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

AIM: To determine the expressions of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9)in hepatocellular carcinoma (HCC) and to investigate the relationship between iNOS and MMP-9 expression and their effects on angiogenesis and progression of HCC.METHODS: 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.

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

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。

Nerve growth factor and inducible nitric oxide synthase expression in the mesencephalon and diencephalon, as well as visual-and auditory-related nervous tissues, in a macaque model of type 2 diabetes

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.

Biliverdin Reductase-A correlates with inducible nitric oxide synthasein in atorvastatin treated aged canine brain

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.

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

瞄准：到可诱导的氮的氧化物 synthase (i NOS ) 和在有导致乙醇的肝损伤的老鼠和他们有肝的关系的内皮的氮的氧化物 synthase (eNOS ) 的表示和活动损坏的学习，原子 factor-kappaB (NF-kappaB ) 和肿瘤坏死 factor-alpha (TNF-alpha ) 的激活在肝的表示。方法：女 Sprague-Dawley 老鼠为 4 或 6 wk 经由胃管饲法与乙醇或 isocaloric 葡萄糖日报一起被给鱼油(0.5 mL ) 。肝损伤用浆液丙氨酸 aminotransferase (中高音) 被估计活动和病理学的分析。肝 malondialdehyde (MDA ) ，氮的氧化物内容， i NOS 和 eNOS 活动是坚定的。在肝的 NF-kappaB p65iiNOS， eNOS 和 TNF-alpha 蛋白质或 mRNA 表示被免疫组织化学或反向的 transcriptase 聚合酶链反应(RT-PCR ) 检测。结果：为 4 wk 的长期的乙醇管饲法在肝引起了脂肪变性，发炎和坏死，并且提高了浆液中高音活动。延长乙醇管理(6 wk ) 提高了肝损坏。这些回答每氧化，没有内容， i NOS 活动和减少的 eNOS 活动伴有增加的类脂化合物。NF-kappaB p65， i NOS 和 TNF-alpha 蛋白质或 mRNA 表示显著地在长期的乙醇管饲法以后被导致，而 eNOS mRNA 表示仍然保持未改变。提高的 i NOS 活动和表示断然与肝损坏，特别 necro 发炎， NF-kappaB 的激活，和 TNF-alpha mRNA 表示被相关。结论：i NOS 表示和活动在老鼠在长期的乙醇暴露以后在肝被导致，它与肝损坏，特别 necro 发炎， NF-kappaB 的激活和 TNF-alpha 表示被相关。eNOS 活动被减少，但是它的 mRNA 表示没被影响。

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 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.

Inducible nitric oxide synthase polymorphism is associated with the increased risk of differentiated gastric cancer in a Japanese population

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.

Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury

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.

EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

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.

Clinical Significance of a Myeloperoxidase Gene Polymorphism and Inducible Nitric Oxide Synthase Expression in Cirrhotic Patients with Hepatopulmonary Syndrome

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.

Expression and role of inducible nitric oxide synthase in ischemia-reperfusion liver in rats

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.

Prednisolone inhibits SaOS2 osteosarcoma cell proliferation by activating inducible nitric oxide synthase

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.

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

瞄准：为了评估反应的氧种类(ROS ) 和可诱导的氮的氧化物的表示的生产，在老鼠的 synthase (i NOS ) 孤立肾脏钩端螺旋体和包柔氏螺旋体属 burgdorferi 刺激的 Kupffer 房间(KC ) 。方法：老鼠 Kupffer 房间被肝的灌注用 0.05% 胶原酶分开，并且由 Percoll 坡度净化了。净化的 Kupffer 房间是有活着的 L 的测试在试管内。interogans 和 B。burgdorferi 准备。ROS 的生产被化合光决定，而 i NOS 蛋白质表达式被西方的污点试金用 anti-iNOS 抗体计算。结果：B。burgdorferi 并且到一更少的程度 L。interrogans 与一座山峰导致了 ROS 生产在感染以后的 35 min。化合光信号日益增多地减少了并且由孵化的 180 min 是无法发现的。Leptospirae 和 borreliae 在在 6 个小时达到顶点并且仍然是明显的 Kupffer 房间导致了增加的 i NOS 表情在感染以后的 22 h。结论：螺旋菌的两个类在老鼠 Kupffer 细胞导致了 ROS 和 i NOS 生产。自从在 leptospiral 以及在 borrelial 感染的肝损坏的原因，仍然是未知的，我们建议那螺旋体和肝的包柔氏螺旋体属损坏能被氧激进分子开始调停，并且然后被氮的氧化物部分地至少维持。

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.

Lanthanum Chloride Inhibiting Expression of Inducible Nitric Oxide Synthase in RAW264.7 Macrophages Induced by Lipopolysaccharide

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.

Spatio-temporal expression of inducible nitric oxide synthase in and surrounding a region of rat frontal lobe damaged with a sharp instrument

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.

Spatiotemporal expression of inducible nitric oxide synthase and cyclooxygenase 2 in the spinal cord during early stage sciatic nerve crush 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.

Cervical sympathetic trunk transection affects inducible nitric oxide synthase expression in rat hippocampus following focal cerebral ischemia/reperfusion injury

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.

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.

Requirement for endogenous heat shock factor 1 in inducible nitric oxide synthase induction in murine microglia

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.