taken together,our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells,and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

Growth hormone regulates intestinal ion transport through a modulation of the constitutive nitric oxide synthase-nitric oxide-cAMP pathway

AIM： Growth hormone （GH） directly interacts with the enterocyte stimulating ion absorption and reducing ion secretion induced by agonists of cAMR Since nitric oxide （NO） is involved in the regulation of transepithelial ion transport and acts as a second messenger for GH hemodynamic effects, we tested the hypothesis that NO may be involved in the resulting effects of GH on intestinal ion transport. METHODS： Electrical parameters reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers and exposed to GH and cholera toxin （CT） alone or in combination, in the presence or absence of the NO synthase （NOS） inhibitor, Nω-nitro-L-arginine methyl ester （L-NAME）. Similar experiments were conducted to determine cAMP and nitrite/nitrate concentrations. NOS expression was assayed by Western blot analysis. RESULTS： L-NAME causes total abrogation of absorptive and antisecretory effects by GH on intestinal ion transport, In addition, L-NAME was able to inhibit the GH-effects on intracellular cAMP concentration under basal conditions and in response to CT, GH induced a Ca^2＋ -dependent increase of nitrites/nitrates production, indicating the involvement of the constitutive rather than the inducible NOS isoform, which was directly confirmed by Western blot analysis. CONCLUSION： These results suggest that the GH effects on intestinal ion transport, either under basal conditions or in the presence of cAMP-stimulated ion secretion, are mediated at an intracellular level by the activity of cNOS.

Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

Association of Glu298Asp Polymorphism of the En-dothelial Nitric Oxide Synthase Gene with Essential Hypertension in Elderly People

Objective: To investigate the association of Glu298Asp polymorphism of theeNOS gene with essential hypertension in elderly people. Methods: Ninety-five cases of essentialhypertension were randomly chosen from outpatients and inpatients as the study group, and an equalnumber of sexes, age-matched healthy people as the control group. Their height, weight and bloodpressure were recorded and their fasting plasma lipid concentrations were measured. Glu298Asppolymorphism of the eNOS gene was measured using the methods of PCR and RFLP. Results: Theconstituent ratio of Genotype Glu/Asp in the study group (26.3%) was higher than that in the controlgroup (12.6%, x^2 = 5. 67, P<0.05), the allelic frequency of 298Asp in the study group (13.2%) wassignificantly higher than that in the control group (6.3%, x^2 = 5.06, P<0.05). Conclusion: Glu298Asp variant of the eNOS gene may be an independent predictor in essential hypertension.

Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats

Aim： To investigate the effect of icariin on erectile function and the expression of nitric oxide synthase （NOS） isoforms in castrated rats. Methods： Thirty-two adult male Wistar rats were randomly divided into one shamoperated group （A） and three castrated groups （B, C and D）. One week after surgery, rats were treated with normal saline （groups A and B） or oral icariin （1 mg/[kg·day] for group C and 5 mg/[kg·day] for group D） for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure （ICP） during electrostimulation of the cavernosal nerve. The serum testosterone （ST） levels, the percent of smooth muscle （PSM） in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase （nNOS）, inducible nitric oxide synthase （iNOS）, endothelial nitric oxide synthase （eNOS） and phosphodiesterase V （PDES） in corpus cavernosum （CC） were also evaluated. Results： ICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A （P 〈 0.01）. However, ICE PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B （P 〈 0.05）. Changes in ST and the expression of eNOS and PDE5 were not significant （P 〉 0.05） in groups C and D compared with those in group B. Conclusion： Oral treatment with icariin （〉 98.6 % purity） for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.

Distribution of nitric oxide synthase in stomach myenteric plexus of rats

AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus. METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique. RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus.The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62+/-38 cells mm(2) (antrum), 43+/-32 cells/mm(2) (body), and 32+/-28 cells mm(2) (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels. CONCLUSION: Nitric oxide might be involved in the modulation of motility, secretion and blood circulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma

AIM： To determine the expressions of inducible nitric oxide synthase （iNOS） and matrix metalloproteinase-9 （MMP-9） in hepatocellular carcinoma （HCC） and to investigate the relationship between iNOS and MMP-9 expression and their effects on angiogenesis and progression of HCC.METHODS： In this study, we examined iNOS, MMP-9, and CD34 expression in specimens surgically removed from 32 HCC patients and 7 normal liver tissues by immunohistochemical staining. Meanwhile, microvessel density （MVD） was determined as a marker of angiogenesis by counting CD34-positive cells. RESULTS： The positive rates of iNOS and MMP-9 expression were 71.88% （23/32） and 78.13% （25/32） in HCC. MMP-9 expression was significantly correlated with tumor size, capsule status, TNM stage, and risk of HCC recurrence （P = 0.032, P= 0.033, P= 0.007, and P= 0.001, respectively）. There was also a significant relationship between iNOS expression and capsule status and risk of HCC recurrence （P = 0.049 and P = 0.004, respectively）, but no correlation between iNOS expression and tumor size and TNM stage. There was a positive association between MVD and TNM stage and risk of HCC recurrence （P = 0.037 and P = 0.000, respectively）. The count of MVD was significantly different in different iNOS and MMP-9 immunoreactivity groups （F= 17.713 and 17.097, P= 0.000 and P = 0.000, respectively）. The examination of Spearman＇s rank correlation coefficient showed that there was a significant positive correlation between MVD and iNOS, MMP-9 immunoreactivity （r = 0.754 and 0.751, P= 0.000 and P=-0.000, respectively）. There was also a significant association between MMP-9 and iNOS expression in HCC （P = 0.010）. CONCLUSION： Nitric oxide （NO） produced by iNOS could modulate MMP-9 production and therefore contribute totumor cell angiogenesis and invasion and metastasis in HCC. The strong expression of iNOS and MMP-9 in HCC may be helpful in evaluating the recurrence of HCC, predicting poor prognosis. For patients with strong expression of MMP-9 and iNOS, the optimal treatment scheme needs to be selected.

