Aim Inducible nitric oxide synthase (iNOS) makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide (LPS) induces iNOS expression through activation of the inhibitor of K...Aim Inducible nitric oxide synthase (iNOS) makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide (LPS) induces iNOS expression through activation of the inhibitor of KB- α (IKB-α) -nuclear factor-KB (NF-KB) cascade, whereas interferon-γ (IFN-γ) acts through Janus kinase ( JAK)- signal transducer and activator of transcription 1 ( STAT1 ) signals. Heat shock factor 1 ( HSF1 ), a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6). But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide (NO) content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation (CHIP) was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.展开更多
Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug...Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are still unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/ tyrosine kinase activity is able to modulate cell signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebellum and liver of the same animals. We demonstrated that atorvastatin treatment (80 mg/day for 14.5 months) to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebellum. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as well as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not associated with higher oxidative/nitrosative stress levels. We hypothesize that biliverdin reductase-A-dependent inducible nitric oxide synthase regulation strongly contributes to the cognitive improvement observed following atorvastatin treatment.展开更多
AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differen...AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differentiation were used.SaOS2 show a rather mature phenotype,while U2 OS are negative for almost all osteoblastic markers.The cells were exposed to different concentrations of prednisolone(1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase(i NOS) l-N6-(iminoethyl)-lysine-HCl(L-NIL).Cell growth was assessed by counting viable cells.The production of nitric oxide(NO) was measured in the conditioned media by the Griess method.The production of reactive oxygen species was quantified using 2'-7'-dichlorofluorescein diacetate.Western blot with specific antibodies against NOSs was performed on cell extracts.RESULTS:Prednisolone inhibited SaOS2 cell growth in a dose dependent manner.No significant effects were observed in U2OS.The inhibition of SaOS2 growth is not due to oxidative stress,because antioxidants do not rescue cell proliferation.Since high concentrations of NO inhibit bone formation,we also measured NO and found it induced in SaOS2,but not in U2 OS,exposed to prednisolone,because of the upregulation of i NOS as detected by western blot.Therefore,we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL.L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L.At the same concentrations,we found that L-NIL rescued SaOS2 growth after exposure to prednisolone.In U2 OS cells,prednisolone did not induce NO production nor affected cell growth.All together,these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.CONCLUSION:Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of i NOS,while no effect was exerted on U2OS.展开更多
The present study detected distribution and expression of nerve growth factor and inducible nitric oxide synthase in the mesencephalon and diencephalon, as well as visual- and auditory-related nervous tissues, in a ma...The present study detected distribution and expression of nerve growth factor and inducible nitric oxide synthase in the mesencephalon and diencephalon, as well as visual- and auditory-related nervous tissues, in a macaque model of type 2 diabetes using immunohistochemistry. Results showed that nerve growth factor expression decreased, but inducible nitric oxide synthase expression increased, in the mesencephalon and diencephalon, as well as visual- and auditory- related nervous tissues. These results suggested that nerve growth factor and inducible nitric oxide synthase play an important role in regulating the development of diabetic visual- and auditory-related diseases.展开更多
BACKGROUND:Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and ...BACKGROUND:Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March 2007 to March 2008. MATERIALS: A total of 120 healthy, adult Sprague Dawley rats were selected for this study. Rat models of moderate acute thoracic (T9) spinal cord injury were established using the modified Allen method. Shuxuening injection was obtained from Zhenbaodao Pharmaceutical Co., Ltd., China. Methylprednisolone was purchased from North China Pharmaceutical Co., Ltd. METHODS: All rats were equally and randomly divided into four groups. Only the spinal cord was exposed in the sham operation group rats. In the trauma group, rats were not treated with drugs following spinal cord injury. Rats in the hormone group were intraperitoneally injected with 30 mg/kg methylprednisolone following spinal cord injury. Rats in the ginkgo biloba leaf extract group were intraperitoneally infused with a 1.0 mL/kg Shuxuening injection per day. MAIN OUTCOME MEASURES: At 1 hour, as well as 1, 3, 5, 7, and 14 days after spinal cord injury, iNOS- and Bcl-2/Bax-positive cells were quantified with immunohistochemistry. Pathological changes were detected using hematoxylineosin staining under an optical microscope. RESULTS: Spinal cord injury in the ginkgo biloba leaf extract and hormone groups was milder compared with the trauma group. Demyelination was significantly ameliorated and the necrotic cavity was obviously reduced in the injured spinal cord of rats in the ginkgo biloba leaf extract and hormone groups at each time point. iNOS expression was increased in the injured spinal cord, and reached a peak at 5 days. The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P 〈 0.05-0.01). The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P 〈 0.05). Bcl-2 expression reached a peak at 3 days, and Bax expression reached a peak at 5 days following rat spinal cord injury. Bcl-2 expression was increased, but Bax expression was decreased in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P 〈 0.05-0.01). Bcl-2 expression was greater, but Bax expression was reduced in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P 〈 0.05). CONCLUSION: Ginkgo biloba leaf extract exhibits neuroprotective effects by upregulating Bcl-2 expression, downregulating Bax expression, and significantly inhibiting high expressions of iNOS in the injured spinal cord. The neuroprotective effects of ginkgo biloba leaf extract are greater compared with methylprednisolone at 1 week after spinal cord injury.展开更多
