Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of a...Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.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
Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rock...Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.展开更多
BACKGROUND Although the associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)induces more rapid liver regeneration than portal vein embolization,the mechanism remains unclear.AIM To assess...BACKGROUND Although the associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)induces more rapid liver regeneration than portal vein embolization,the mechanism remains unclear.AIM To assess the influence of inflammatory cytokines and endothelial nitric oxide synthase(eNOS)activation on liver regeneration in ALPPS.METHODS The future liver remnant/body weight(FLR/BW)ratio,hepatocyte proliferation,inflammatory cytokine expression,and activation of the Akt-eNOS pathway were evaluated in rat ALPPS and portal vein ligation(PVL)models.Hepatocyte proliferation was assessed based on Ki-67 expression,which was confirmed using immunohistochemistry.The serum concentrations of inflammatory cytokines were measured using enzyme linked immune-solvent assays.The Akt-eNOS pathway was assessed using western blotting.To explore the role of inflammatory cytokines and NO,Kupffer cell inhibitor gadolinium chloride(GdCl3),NOS inhibitor N-nitro-arginine methyl ester(L-NAME),and NO enhancer molsidomine were administered intraperitoneally.RESULTS The ALPPS group showed significant FLR regeneration(FLR/BW:1.60%±0.08%,P<0.05)compared with that observed in the PVL group(1.33%±0.11%)48 h after surgery.In the ALPPS group,serum interleukin-6 expression was suppressed using GdCl3 to the same extent as that in the PVL group.However,the FLR/BW ratio and Ki-67 labeling index were significantly higher in the ALPPS group administered GdCl3(1.72%±0.19%,P<0.05;22.25%±1.30%,P<0.05)than in the PVL group(1.33%±0.11%and 12.78%±1.55%,respectively).Phospho-Akt Ser473 and phospho-eNOS Ser1177 levels were enhanced in the ALPPS group compared with those in the PVL group.There was no difference between the ALPPS group treated with L-NAME and the PVL group in the FLR/BW ratio and Ki-67 labeling index.In the PVL group treated with molsidomine,the FLR/BW ratio and Ki-67 labeling index increased to the same level as in the ALPPS group.CONCLUSION Early induction of inflammatory cytokines may not be pivotal for accelerated FLR regeneration after ALPPS,whereas Akt-eNOS pathway activation may contribute to accelerated regeneration of the FLR.展开更多
AIM To observe the nitric oxide synthase (NOS) distribution in the esophageal mucosa andhemodynamic changes in cirrhotic rats.METHODS NOS distribution in the loweresophagus of rats with carbon tetrachlorideinduced cir...AIM To observe the nitric oxide synthase (NOS) distribution in the esophageal mucosa andhemodynamic changes in cirrhotic rats.METHODS NOS distribution in the loweresophagus of rats with carbon tetrachlorideinduced cirrhosis was assessed by using NADPHdiaphorase (NADPH-d ) histochemical method.Concentration of NO in serum were measured byfluorometric assay. Mean arterial pressure(MAP), cardiac output (CO), cardiac index (CI),splanchnic vascular resistance (SVR ), andsplanchnic blood flow (SBF ) were alsodetermined using 5’CO-labeled microspheretechnique.RESULTS Intensity of NOS staining in theesophageal epithelium of cirrhotic rats wassignificantly stronger than that in controls.There was a NOS--positive staining area in theendothelia of esophageal submucosal vessels ofcirrhotic rats, but the NOS staining was negativein normal rats. NO concentration of serum incirrhotic rats were significantly higher incomparison with that of controls. Cirrhotic ratshad significantly lower MAP, SVR and higherSBF than those of the controls.CONCLUSION SPlanchnic hyperdynamiccirculatory state was observed in rats withcirrhosis. The endogenous NO may play animportant role in development of esophagealvarices and in changes of hemodynamics incirrhosis.展开更多
Studies have shown that hyperglycemia aggravates brain damage by affecting vascular endothelial function. However, the precise mechanism remains unclear. Male Sprague-Dawley rat models of diabetes were established by ...Studies have shown that hyperglycemia aggravates brain damage by affecting vascular endothelial function. However, the precise mechanism remains unclear. Male Sprague-Dawley rat models of diabetes were established by a high-fat diet combined with an intraperitoneal injection of streptozotocin. Rat models of traumatic brain injury were established using the fluid percussion method. Compared with traumatic brain injury rats without diabetic, diabetic rats with traumatic brain injury exhibited more severe brain injury, manifested as