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.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies.RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillariesrunning in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed.CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype-specific differences in the subcellular distributions of NOS isoforms.

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.

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.

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.

Identification and Pathogen Stimulation Patterns of Neuronal Nitric Oxide Synthase(nNOS)in Black Rockfish(Sebastes schlegelii)

Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

This study investigated the changes in human umbilical vein endothelial cells （HUVECs） induced by overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS （eNOS） activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid （P〈0.01）. The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells （P〈0.01 and P〈0.01, respectively）. Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

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.

Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanolinduced liver injury

AIM： To study the expression and activity of inducible nitric oxide synthase （iNOS） and endothelial nitric oxide synthase （eNOS） in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-KB （NF-κB） and tumor necrosis factor-α （TNF-α） expression in the liver. METHODS： Female Sprague-Dawley rats were given fish oil （0.5 mL） along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase （ALT） activity and pathological analysis. Liver malondialdehyde （MDA）, nitric oxide contents, iNOS and eNOS activity were determined. NF-κB p65, iNOS, eNOS and TNF-α protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction （RT-PCR）. RESULTS： Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration （6 wk） enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-κB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-KB, and TNF-α mRNA expression. CONCLUSION： iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-KB and TNF-α expression, eNOS activity is reduced, but its mRNA expression is not affected.

Impact of endothelial nitric oxide synthase activation on accelerated liver regeneration in a rat ALPPS model

BACKGROUND Although the associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)induces more rapid liver regeneration than portal vein embolization,the mechanism remains unclear.AIM To assess the influence of inflammatory cytokines and endothelial nitric oxide synthase(eNOS)activation on liver regeneration in ALPPS.METHODS The future liver remnant/body weight(FLR/BW)ratio,hepatocyte proliferation,inflammatory cytokine expression,and activation of the Akt-eNOS pathway were evaluated in rat ALPPS and portal vein ligation(PVL)models.Hepatocyte proliferation was assessed based on Ki-67 expression,which was confirmed using immunohistochemistry.The serum concentrations of inflammatory cytokines were measured using enzyme linked immune-solvent assays.The Akt-eNOS pathway was assessed using western blotting.To explore the role of inflammatory cytokines and NO,Kupffer cell inhibitor gadolinium chloride(GdCl3),NOS inhibitor N-nitro-arginine methyl ester(L-NAME),and NO enhancer molsidomine were administered intraperitoneally.RESULTS The ALPPS group showed significant FLR regeneration(FLR/BW:1.60%±0.08%,P<0.05)compared with that observed in the PVL group(1.33%±0.11%)48 h after surgery.In the ALPPS group,serum interleukin-6 expression was suppressed using GdCl3 to the same extent as that in the PVL group.However,the FLR/BW ratio and Ki-67 labeling index were significantly higher in the ALPPS group administered GdCl3(1.72%±0.19%,P<0.05;22.25%±1.30%,P<0.05)than in the PVL group(1.33%±0.11%and 12.78%±1.55%,respectively).Phospho-Akt Ser473 and phospho-eNOS Ser1177 levels were enhanced in the ALPPS group compared with those in the PVL group.There was no difference between the ALPPS group treated with L-NAME and the PVL group in the FLR/BW ratio and Ki-67 labeling index.In the PVL group treated with molsidomine,the FLR/BW ratio and Ki-67 labeling index increased to the same level as in the ALPPS group.CONCLUSION Early induction of inflammatory cytokines may not be pivotal for accelerated FLR regeneration after ALPPS,whereas Akt-eNOS pathway activation may contribute to accelerated regeneration of the FLR.

Expression of Endothelial Nitric Oxide Synthase Traffic Inducer in the Placenta of Pregnancy Induced Hypertension

The expression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） in the placenta of the patients with pregnancy induced hypertension （PIH） was detected and its role in the pathogenesis of PIH was studied. The pathological changes in placental vessels were observed by HE staining. NO2-/NO3- , the stable metabolic end products of NO, was measured with nitrate reductase. The eNOS activity in placental tissues was assayed by spectrophotometry. Western blot analysis was applied to detect NOSTRIN expression. The incidence of thickening and fibronoid necrosis of placental vessels was significantly higher in women with PIH than in the normal group （P〈 0.01）. The levels of placental NO2-/NO3- in PIH patients （27. 53±7.48 μmol/mg） were significantly lower than in normal group （54. 27±9.53 μmol/mg, P〈0.01）. The activity of eNOS was significantly decreased in PIH group （12. 826±3.61 U/mg） as compared with that in normal group （21. 72±3.83 U/mg, P〈0.01）. Western blot analysis revealed that both groups expressed 58 kD NOSTRIN, but the protein level was significantly higher in women with PIH than in the normal group （P〈0.01）. A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in placental tissue of women with PIH （r=-0.57, P〈0.01）. It was concluded that the level of NOSTRIN expression in placenta of women with PIH was increased, which may play an important role in the pathogenesis of PIH.

Potential involvement of nitric oxide synthase but not inducible nitric oxide synthase in the development of experimental corneal neovascularization

AIM: To investigate the effect of nitric oxide and its synthetase on experimental corneal neovascularization (CRNV). METHODS: CRNV was induced by alkali injury in mice, nitric oxide synthetase (NOS) was inhibited by NG-nitro-L-arginine (L-NAME) and inducible nitric oxide synthetase (iNOS) was inhibited by aminoguanidine hemisulfate salt (AG). The inhibitory effect was detected at day 2 and 4 after corneal alkali injury by reverse transcription polymerase chain reaction (RT-PCR). CRNV was compared between the control and the treated mice by microscopic observation and corneal whole mount CD31 immunostaining. RESULTS: The inhibition of L-NAME to NOS and AG to iNOS after corneal injury was confirmed by RT-PCR (P <0.05). Compared with control mice, L-NAME treated mice exhibited significantly decreased CRNV areas (P<0.05). In contrast, AG treatment failed to attenuate alkali induced CRNV (P>0.05). CONCLUSION: Our findings suggest that NOS but not iNOS plays a critical role in alkali injury induced CRNV.

EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON THE GENE EXPRESSION OF NITRIC OXIDE SYNTHASE IN ISCHEMIC RAT BRAINS

The effect of Radix Salviae Miltiorrhizae (RSM) on the gene expression of nitric oxide synthase (NOS) in rat brains during ischemia was studied with in situ hybridization and the results were analyzed with IBAS 2000 Image Analysis System. It was found that NOS gene expression of cerebral cortex and caudate-putamen was markedly increased in 24 hours in ischemia group (P

Temporal expression of hepatic inducible nitric oxide synthase in liver cirrhosis

AIM: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. We have found inducible nitric oxide synthase (iNOS) can be induced in hepatocytes of cirrhotic liver. This study further investigated the temporal expression and activity of hepatic iNOS in cirrhosis development. METHODS: Cirrhosis was induced in rats by chronic bile duet ligatjon (BDL). At different time points after the operation, samples were collected to examine NO concentration, liver function, and morphological changes. Hepatocytes were isolated for determination of iNOS mRNA, protein and enzymatic activity. RESULTS: Histological examination showed early cirrhosis 1-2 wk after BDL, with advanced cirrhosis at 3-4 wk. Bilirubin increased dramatically 3 d after BDL, but decreased by 47% on d 14. Three weeks after BDL, it elevated again. Systemic NO concentration did not increase significantly until 4 wk after BDL, when ascites developed. Hepatocyte iNOS mRNA expression was identified 3 d after BDL, and enhanced with time to 3 wk, but reduced thereafter. iNOS protein showed a similar pattern to mRNA expression. iNOS activity decreased from d 3 to d 7, but increased again thereafter till d 21. CONCLUSION: Hepatic iNOS can be induced in the early stage, which increases with time as cirrhosis develops. lts enzymatic activity is significantly correlated with protein expression and histological alterations of the liver, but not with systemic NO levels, nor with absolute values of liver function markers.

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.

Effects of IGF-Ⅱ on promoting proliferation and regulating nitric oxide synthase gene expression in mouse osteoblast-like cell

Objective: To investigate the effects of insulin-like growth factor Ⅱ (IGF-Ⅱ) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells. Methods: Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-Ⅱ. After the cells were treated with IGF-Ⅱ at different concentrations for different time duration,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation,and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs. Results: After the MC3T3-E 1 cells were treated with IGF-Ⅱ at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24,48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-Ⅱ for 48 h, and with 1, 10 and 100 ng/ml IGF-Ⅱ for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-Ⅱ for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-Ⅱ dosages for different time duration did not show any differences compared with the normal control (P>0.05).Conclusion: IGF-Ⅱ at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-Ⅱ. Higher concentration of IGF-Ⅱ could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-Ⅱ maintenance of the low NO levels in MC3T3-E 1 cells.

Effect of Helicobacterpyloriinfection on gastric mucosal pathologic change and level of nitric oxide and nitric oxide synthase

AIM： To investigate the level of nitric oxide （NO） and nitrous oxide synthase （NOS） enzyme and its effect on gastric mucosal pathologic change in patients infected with Helicobacter pylori （H pylori）, and to study the pathogenic mechanism of H pylori. METHODS： The mucosal tissues of gastric antrum were taken by endoscopy, then their pathology, H pylori and anti-CagA-IgG were determined. Fifty H pylori positive cases and 35 H pylori negative cases were randomly chosen. Serum level of NO and NOS was detected. RESULTS： One hundred and seven cases （71.33%） were anti-CagA-IgG positive in 150 H pyloripositive cases. The positive rate was higher especially in those with preneoplastic diseases, such as atrophy, intestinal metaplasia and dysplasia. The level of NO and NOS in positive group was higher than that in negative group, and apparently lower in active gastritis than in pre-neoplastic diseases such as atrophy, intestinal metaplasia and dysplasia. CONCLUSION： H pylori is closely related with chronic gastric diseases, and type I H pylori may be the real factor for H pylori-related gastric diseases. Infection with H pylori can induce elevation of NOS, which produces NO.

Role of Nitric Oxide and Nitric Oxide Synthases in Ischemia-reperfusion Injury in Rat Organotypic Hippocampus Slice

To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.

Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells

AIM: To investigate the link between chronic biliary inflammation and carcinogenesis using hamster gallbladder epithelial cells. METHODS: Gallbladder epithelial cells were isolated from hamsters and cultured with a mixture of inflammatory cytokines including interleukin-1β,interferon-γ,and tumor necrosis factor-α. Inducible nitric oxide synthase (iNOS) expression,nitric oxide (NO) generation,and DNA damage were evaluated. RESULTS: NO generation was increased significantly following cytokine stimulation,and suppressed by an iNOS inhibitor. iNOS mRNA expression was demonstrated in the gallbladder epithelial cells during exposure to inflammatory cytokines. Furthermore,NO-dependent DNA damage,estimated by the comet assay,was significantly increased by cytokines,and decreased to control levels by an iNOS inhibitor. CONCLUSION: Cytokine stimulation induced iNOS expression and NO generation in normal hamster gallbladder epithelial cells,which was sufficient to cause DNA damage. These results indicate that NO-mediated genotoxicity induced by inflammatory cytokines through activation of iNOS may be involved in the process of biliary carcinogenesis in response to chronic inflammation of the biliary tree.

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.