BACKGROUND: Inducible nitric oxide synthase (iNOS) cannot be detected in the neurons and glial cells of normal rats, but iNOS can be found in some neurons and glial cells of rats following ischemic, traumatic, neur...BACKGROUND: Inducible nitric oxide synthase (iNOS) cannot be detected in the neurons and glial cells of normal rats, but iNOS can be found in some neurons and glial cells of rats following ischemic, traumatic, neurotoxic or inflammatory damage. OBJECTIVE: To investigate iNOS expression and iNOS-positive cell types at various time points following damage to the rat frontal lobe using a sharp instrument. DESIGN: A nerve molecular biology, randomized, controlled study. TIME AND SETTING: This experiment was performed at the Department of Human Anatomy, Institute of Neurobiology, Medical School of Nantong University, between April 2006 and December 2007. MATERIALS: Rabbit anti-iNOS antibody (Santa Cruz, USA), biotin labeled goat anti-rabbit antibody (Sigma, USA), reverse transcription kit (Biouniquer, Hong Kong, China) and horseradish peroxidase labeled goat anti-rabbit antibody (Pierce, USA) were used for this study. METHODS: A total of 112 healthy rats aged 3 months were randomly assigned into a sham operation group (n = 28) and a damage group (n = 84). Rat models of frontal lobe damage were induced in the damage group using a sharp instrument to make an incision in the frontal lobe cortex. In the sham operation group, the rat bone window was opened but brain tissues were left intact. MAIN OUTCOME MEASURES: Parameters were measured at 3, 6, 12, 24, 72, 120 and 168 hours following damage in both groups. Pathological changes were observed using Nissl staining and hematoxylin-eosin staining. Expression of iNOS mRNA, iNOS protein and iNOS-positive cells were examined by RT-PCR, Western blot analysis and immunohistochemistry, respectively. RESULTS: A large number of inflammatory cells infiltrated the damaged region 12 and 24 hours following damage, iNOS mRNA and iNOS protein expression increased in and around the damaged region 3 hours following damage, reached a peak at 24 hours, and then gradually decreased. The changes in iNOS-positive cell number reflected the changes in iNOS mRNA and iNOS protein expression after damage, iNOS was mainly found in neural cells at 3 and 6 hours, in macrophages at 12 and 24 hours, and in glial cells at 72 and 120 hours after damage. iNOS-positive cells were few in and surrounding the damaged region at 168 hours. There were a few iNOS-positive neural cells in the rat frontal lobe cortex in the sham operation group. CONCLUSION: Neurons, macrophages and glial cells can express iNOS following rat frontal lobe damage caused by a sharp instrument. The levels of iNOS expression, and the cell types expressing iNOS, change with time.展开更多
BACKGROUND: Previous studies have shown that inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury...BACKGROUND: Previous studies have shown that inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE: To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING: The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS: Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody (Transduction Laboratory, USA), as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG (Santa Cruz Biotechnology, USA) were used in the present study.METHODS: A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group (n = 32), crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time (6,12, 24, and 72 hours), respectively (n = 8). Sham surgery (n = 8) and normal control (n = 8) groups were also established.MAIN OUTCOME MEASURES: iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS: iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury (P〈0.05) and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side (P〈 0.05).CONCLUSION: iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.展开更多
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...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 duct ligation (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. Its 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.展开更多
Nitric oxide(NO)and its reaction products were key players in the pathophysiology of sepsis and shock.The present study was designed to explore the effects of lanthanum chloride(LaCl3)on inducible nitric oxide syn...Nitric oxide(NO)and its reaction products were key players in the pathophysiology of sepsis and shock.The present study was designed to explore the effects of lanthanum chloride(LaCl3)on inducible nitric oxide synthase(iNOS)expression,at both gene and protein levels,in RAW264.7 macrophages induced by Lipopolysaccharide(LPS).Reverse transcription polymerase chain reaction(RT-PCR),immunofluorescence,and western blot were employed to measure iNOS gene expression,localization,and protein expression respectively.NO production in culture supernatants was detected by the nitrate reductase method.The results showed that LaCl3 significantly attenuated the iNOS gene and protein expression,as well as NO production in RAW264.7cells induced by LPS.展开更多
Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon ...Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.展开更多