Nitric oxide synthase and heme oxygenase expressions in human liver cirrhosis

AIM： Portal hypertension is a common complication of liver cirrhosis. Intrahepatic pressure can be elevated in several ways. Abnormal architecture affecting the vasculature, an increase in vasoconstrictors and increased circulation from the splanchnic viscera into the portal system may all contribute. It follows that endogenous vasodilators may be able to alleviate the hypertension. We therefore aimed to investigate the levels of endogenous vasodilators, nitric oxide （NO） and carbon monoxide （CO） through the expression of nitric oxide synthase （NOS） and heme oxygenase （HO）. METHOD： Cirrhotic （n = 20） and non-cirrhotic （n = 20） livers were obtained from patients who had undergone surgery. The mRNA and protein expressions of the various isoforms of NOS and HO were examined using competitive PCR, Western Blot and immunohistochemistry. RESULTS： There was no significant change in either inducible NOS （iNOS） or neuronal NOS （nNOS） expressions while endothelial NOS （eNOS） was up- regulated in cirrhotic livers. Concomitantly, caveolin-1, an established down-regulator of eNOS, was upregulated. Inducible HO-1 and constitutive HO-2 were found to show increased expression in cirrhotic livers albeit in different Iocalizations. CONCLUSION： The differences of NOS expression might be due to their differing roles in maintaining liver homeostasis and/or involvement in the pathology of cirrhosis. Sheer stress within the hypertensive liver may induce increased expression of eNOS. In turn, caveolin-1 is also increased. Whether this serves as a defense mechanism against further cirrhosis or is a consequence of cirrhosis, is yet unknown. The elevated expression of HO-1 and HO-2 suggest that CO may compensate in its role as a vasodilator albeit weakly. It is possible that CO and NO have parallel or coordinated functions within the liver and may work antagonistically in the pathophysiology of portal hypertension.

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM： TO investigate the dynamic change and role of neuronal nitric oxide synthase （nNOS） and inducible nitric oxide synthase （iNOS） in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis （NEC） is associated with the levels of nitric oxide synthase （NOS） in the mucosa of the affected intestine tissue. METHODS： Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide （LPS）. Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS： The LPS-injected increase in injury scores pups showed a significant versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION： nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

Role of brain-derived neurotrophic factor and neuronal nitric oxide synthase in stress-induced depression

BACKGROUND： Accumulated evidence indicates an important role for hippocampal dendrite atrophy in development of depression, while brain-derived neurotrophic factor （BDNF） participates in hippocampal dendrite growth. OBJECTIVE： To discuss the role of BDNF and neuronal nitric oxide synthase （nNOS） in chronic and unpredictable stress-induced depression and the pathogenesis of depression. DESIGN, TIME AND SETTING： Randomized, controlled animal experiment. The experiment was carded out from October 2006 to May 2007 at the Department of Animal Physiology, College of Life Science, Shaanxi Normal University. MATERIALS： Thirty-seven male Sprague-Dawley rats weighing 250-300 g at the beginning of the experiment were obtained from Shaanxi Provincial Institute of Traditional Chinese Medicine （Xi＇an, China）. BDNF antibody and nNOS antibody were provided by Santa Cruz （USA）. K252a （BDNF inhibitor） and 7-NI （nNOS inhibitor） were provided by Sigma （USA）. METHODS： Animals were randomly divided into five groups： Control group, chronic unpredicted mild stress （CUMS） group, K252a group, K252a＋7-NI group and 7-NI＋CUMS group. While the Control, K252a and K252a＋7-NI groups of rats not subjected to stress had free access to food and water, other groups of rats were subjected to nine stressors randomly applied for 21 days, with each stressor applied 2-3 times. On days 1, 7, 14 and 21 during CUMS, rats received microinjection of 1 μL of physiological saline in the Control and CUMS groups, 1 ~ L of K252a in the K252a group, 1 μL of K252a and 7-NI in the K252a＋7-NI group, and 1 μL of 7-NI in the 7-NI＋CUMS group. We observed a variety of alterations in sucrose preference, body weight change, open field test and forced swimming test, and observed the expression of BDNF and nNOS in rat hippocampus by immunohistochemistry; MAIN OUTCOME MEASURES： ① A variety.of behavioral alterations of rats; ② The expression of BDNF and nNOS in rat hippocampus. RESULTS： Compared with the Control group, the behavior of the CUMS rats was significantly depressed, the expression of BDNF decreased （P 〈 0.01） but the expression of nNOS increased （P 〈 0.01）. The behavior of rats given intra-hippocampal injection of BDNF inhibitor was significantly depressed and the expression of nNOS was significantly increased （P 〈 0.01）. Intra-hippocampal injections of an nNOS inhibitor reversed the depression-like behavioral changes induced by CUMS or intra-hippocampal injection of BDNF inhibitor. CONCLUSION： CUMS induced a decrease in expression of BDNF and an increase in expression of NO in the hippocampus, which may lead to depression.

Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

AIM: To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA,pathological features and clinical staging of gastric cancer.METHODS: The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by in situ hybridization.RESULTS: Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t = 0.4161, P＜0.01).Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (x2 = 10.23, P＜0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r= 0.3426, P＜0.05). However,iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r = 0.3612, P＜0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa.CONCLUSION: In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread. Inactivation of antioncogene p53 and overexpression of iNOS might play a synergetic role in the process of carcinogenesis of human gastric carcinoma.

Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.