increased brain water content and blood-brain barrier permeability, the upregulation of heme oxygenase-1, myeloperoxidase, and Bax, the downregulation of occludin, zona-occludens 1, and Bcl-2 in the penumbra, and reduced modified neurological severity scores. The intraperitoneal injection of a nitric oxide synthase inhibitor N(5)-(1-iminoethyl)-L-ornithine(10 mg/kg) 15 minutes before brain injury aggravated the injury. These findings suggested that nitric oxide synthase plays an important role in the maintenance of cerebral microcirculation, including anti-inflammatory, anti-oxidative stress, and anti-apoptotic activities in diabetic rats with traumatic brain injury. The experimental protocols were approved by the Institutional Animal Care Committee of Harbin Medical University, China(approval No. ky2017-126) on March 6, 2017.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
BACKGROUD: Ethanol can influence neural development and the ability of learning and memory, but its mechanism of the neural toxicity is not clear till now. Endogenous nitric oxide (NO) as a gaseous messenger is proved...BACKGROUD: Ethanol can influence neural development and the ability of learning and memory, but its mechanism of the neural toxicity is not clear till now. Endogenous nitric oxide (NO) as a gaseous messenger is proved to play an important role in the formation of synaptic plasticity, transference of neuronal information and the neural development, but excessive nitro oxide can result in neurotoxicity. OBJECTIVE: To observe the effects of acute alcoholism on the learning and memory ability and the content of neuronal nitric oxide synthase (nNOS) in brain tissue of rats. DESIGN: A randomized controlled animal experiment. SETTING: Department of Physiology, Xinxiang Medical College. MATERIALS: Eighteen male clean-degree SD rats of 18-22 weeks were raised adaptively for 2 days, and then randomly divided into control group (n = 8) and experimental group (n = 10). The nNOS immunohistochemical reagent was provided by Beijing Zhongshan Golden Bridge Biotechnology Co.,Ltd. Y-maze was produced by Suixi Zhenghua Apparatus Plant. METHODS: The experiment was carried out in the laboratory of the Department of Physiology, Xinxiang Medical College from June to October in 2005. ① Rats in the experimental group were intraperitoneally injected with ethanol (2.5 g/kg) which was dissolved in normal saline (20%). The loss of righting reflex and ataxia within 5 minutes indicated the successful model. Whereas rats in the control group were given saline of the same volume. ② Examinations of learning and memory ability: The Y-maze tests for learning and memory ability were performed at 6 hours after the models establishment. The rats were put into the Y-maze separately. The test was performed in a quiet and dark room. There was a lamp at the end of each of three pathways in Y-maze and the base of maze had electric net. All the lamps of the three pathways were turned on for 3 minutes and then turned off. One lamp was turned on randomly, and the other two delayed automatically. In 5 seconds after alternation, pulsating electric current presented in the base of unsafe area to stimulate rat’s feet to run to the safe area. The lighting lasted for 15 seconds as one test. Running from unsafe area to safe area at one time in 10 seconds was justified as successful. Such test was repeated for 10 times for each rat and the successful frequency was recorded. The qualified standard of maze test was that the rat arrived in the safe area 9 times during 10 experiments. The number of trainings for the qualified standard was used to represent the result of spatial learning. ③ Determination of the content of nNOS in brain tissue: After the Y-maze test, the rats were anaesthetized, and blood was let from the incision on right auricle, transcardially perfused via the left ventricle with about 200 mL saline, then fixed by perfusion of 40 g/L paraformaldehyde. Hippocampal CA1 region, corpus striatum and cerebellum were taken to prepare serial freezing coronal sections. The nNOS contents in the brain regions were determined with the immunohistochemical methods to reflect the changes of nitric oxide in brain tissue. MAIN OUTCOME MEASURES: The changes of learning and memory ability and the changes of the nNOS contents in the brain tissue of rats with acute alcoholism were observed. RESULTS: One rat in the experimental group was excluded due to its slow reaction to electric stimulation in the Y-maze test, and the other 17 rats were involved in the analysis of results. ① The training times to reach qualifying standards of Y-maze in the experimental group was more than that in the control group [(34.33 ±13.04), (27.50±8.79) times, P < 0.05]. ② Forms and numbers of nNOS positive neurons in brain tissue: It could be observed