The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects we...The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects were divided into three groups according to their disease/health conditions: the HPS group (cirrhotic patients with HPS; n=63), the non-HPS group (cirrhotic patients without HPS; n=182), and the control group (healthy subjects without liver disease; n=35). The distribution of the MPO–463 G/A genotype and its relationship with iNOS expression in a typical cell block from ascitic fluid were detected by immunohistochemistry and polymerase chain reaction-restricted fragment length polymorphism analysis (PCR-RFLP). In the HPS group, the partial pressure of oxygen in blood and ascitic fluid was significantly decreased (8.95±1.58 kPa and 6.81±0.95 kPa, respectively; both P<0.01), while the partial pressure of carbon dioxide significantly increased (4.62±0.20 kPa and 5.92±0.45 kPa, respectively; P<0.01). MPO and iNOS levels were significantly increased in the HPS group as compared with the non-HPS group. These increases were even more remarkable in ascitic fluid (41.36±11.62 and 13.23±4.81 μg/L; 10.27± 3.20 and 4.95±1.12 μg/L) than in blood (16.66±5.24 and 4.87±1.73 μg/L; 5.79±2.31 and 2.35±0.84 μg/L). The distribution of the MPO genotypes GG, GA, and AA were 76.2%, 22.2% and 1.6% in the HPS group, and 57.7%, 37.9% and 4.4% in the non-HPS group (P<0.05). The expression of iNOS was significantly higher in patients with the G alleles (G/G and G/A) (61.54%, 48/78) than in patients with A alleles (G/A and A/A) (38.46%, 30/78) (P<0.01). It was suggested that the expression levels of iNOS and MPO were correlated with HPS-induced hypoxemia. The MPO-463 G/A mutation might be a protective factor that prevents the development of HPS. The MPO might be involved in the regulation of iNOS expression. In humans, MPO pathways, the iNOS/NO system, and their interaction might have an impact on the occurrence and development of HPS.展开更多
BACKGROUND: The stellate ganglion block (SGB) plays a protective role in focal cerebral ischemia/reperfusion injury. The human SGB can be simulated by transection of the cervical sympathetic trunk (TCST) in rats....BACKGROUND: The stellate ganglion block (SGB) plays a protective role in focal cerebral ischemia/reperfusion injury. The human SGB can be simulated by transection of the cervical sympathetic trunk (TCST) in rats. OBJECTIVE: To observe the effects of TCST on inducible nitric oxide synthase (iNOS) levels and cerebral infarct volume in the hippocampus of rats with cerebral ischemia/reperfusion injury, and to analyze the mechanism of action. DESIGN, TIME AND SETTING: A completely randomized, controlled, neuropathological experiment was performed at the Institute of Neurological Disease, Taihe Hospital, Yunyang Medical College between March and September 2006. MATERIALS: A total of 93 Wistar rats, aged 1718 weeks, of either gender, were used for this study. 2, 3, 5-triphenyl tetrazolium chloride was purchased from Changsha Hongyuan Biological Reagent Company China. Rabbit iNOS antibody and goat anti-rabbit IgG antibody were the products of Wuhan Boster Biological Reagent Co., Ltd., China. METHODS: Ten rats were randomly selected for the sham-operated group. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion (MCAO) using the suture method in the remaining rats. Forty successful rat models were randomly and equally divided into the following two groups: (1) TCST group: subsequent to TCST, MCAO was performed for 2 hours, followed by 24 hours reperfusion; (2) model group: rats underwent experimental procedures similar to the TCST group, with the exception of TCST. Rats in the sham-operated group were subjected to experimental procedures similar to the model group; however, the thread was only introduced to a depth of 10 mm. MAIN OUTCOME MEASURES: Following 24 hours of reperfusion, functional neurological deficits were scored. Brain tissue sections from ten rats of each group were used to measure cerebral infarct volume by TTC staining. Hippocampal tissue sections of an additional ten rats from each group were used to detect iNOS levels using the streptavidin-peroxidase immunohistocbemistry method. Experimental data were expressed as absorbance values. RESULTS: Of the 93 selected rats, 50 were included in the final analysis. After MCAO for 2 hours, followed by 24 hours reperfusion, the absorbance values of iNOS-immunoreactive product were significantly greater in the model group compared to the TCST and sham-operated groups (P 〈 0.05). However, there was no difference between the TCST and sham-operated groups (P 〉 0.05). In addition, cerebral infarct volume in the model group was significantly greater compared to the TCST group (P 〈 0.05). The TCST group performed with lower total functional neurological scores compared to the model group (P 〈 0.05). CONCLUSION: TCST protects the brain against focal cerebral ischemia/reperfusion injury by possibly down-regulating hippocampal iNOS expression.展开更多
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...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.展开更多
AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized int...AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.展开更多
Objective:To investigate the production of nitric oxide(NO) and the expression of indurible nitric oxide synthase (iNOS). and their possible role in abdominal aortic aneurysm (AAA). Methods: A total of 28 patients wit...Objective:To investigate the production of nitric oxide(NO) and the expression of indurible nitric oxide synthase (iNOS). and their possible role in abdominal aortic aneurysm (AAA). Methods: A total of 28 patients with AAA, 10 healthy controls, and 8 patients with arterial occlusive disease were enrolled into this study. Standard colorimetric assay was used to examine NO concentration in plasma from patients with AAA and normal controls, and in cultured smooth muscle cells (SMCs). Expression of iNOS in aortas and cultured SMCs were detected by immunochemistry. The correlation of iNOS expression with age of the patient, size of aneurysm, and degree of inflammation was also investigated by Cochran-Mantel-Haenszel X2 test and Kendall correlation. Results: Expression of iNOS increased significantly in the wall of aneurism in the patients with AAA compared to the healthy controls (P<0. 05) and the patients with occlusive arteries (P<0. 05). iNOS protein and media NOx (nitrite + nitrate) also increased in cultured SMCs from human AAA (n = 4, P<0. 05), while plasma NOx decreased in patients with AAA (n = 25) compared to the healthy controls (n = 20). There was a positive correlation between iNOS protein and the degree of inflammation in aneurismal wall (Kendall coefficient = 0. 5032, P = 0. 0029). Conclusion: SMCs and inflammatory cells are main cellular sources of increased iNOS in AAA, and NO may play a part in pathogenesis in AAA through inflammation, SMCs and oxidative stress.展开更多
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...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: Tn 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-9expression were 71.88% (23/32) and 78.13% (25/32) in HCC. MMP-g 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 = 0.04g 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 to tumor 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.展开更多
瞄准:与肠的损害在新生的老鼠调查神经元的氮的氧化物 synthase ( nNOS )和可诱导的氮的氧化物 synthase ( i NOS )的动态变化和角色并且定义引起坏死小肠结肠炎( NEC )是否在粘膜与氮的氧化物 synthase ( NOS )的层次被联系影响的肠...瞄准:与肠的损害在新生的老鼠调查神经元的氮的氧化物 synthase ( nNOS )和可诱导的氮的氧化物 synthase ( i NOS )的动态变化和角色并且定义引起坏死小肠结肠炎( NEC )是否在粘膜与氮的氧化物 synthase ( NOS )的层次被联系影响的肠织物。方法:Wistar 老鼠在年龄的不到 24 h 与 5 mg/kg 收到了腹膜内注射脂肪的多糖(LPS ) 。回肠纸巾为 NEC 的组织学的评估并且为 nNOS 和 i NOS 的大小在 1, 3, 6, 12 和 24 h 追随者 LPS 挑战被收集。在肠的损害的度和 NOS 的层次之间的关联是坚定的。结果:注射 LPS 的小狗在损害分数对控制显示出重要增加。nNOS 蛋白质和 mRNA 的表示在 LPS 注射以后被减少。在 nNOS 蛋白质和在 24 h 以内的中部的肠的损害的等级之间有否定重要关联。i NOS 蛋白质和 mRNA 的表示显著地在肠的损害的山峰被增加。结论:nNOS 和 i NOS 在导致 LPS 的肠的损害起不同作用。小心应该在 NEC 有关 NOS 禁止者的潜在的治疗学的使用被施加。展开更多
AIM: To examine the association of inducible nitric oxide synthase (iNOS) C150T polymorphism with gastric cancer, as well as with gastric atrophy and H pylori seropositivity. METHODS: A single nucleotide polymorphism ...AIM: To examine the association of inducible nitric oxide synthase (iNOS) C150T polymorphism with gastric cancer, as well as with gastric atrophy and H pylori seropositivity. METHODS: A single nucleotide polymorphism of iNOS C150T was examined for 454 Japanese health checkup examinees (126 males and 328 females) aged 35 to 85 years without a history of cancer and 202 gastric cancer patients (134 males and 68 females) aged 33 to 94 years with pathologically confirmed diagnosis of gastric adenocarcinoma. RESULTS: The iNOS C150T polymorphism was not associated with gastric atrophy or with H pylori seropositivity. The odds ratio (OR) of the C/T + T/T for gastric cancer was increased without statistical significance (OR=1.19, 95% confidence interval (CI): 0.68-2.08). In the differentiated subgroup (n = 113), however, the OR of the C/T genotype for gastric cancer was significant (OR = 2.02, 95% CI: 1.04-3.92) relative to the C/C genotype. In addition, considering the location of gastric cancer (n = 105), there were significant differences between the controls and non-cardia group with the OR of 2.13 (95% CI: 1.08-4.18) for C/T and 1.94 (95% CI: 1.00-3.78) for C/T + T/T. CONCLUSION: The iNOS C150T polymorphism is associated with the risk of H pylori-related gastric cancerin a Japanese population. This polymorphism may play an important role in increasing the risk of gastric cancer in Asian countires with the highest rates of gastric cancer.展开更多
OBJECTIVE: To investigate the expression and the role of iNOS expression in hepatic ischemia-reperfusion (L/R) injury. METHODS: Male Wistar rats were subjected to 30-minute hepatic ischemia, then iNOS protein and iNOS...OBJECTIVE: To investigate the expression and the role of iNOS expression in hepatic ischemia-reperfusion (L/R) injury. METHODS: Male Wistar rats were subjected to 30-minute hepatic ischemia, then iNOS protein and iNOS mRNA expression in liver tissue was assessed by Western blot and RT-PCR analysis respectively at different time points after reperfusion. The effects of L-NAME (Nω-nitro-L-arginine methyl ester, a nonselective NOS inhibitor) or AE-ITU (aminoethytl-isothiourea, a relative selective inhibitor of iNOS) treatment were also evaluated. RESULTS: High levels of iNOS protein and mRNA expression were detected in the liver tissue subjected to I/R, but not in the sham-operated rats. iNOS protein and iNOS mRNA expression reached a maximum on the first day after reperfusion and decreased later. The levels of iNOS protein and iNOS mRNA disappeared on 7th, 3rd day after reperfusion respectively. The high iNOS expression was correlated with hepatic dysfunction. L-NAME administration worsened hepatic dysfunction induced by hepatic I/R. In contrast, AE-ITU administration showed mild protective effects against hepatic dysfunction induced by hepatic I/R. CONCLUSION: Ischemia-reperfusion may induce or up-regulate the expression of iNOS protein and iNOS mRNA, which is detrimental to hepatic I/R injury.展开更多
文摘Aim Inducible nitric oxide synthase (iNOS) makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide (LPS) induces iNOS expression through activation of the inhibitor of KB- α (IKB-α) -nuclear factor-KB (NF-KB) cascade, whereas interferon-γ (IFN-γ) acts through Janus kinase ( JAK)- signal transducer and activator of transcription 1 ( STAT1 ) signals. Heat shock factor 1 ( HSF1 ), a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6). But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide (NO) content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation (CHIP) was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.