under light microscope that in the hippocampal CA1 region, there were fewer nNOS positive neurons, which were lightly stained, and the processes were not clear enough; But the numbers of the positive neurons which were deeply stained as buffy were obviously increased in the experimental group, the cell body and cytoplasm of process were evenly stained, but the nucleus was not stained. The nNOS positive neurons in corpus striatum had similar forms and size in the experimental group and control group. The form of the nNOS positive neurons in cerebellum were similar between the two groups. The numbers of nNOS positive neurons in hippocampal CA1 region and corpus striatum in the experimental group [(18.22±7.47), (11.38±5.00) cells/high power field] were obviously higher than those in the control group [(10.15±4.24), (6.15±3.69) cells/high power field. The number of nNOS positive neurons in cerebellum had no significant difference between the two groups [(49.56±18.84), (44.43±15.42) cells/high power field, P > 0.05]. CONCLUSION: Acute alcoholism may impair learning and memory ability, and nitric oxide may be involved in mediating the neurotoxic role of ethanol.展开更多
Purpose: The purpose of this study was to develop a method to quantitatively assess the effect of nitric oxide synthase (NOS) inhibition on tumor vascular activity using dynamic contrast-enhanced computed tomography (...Purpose: The purpose of this study was to develop a method to quantitatively assess the effect of nitric oxide synthase (NOS) inhibition on tumor vascular activity using dynamic contrast-enhanced computed tomography (DCE-CT) and to investigate its usefulness using animal experiments. Mate-rials and Methods: The DCE-CT studies were performed in anesthetized Fisher rats bearing tumors using a 4-row multi-slice CT. The scanning started 4 s before a bolus injection of iodinated contrast agent (CA) (150 mgI/kg) from the tail vein using an automatic injector and lasted 60 s at 1-s in-tervals. The contrast enhancement (CE) images were generated by subtracting the CT images before and after the administration of CA. First, the DCE-CT studies were performed before and 15, 30, and 45 min after administration of N-nitro-L-arginine (L-NNA) (1, 3, and 10 mg/kg) or vehicle, and the relative CE values were calculated by normalizing the CE image at each time point by that obtained from the first DCE-CT study. Second, we investigated the case when L-arginine (L-ARG) (200 mg/kg) and L-NNA (1, 3, and 10 mg/kg) were administered after the first and second DCE-CT studies, respectively. Third, we investigated the case when L-NNA (1, 3, and 10 mg/kg) and L-ARG (200 mg/kg) were administered after the first and second DCE-CT studies, respectively. Finally, we investigated the case when L-NNA (1, 3, and 10 mg/kg) and L-ARG (200 mg/kg) were administered simultaneously after the first DCE-CT study. Results: The relative CE value significantly decreased after L-NNA administration in a dose-dependent manner (p-values = 0.0074 and <0.0001 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 15 min, 0.0003 and <0.0001 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 30 min, and 0.0367 and 0.0004 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 45 min). When L-ARG was administered prior to the administration of 1 mg/kg L-NNA, the relative CE value at 45 min was significantly higher than that at 15 min. When L-ARG was administered after L-NNA administration, there was no significant difference between the relative CE values at 15 min and 45 min. These results suggest that when using L-NNA in combination with L-ARG, their effect on tumor vascular activity differs depending on the order of their administration. When L-NNA and L-ARG were administered simultaneously, there was a tendency for the relative CE value to be higher than that when only L-NNA was administered, at all injected doses of L-NNA. Conclusion: Our method using DCE-CT is useful for monitoring the effect of NOS inhibition on tumor vascular activity and for determining the optimal injected dose and timing of NOS inhibitors for anticancer therapy.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.However...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 IgG 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