基金Funding for the canine atorvastatin study was through the Alzheimer's Association IIRG-03-5673 to Elizabeth Head
文摘Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are still unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/ tyrosine kinase activity is able to modulate cell signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebellum and liver of the same animals. We demonstrated that atorvastatin treatment (80 mg/day for 14.5 months) to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebellum. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as well as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not associated with higher oxidative/nitrosative stress levels. We hypothesize that biliverdin reductase-A-dependent inducible nitric oxide synthase regulation strongly contributes to the cognitive improvement observed following atorvastatin treatment.
文摘AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differentiation were used.SaOS2 show a rather mature phenotype,while U2 OS are negative for almost all osteoblastic markers.The cells were exposed to different concentrations of prednisolone(1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase(i NOS) l-N6-(iminoethyl)-lysine-HCl(L-NIL).Cell growth was assessed by counting viable cells.The production of nitric oxide(NO) was measured in the conditioned media by the Griess method.The production of reactive oxygen species was quantified using 2'-7'-dichlorofluorescein diacetate.Western blot with specific antibodies against NOSs was performed on cell extracts.RESULTS:Prednisolone inhibited SaOS2 cell growth in a dose dependent manner.No significant effects were observed in U2OS.The inhibition of SaOS2 growth is not due to oxidative stress,because antioxidants do not rescue cell proliferation.Since high concentrations of NO inhibit bone formation,we also measured NO and found it induced in SaOS2,but not in U2 OS,exposed to prednisolone,because of the upregulation of i NOS as detected by western blot.Therefore,we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL.L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L.At the same concentrations,we found that L-NIL rescued SaOS2 growth after exposure to prednisolone.In U2 OS cells,prednisolone did not induce NO production nor affected cell growth.All together,these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.CONCLUSION:Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of i NOS,while no effect was exerted on U2OS.
基金supported by the Program for Changjiang Scholars and Innovative Research Teaming University, No. IRT0848Sichuan Province International Technology Cooperation and Communication Research Programs, No. 2010HH0013+2 种基金Sichuan Province Basic Research Program, No. 2011JY0054the National Key Research Program of China, No. 2011ZX09301-001, 2011ZX09307-301-3Science and Technology Support Programs of Sichuan Province, No. 2011JO0040, 2011ZO0034
文摘The present study detected distribution and expression of nerve growth factor and inducible nitric oxide synthase in the mesencephalon and diencephalon, as well as visual- and auditory-related nervous tissues, in a macaque model of type 2 diabetes using immunohistochemistry. Results showed that nerve growth factor expression decreased, but inducible nitric oxide synthase expression increased, in the mesencephalon and diencephalon, as well as visual- and auditory- related nervous tissues. These results suggested that nerve growth factor and inducible nitric oxide synthase play an important role in regulating the development of diabetic visual- and auditory-related diseases.
文摘BACKGROUND:Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March 2007 to March 2008. MATERIALS: A total of 120 healthy, adult Sprague Dawley rats were selected for this study. Rat models of moderate acute thoracic (T9) spinal cord injury were established using the modified Allen method. Shuxuening injection was obtained from Zhenbaodao Pharmaceutical Co., Ltd., China. Methylprednisolone was purchased from North China Pharmaceutical Co., Ltd. METHODS: All rats were equally and randomly divided into four groups. Only the spinal cord was exposed in the sham operation group rats. In the trauma group, rats were not treated with drugs following spinal cord injury. Rats in the hormone group were intraperitoneally injected with 30 mg/kg methylprednisolone following spinal cord injury. Rats in the ginkgo biloba leaf extract group were intraperitoneally infused with a 1.0 mL/kg Shuxuening injection per day. MAIN OUTCOME MEASURES: At 1 hour, as well as 1, 3, 5, 7, and 14 days after spinal cord injury, iNOS- and Bcl-2/Bax-positive cells were quantified with immunohistochemistry. Pathological changes were detected using hematoxylineosin staining under an optical microscope. RESULTS: Spinal cord injury in the ginkgo biloba leaf extract and hormone groups was milder compared with the trauma group. Demyelination was significantly ameliorated and the necrotic cavity was obviously reduced in the injured spinal cord of rats in the ginkgo biloba leaf extract and hormone groups at each time point. iNOS expression was increased in the injured spinal cord, and reached a peak at 5 days. The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P 〈 0.05-0.01). The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P 〈 0.05). Bcl-2 expression reached a peak at 3 days, and Bax expression reached a peak at 5 days following rat spinal cord injury. Bcl-2 expression was increased, but Bax expression was decreased in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P 〈 0.05-0.01). Bcl-2 expression was greater, but Bax expression was reduced in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P 〈 0.05). CONCLUSION: Ginkgo biloba leaf extract exhibits neuroprotective effects by upregulating Bcl-2 expression, downregulating Bax expression, and significantly inhibiting high expressions of iNOS in the injured spinal cord. The neuroprotective effects of ginkgo biloba leaf extract are greater compared with methylprednisolone at 1 week after spinal cord injury.