Objective To investigate the distribution of nitric oxide synthase (NOS), nerve growth factor receptor (NGFR) and interstitial cells of Cajal (ICCs) in Hirschsprung's disease (HD). Methods The distribution of NOS,...Objective To investigate the distribution of nitric oxide synthase (NOS), nerve growth factor receptor (NGFR) and interstitial cells of Cajal (ICCs) in Hirschsprung's disease (HD). Methods The distribution of NOS, NGFR and ICCs was studied by using NADPH diaphorase histochemistry, immunohistochemistry with a monoclonal antibody to human NGFR and the specific polyclonal antibody against c-kit in 8 normal controls and 10 cases of HD. Results NOS and NGFR were abundantly present in the myenteric plexus and in the nerve fibers of musculature. ICCs were intensively distributed in the surface of circular musculature and around the myenteric plexus to form a network in normal control colon. In contrast, NOS and NGFR were scarce or absent in the myenteric plexus and in the nerve fibers of musculature, while the hypertrophic nerve trunks were NGFR positive, ICCs were scarcely distributed and the network was disrupted in the aganglionic colon in HD. Conclusion These findings suggest the involvement of NOS, NGFR and ICCs in the pathophysiology of HD.展开更多
Background: To investigate the effects of unsafe decompression on rat pulmonary endothelial function and its relevant mechanisms.Methods: Sixty male Sprague-Dawley(SD) rats were randomly divided into a control group(n...Background: To investigate the effects of unsafe decompression on rat pulmonary endothelial function and its relevant mechanisms.Methods: Sixty male Sprague-Dawley(SD) rats were randomly divided into a control group(n=30) and a decompression sickness(DCS) group(n=30). The DCS model was established by placing the rats in the DCS group in a pressurized cabin where they were exposed to a 600 k Pa compressed air environment for 60 min, and the pressure was then reduced by 100 k Pa/min until it reached atmospheric pressure. After the surviving rats in the DCS group and the rats in the control group were anesthetized, their pulmonary arteries were stripped to test the in vitro pulmonary artery endothelium-dependent vasodilation capacity. Western blotting was used to measure the expression and dissociation of endothelial nitric oxide synthase(e NOS) in pulmonary artery tissues and all protein nitration levels in pulmonary artery tissues; reactive oxygen species(ROS) formation was measured via in vitro pulmonary artery superoxide anion probe dihydroethidium(DHE) staining.Results: After experiencing unsafe decompression, 10 of the 30 rats in the DCS group died. The pulmonary artery endothelium-dependent vasodilation capacity in the surviving rats decreased significantly(P<0.05). The difference in e NOS expression between the DCS group and the control group was statistically insignificant(P>0.05), but the ratio of e NOS monomer/dimer in the DCS group was significantly higher than that in the control group(P<0.05). All protein tyrosine nitration levels in the pulmonary artery tissues of the DCS group were significantly higher than those of the control group(P<0.05). The results of DHE staining showed that the amount of ROS formation in the pulmonary arteries of the DCS group was significantly higher than that of the control group(P<0.05).Conclusion: Unsafe decompression during a simulated submarine escape process can lead to e NOS dimer uncoupling in the pulmonary artery endothelium. The dissociated e NOS monomer cannot synthesize nitric oxide(NO) and thus affect the endothelium-dependent vasodilation capacity. The e NOS monomer can promote peroxynitrite(ONOO–) synthesis, leading to an increase in protein tyrosine nitration levels in pulmonary artery tissues and causing disorder in cell cycle regulation. The e NOS monomer can also cause an increase in the formation of ROS and thus mediate peroxidation damage.展开更多
BACKGROUND: Melatonin is a kind of hormones derived from pineal gland. Recent researches demonstrate that melatonin is characterized by anti-oxidation, anti-senility and destroying free radicals. While, effect and pat...BACKGROUND: Melatonin is a kind of hormones derived from pineal gland. Recent researches demonstrate that melatonin is characterized by anti-oxidation, anti-senility and destroying free radicals. While, effect and pathogenesis of pineal gland on learning ability should be further studied. OBJECTIVE: To investigate the effects of pinealectomy on learning abiliy, distribution of cholinesterase and expression of neuronal nitric oxide synthase (nNOS) in cerebral cortex of rats and probe into the effect of melatonin on learning ability, central cholinergic system and nNOS expression. DESIGN: Randomized grouping design and animal study. SETTING: Department of Neurology, the 187 Hospital of Chinese PLA. MATERIALS: A total of 12 male SD rats, of normal learning ability testing with Y-tape maze, of clean grade, weighing 190-210 g, aged 6 weeks, were selected in this study. METHODS: The experiment was carried out in the Department of Neurology, Zhujiang Hospital from July 1997 to June 2000. All SD rats were divided into experimental group (n =6, pinealectomy) and control group (n =6, sham operation). Seven days later, rats in both two groups were continuously fed for 33 days. ① Learning ability test: The learning ability of rats was tested by trisection Y-type maze and figured as attempting times. ② Expression of acetylcholinesterase (AchE) was detected by enzyme histochemistry and nNOS was measured by SABC method. ③ Quantitative analysis of AchE fibers: AchE fibers density in unit area (surface density) was surveyed with Leica Diaplan microscope and Leica Quantimet 500+ image analytic apparatus and quantitative parameter was set up for AchE fibers covering density (μm2) per 374 693.656 μm2, moreover, the AchE fibers density was measured in Ⅱ-Ⅳ layers of motor and somatosensory cortex (showing three layers per field of vision at one time), in radiative, lacunaria and molecular layers of CA1, CA2 and CA3 areas, and in lamina multiforms of dentate gyrus. Three tissue slices were picked up randomly in the same part of each rat, together six tissue slices for nNOS expression and four near view (× 400) were selected in the parts of right neocortex, medial septal nucleus-diagonal band nucleus (SM-DB), corpus striatus and hippocampus to count nNOS-positive cells. MAIN OUTCOME MEASURES: Learning ability; distribution and quantitative analysis of AchE fibers; expression of nNOS in various cerebral areas. RESULTS: The twelve rats were all involved in the final analysis. ① Learning ability test: The learning abilities before operation in the experimental group [(14.67±4.97) times] were consistent with those in the control group [(14.33±4.32) times, P > 0.05], the learning abilities in the experimental group at 40 days after pinealectomy [(28.67±2.42) times] were obviously more than those before pinealectomy and those in the control group after operation [(13.83±8.33) times, P < 0.01]. ② Results of AchE-positive fibers density in cerebral cortex of rats: The AChE-positive fibers densities in motor and somatosensory cortex, CA1, CA2 and CA3 areas of hippocampus and in lamina multiforms of dentate gyrus in the experimental group were obviously lower than those in the control group [experimental group: (15 244±1 339), (14 764±1 391), (12 991±970), (15 077±1 020), (19 546±1 489), (19 337±1 378) μm2; control group: (21 001±1 021), (17 930±2 225), (17 260±1 342), (18 911±1 048), (24 108±1 671), (22 917±1 909) μm2, P < 0.01]. ③ Expression of nNOS in various cerebral areas: nNOS-positive cells in cerebral cortex of rats of the experimental group were more, furthermore the ones in somatosensory cortex were slightly more in motor cortex and the number (5.90±0.68) was more than that in the control group (3.68±0.39,P < 0.05). The nNOS-positive cells in SM-DB (16.21±2.03) were markedly more than those in the control group (9.32±1.05,P < 0.01). The nNOS-positive cells in hippocampus (4.27±0.75) and in corpus striatus (9.35±2.58) were not different with those in the control group (3.94±0.53, 8.96±2.31, P > 0.05). CONCLUSION: Decrease of melatonin due to pinealectomy of rats can result in learning disorder, which may be related to trauma of cholinergic neuron in cerebral cortex which were caused by nitric oxide neurotoxicity arose from the overexpression of nNOS in cerebral neocortex and SM-DB.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
BACKGROUND: Some researches showed that motoneurons in spinal cord anterior horn wound die following brachial plexus injury, but the concrete mechanism of motoneurons death remains unclear. OBJECTIVE: To observe the e...BACKGROUND: Some researches showed that motoneurons in spinal cord anterior horn wound die following brachial plexus injury, but the concrete mechanism of motoneurons death remains unclear. OBJECTIVE: To observe the expression of nitric oxide synthase (NOS) and survival of C7 motoneurons in spinal cord of rats after selective brachial plexus injury. DESIGN: A randomized controlled animal experiment. SETTING:Department of Anatomy, Sun Yet-sen Medical College, Sun Yet-sen University. MATERIALS: Totally 35 adult healthy male Sprague-Dawley rats with the body mass of 200-300 g were provided by Experimental Animal Center, Sun Yet-sen Medical College, Sun Yat-sen University. The rats were divided into control group (n =5) and experimental group(n =30) by random number table method, and the experimental group was divided into three injury subgroups: anterior root avulsion group, dorsal root transection group and spinal cord hemisection group, 10 rats in each group. There were horse anti-neuronal NOS (nNOS) polycolonal antibody (Sigma company) and nicotina mideadeninedinucleotide phosphate (NADPH-d) (Sigma Company). METHODS: The experiment was performed at Department of Anatomy, Sun Yet-sen Medical College, Sun Yet-sen University between September 2004 and April 2005. ①After anesthetizing the rats, the spinous process of second thoracic vertebra as a marker, the vertebra was exposed from C5 to T1 and the lamina of vertebra was unclenched, and