基金Supported by:Natural Science Research Plan for Jiangsu Colleges,No. 05KJD180165
文摘BACKGROUND: Inducible nitric oxide synthase (iNOS) cannot be detected in the neurons and glial cells of normal rats, but iNOS can be found in some neurons and glial cells of rats following ischemic, traumatic, neurotoxic or inflammatory damage. OBJECTIVE: To investigate iNOS expression and iNOS-positive cell types at various time points following damage to the rat frontal lobe using a sharp instrument. DESIGN: A nerve molecular biology, randomized, controlled study. TIME AND SETTING: This experiment was performed at the Department of Human Anatomy, Institute of Neurobiology, Medical School of Nantong University, between April 2006 and December 2007. MATERIALS: Rabbit anti-iNOS antibody (Santa Cruz, USA), biotin labeled goat anti-rabbit antibody (Sigma, USA), reverse transcription kit (Biouniquer, Hong Kong, China) and horseradish peroxidase labeled goat anti-rabbit antibody (Pierce, USA) were used for this study. METHODS: A total of 112 healthy rats aged 3 months were randomly assigned into a sham operation group (n = 28) and a damage group (n = 84). Rat models of frontal lobe damage were induced in the damage group using a sharp instrument to make an incision in the frontal lobe cortex. In the sham operation group, the rat bone window was opened but brain tissues were left intact. MAIN OUTCOME MEASURES: Parameters were measured at 3, 6, 12, 24, 72, 120 and 168 hours following damage in both groups. Pathological changes were observed using Nissl staining and hematoxylin-eosin staining. Expression of iNOS mRNA, iNOS protein and iNOS-positive cells were examined by RT-PCR, Western blot analysis and immunohistochemistry, respectively. RESULTS: A large number of inflammatory cells infiltrated the damaged region 12 and 24 hours following damage, iNOS mRNA and iNOS protein expression increased in and around the damaged region 3 hours following damage, reached a peak at 24 hours, and then gradually decreased. The changes in iNOS-positive cell number reflected the changes in iNOS mRNA and iNOS protein expression after damage, iNOS was mainly found in neural cells at 3 and 6 hours, in macrophages at 12 and 24 hours, and in glial cells at 72 and 120 hours after damage. iNOS-positive cells were few in and surrounding the damaged region at 168 hours. There were a few iNOS-positive neural cells in the rat frontal lobe cortex in the sham operation group. CONCLUSION: Neurons, macrophages and glial cells can express iNOS following rat frontal lobe damage caused by a sharp instrument. The levels of iNOS expression, and the cell types expressing iNOS, change with time.
基金the National Natural Science Foundation of China,No. 30860290the Scientific Research Foundation of Department of Health of Hunan Province of China,No. C2007038+2 种基金the Scientific Research Program of Department of Education of Hunan Province,No. 08C816the Nanometer Specific Foundation of Shanghai of China,No. 1052nm05800the Doctor Innovation Foundation of Medical College of Shanghai Jiao Tong University of China,No. BXJ201043
文摘BACKGROUND: Previous studies have shown that inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE: To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING: The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS: Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody (Transduction Laboratory, USA), as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG (Santa Cruz Biotechnology, USA) were used in the present study.METHODS: A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group (n = 32), crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time (6,12, 24, and 72 hours), respectively (n = 8). Sham surgery (n = 8) and normal control (n = 8) groups were also established.MAIN OUTCOME MEASURES: iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS: iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury (P〈0.05) and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side (P〈 0.05).CONCLUSION: iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.
文摘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 duct ligation (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. Its 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.
基金Project supported by the National Natural Science Foundation of China(30660182)the Program for Innovative ResearchTeam of Nanchang University
文摘Nitric oxide(NO)and its reaction products were key players in the pathophysiology of sepsis and shock.The present study was designed to explore the effects of lanthanum chloride(LaCl3)on inducible nitric oxide synthase(iNOS)expression,at both gene and protein levels,in RAW264.7 macrophages induced by Lipopolysaccharide(LPS).Reverse transcription polymerase chain reaction(RT-PCR),immunofluorescence,and western blot were employed to measure iNOS gene expression,localization,and protein expression respectively.NO production in culture supernatants was detected by the nitrate reductase method.The results showed that LaCl3 significantly attenuated the iNOS gene and protein expression,as well as NO production in RAW264.7cells induced by LPS.
基金This work was supported by a grant from the Scientific Research Foundation of Ministry of Public Health of PR China (No. 96-1-204).
文摘Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.
基金supported by a grant from Heilongjiang Provincial Science and Technology Breakthrough Project Foundation (No. GB07C32506)
文摘The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects were divided into three groups according to their disease/health conditions: the HPS group (cirrhotic patients with HPS; n=63), the non-HPS group (cirrhotic patients without HPS; n=182), and the control group (healthy subjects without liver disease; n=35). The distribution of the MPO–463 G/A genotype and its relationship with iNOS expression in a typical cell block from ascitic fluid were detected by immunohistochemistry and polymerase chain reaction-restricted fragment length polymorphism analysis (PCR-RFLP). In the HPS group, the partial pressure of oxygen in blood and ascitic fluid was significantly decreased (8.95±1.58 kPa and 6.81±0.95 kPa, respectively; both P<0.01), while the partial pressure of carbon dioxide significantly increased (4.62±0.20 kPa and 5.92±0.45 kPa, respectively; P<0.01). MPO and iNOS levels were significantly increased in the HPS group as compared with the non-HPS group. These increases were even more remarkable in ascitic fluid (41.36±11.62 and 13.23±4.81 μg/L; 10.27± 3.20 and 4.95±1.12 μg/L) than in blood (16.66±5.24 and 4.87±1.73 μg/L; 5.79±2.31 and 2.35±0.84 μg/L). The distribution of the MPO genotypes GG, GA, and AA were 76.2%, 22.2% and 1.6% in the HPS group, and 57.7%, 37.9% and 4.4% in the non-HPS group (P<0.05). The expression of iNOS was significantly higher in patients with the G alleles (G/G and G/A) (61.54%, 48/78) than in patients with A alleles (G/A and A/A) (38.46%, 30/78) (P<0.01). It was suggested that the expression levels of iNOS and MPO were correlated with HPS-induced hypoxemia. The MPO-463 G/A mutation might be a protective factor that prevents the development of HPS. The MPO might be involved in the regulation of iNOS expression. In humans, MPO pathways, the iNOS/NO system, and their interaction might have an impact on the occurrence and development of HPS.
基金Supported by: the Excellent Middle-aged and Youth Talent Project of Education Department of Hubei Province, No. 2002B03001
文摘BACKGROUND: The stellate ganglion block (SGB) plays a protective role in focal cerebral ischemia/reperfusion injury. The human SGB can be simulated by transection of the cervical sympathetic trunk (TCST) in rats. OBJECTIVE: To observe the effects of TCST on inducible nitric oxide synthase (iNOS) levels and cerebral infarct volume in the hippocampus of rats with cerebral ischemia/reperfusion injury, and to analyze the mechanism of action. DESIGN, TIME AND SETTING: A completely randomized, controlled, neuropathological experiment was performed at the Institute of Neurological Disease, Taihe Hospital, Yunyang Medical College between March and September 2006. MATERIALS: A total of 93 Wistar rats, aged 1718 weeks, of either gender, were used for this study. 2, 3, 5-triphenyl tetrazolium chloride was purchased from Changsha Hongyuan Biological Reagent Company China. Rabbit iNOS antibody and goat anti-rabbit IgG antibody were the products of Wuhan Boster Biological Reagent Co., Ltd., China. METHODS: Ten rats were randomly selected for the sham-operated group. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion (MCAO) using the suture method in the remaining rats. Forty successful rat models were randomly and equally divided into the following two groups: (1) TCST group: subsequent to TCST, MCAO was performed for 2 hours, followed by 24 hours reperfusion; (2) model group: rats underwent experimental procedures similar to the TCST group, with the exception of TCST. Rats in the sham-operated group were subjected to experimental procedures similar to the model group; however, the thread was only introduced to a depth of 10 mm. MAIN OUTCOME MEASURES: Following 24 hours of reperfusion, functional neurological deficits were scored. Brain tissue sections from ten rats of each group were used to measure cerebral infarct volume by TTC staining. Hippocampal tissue sections of an additional ten rats from each group were used to detect iNOS levels using the streptavidin-peroxidase immunohistocbemistry method. Experimental data were expressed as absorbance values. RESULTS: Of the 93 selected rats, 50 were included in the final analysis. After MCAO for 2 hours, followed by 24 hours reperfusion, the absorbance values of iNOS-immunoreactive product were significantly greater in the model group compared to the TCST and sham-operated groups (P 〈 0.05). However, there was no difference between the TCST and sham-operated groups (P 〉 0.05). In addition, cerebral infarct volume in the model group was significantly greater compared to the TCST group (P 〈 0.05). The TCST group performed with lower total functional neurological scores compared to the model group (P 〈 0.05). CONCLUSION: TCST protects the brain against focal cerebral ischemia/reperfusion injury by possibly down-regulating hippocampal iNOS expression.
基金supported by the Natural Science Foundation of ChinaNos.81971212 (to FG)+7 种基金81601129 (to XXX)the Open Fund of the Key Laboratory of Medical ElectrophysiologyMinistry of Education&Medical Electrophysiological Key Laboratory of Sichuan ProvinceInstitute of Cardiovascular ResearchSouthwest Medical UniversityNo.KeyME-2018-07 (to FG)Liaoning Province Xingliao Talent Program ProjectNo.XLYC1907164 (to FG)
文摘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.
基金Supported by the National Natural Science(No.81660217)National College Students Innovation and Entrepreneurship Training Program Project (No.201911810004)。
文摘AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.
基金Supported by the National Natural Science Foundation of China (No. 39800177)
文摘Objective:To investigate the production of nitric oxide(NO) and the expression of indurible nitric oxide synthase (iNOS). and their possible role in abdominal aortic aneurysm (AAA). Methods: A total of 28 patients with AAA, 10 healthy controls, and 8 patients with arterial occlusive disease were enrolled into this study. Standard colorimetric assay was used to examine NO concentration in plasma from patients with AAA and normal controls, and in cultured smooth muscle cells (SMCs). Expression of iNOS in aortas and cultured SMCs were detected by immunochemistry. The correlation of iNOS expression with age of the patient, size of aneurysm, and degree of inflammation was also investigated by Cochran-Mantel-Haenszel X2 test and Kendall correlation. Results: Expression of iNOS increased significantly in the wall of aneurism in the patients with AAA compared to the healthy controls (P<0. 05) and the patients with occlusive arteries (P<0. 05). iNOS protein and media NOx (nitrite + nitrate) also increased in cultured SMCs from human AAA (n = 4, P<0. 05), while plasma NOx decreased in patients with AAA (n = 25) compared to the healthy controls (n = 20). There was a positive correlation between iNOS protein and the degree of inflammation in aneurismal wall (Kendall coefficient = 0. 5032, P = 0. 0029). Conclusion: SMCs and inflammatory cells are main cellular sources of increased iNOS in AAA, and NO may play a part in pathogenesis in AAA through inflammation, SMCs and oxidative stress.
文摘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: Tn 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-9expression were 71.88% (23/32) and 78.13% (25/32) in HCC. MMP-g 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 = 0.04g 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 to tumor 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.
文摘瞄准:与肠的损害在新生的老鼠调查神经元的氮的氧化物 synthase ( nNOS )和可诱导的氮的氧化物 synthase ( i NOS )的动态变化和角色并且定义引起坏死小肠结肠炎( NEC )是否在粘膜与氮的氧化物 synthase ( NOS )的层次被联系影响的肠织物。方法:Wistar 老鼠在年龄的不到 24 h 与 5 mg/kg 收到了腹膜内注射脂肪的多糖(LPS ) 。回肠纸巾为 NEC 的组织学的评估并且为 nNOS 和 i NOS 的大小在 1, 3, 6, 12 和 24 h 追随者 LPS 挑战被收集。在肠的损害的度和 NOS 的层次之间的关联是坚定的。结果:注射 LPS 的小狗在损害分数对控制显示出重要增加。nNOS 蛋白质和 mRNA 的表示在 LPS 注射以后被减少。在 nNOS 蛋白质和在 24 h 以内的中部的肠的损害的等级之间有否定重要关联。i NOS 蛋白质和 mRNA 的表示显著地在肠的损害的山峰被增加。结论:nNOS 和 i NOS 在导致 LPS 的肠的损害起不同作用。小心应该在 NEC 有关 NOS 禁止者的潜在的治疗学的使用被施加。
基金Supported by a Grant-in-Aid for Scientific Research on Special Priority Areas of Cancer from the Japanese Ministry of Education, Culture, Sports, Science and Technology
文摘AIM: To examine the association of inducible nitric oxide synthase (iNOS) C150T polymorphism with gastric cancer, as well as with gastric atrophy and H pylori seropositivity. METHODS: A single nucleotide polymorphism of iNOS C150T was examined for 454 Japanese health checkup examinees (126 males and 328 females) aged 35 to 85 years without a history of cancer and 202 gastric cancer patients (134 males and 68 females) aged 33 to 94 years with pathologically confirmed diagnosis of gastric adenocarcinoma. RESULTS: The iNOS C150T polymorphism was not associated with gastric atrophy or with H pylori seropositivity. The odds ratio (OR) of the C/T + T/T for gastric cancer was increased without statistical significance (OR=1.19, 95% confidence interval (CI): 0.68-2.08). In the differentiated subgroup (n = 113), however, the OR of the C/T genotype for gastric cancer was significant (OR = 2.02, 95% CI: 1.04-3.92) relative to the C/C genotype. In addition, considering the location of gastric cancer (n = 105), there were significant differences between the controls and non-cardia group with the OR of 2.13 (95% CI: 1.08-4.18) for C/T and 1.94 (95% CI: 1.00-3.78) for C/T + T/T. CONCLUSION: The iNOS C150T polymorphism is associated with the risk of H pylori-related gastric cancerin a Japanese population. This polymorphism may play an important role in increasing the risk of gastric cancer in Asian countires with the highest rates of gastric cancer.
基金This study was supported partly by the Grant-In-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 11470334) to M.Takeda.
文摘OBJECTIVE: To investigate the expression and the role of iNOS expression in hepatic ischemia-reperfusion (L/R) injury. METHODS: Male Wistar rats were subjected to 30-minute hepatic ischemia, then iNOS protein and iNOS mRNA expression in liver tissue was assessed by Western blot and RT-PCR analysis respectively at different time points after reperfusion. The effects of L-NAME (Nω-nitro-L-arginine methyl ester, a nonselective NOS inhibitor) or AE-ITU (aminoethytl-isothiourea, a relative selective inhibitor of iNOS) treatment were also evaluated. RESULTS: High levels of iNOS protein and mRNA expression were detected in the liver tissue subjected to I/R, but not in the sham-operated rats. iNOS protein and iNOS mRNA expression reached a maximum on the first day after reperfusion and decreased later. The levels of iNOS protein and iNOS mRNA disappeared on 7th, 3rd day after reperfusion respectively. The high iNOS expression was correlated with hepatic dysfunction. L-NAME administration worsened hepatic dysfunction induced by hepatic I/R. In contrast, AE-ITU administration showed mild protective effects against hepatic dysfunction induced by hepatic I/R. CONCLUSION: Ischemia-reperfusion may induce or up-regulate the expression of iNOS protein and iNOS mRNA, which is detrimental to hepatic I/R injury.