spinal dura mater was carved to expose the spinal nerve dorsal roots of C5-T1. The right ventral root of C7 was avulsed, and the residual root was removed in anterior root avulsion group. The right ventral root of C7 was avulsed and the right dorsal roots of brachial plexus (C5-T1) were cut off in dorsal root transection group. In spinal cord hemisection group, the hemisection between the C5 and C6 spinal segment on right side and avulsion of right ventral root of C7 were made. In the control group, the vertebra from C5 to T1 was unclenched and the skin of wound was sutured. ②Three weeks after operation, behavior of rats was observed. The rats were killed after anesthesia. The C7 segment of spinal cord was removed and treated with NADPH-d staining, neutral red counterstaining and NOS immunohistochemistry staining to detect the expression of NOS. MAIN OUTCOME MEASURES: The expression of NOS and survival of C7 motoneurons in spinal cord of rats 3 weeks after operation. RESULTS: Among the 35 included rats, 3 rats died 2 weeks following operation, so totally 32 rats were involved in the result analysis. ①NADPH-d positive neurons of in anterior horn of C7 in the three groups: The NADPH-d positive neurons could be found in anterior horn of C7 in the three groups. The percentage of that in anterior root avulsion group to that of non-injury side of spinal cord was(20.98±2.65)%, (29.43±6.81)% in dorsal root transection group and (31.74±6.80)% in spinal cord hemisection group. There was significant difference among the three injury groups(F =5.135,P =0.016). There was significant difference in anterior root avulsion group with dorsal root transection group and spinal cord hemisection group (t =2.562,3.167,P < 0.05). There was no significant difference between the dorsal root transection group and spinal cord hemisection group (P =0.534). ②survival rate of motoneurons in anterior horn of C7: There were dead motoneurons in the three injury groups, the percentages of surviving motoneurons to that of non-injured side of spinal cord were (69.22±4.04)%,(62.01±3.82)% and (56.74±6.86)%, respectively. There were significant differences among the three groups (F =9.508,P =0.002). The anterior root avulsion group was significantly different from the other two groups(t =2.764,4.587,P < 0.05). There was no significant difference between the dorsal root transection group and spinal cord hemisection group(P =0.073). CONCLUSION: The selective brachial plexus injury can induce the up-regulation of NOS expression in motorneurons of spinal cord anterior horn and block descending pathway of cortex to cause the more significant up-regulation of NOS and low survival rate in motoneurons. It indicates that descending pathway of cortex can inhibit the NOS expression in motorneurons of spinal cord anterior horn, and the high NOS expression might induce the death of motorneurons in spinal cord anterior horn.展开更多
BACKGROUND: Nitric oxide synthase (NOS) inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ...BACKGROUND: Nitric oxide synthase (NOS) inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids (EAA) during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine (L-NA) reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear.OBJECTIVE: By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid (GABA) in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism. DESIGN: A randomized and controlled experiment.SETTING: Department of Pharmacology, Hebei Academy of Medical Sciences.MATERIALS: A total of 42 male healthy SD rats (grade Ⅱ, weighting 250-300 g) were provided by the Experimental Animal Center of Hebei Province (Certification: 04036). Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Sigma Chemicals Co, St Louis, MO, USA. HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC.METHODS: The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups: sham-operated group (n = 6), ischemic group (n = 18), L-NA group (n = 18). The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared, but the filament was not inserted. Each group was further divided into 3 subgroups (n = 6 for each): drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion (MCAO) respectively. L-NA (20 mg/kg, ip) was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain (IV%) in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett’s test.MAIN OUTCOME MEASURES: ① The volume of cerebral infarction; ② The contents of aspartate, glutamate, glycine and GABA in brain tissue after cerebral ischemia.RESULTS: All 42 rats were involved in the final analysis. ① Infarcted volume: Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was (20.13±3.59)% and (23.12±5.84)% in L-NA group, which was not similar to that in ischemic group [(22.10±3.98)%, (25.38±5.37)%, P > 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [(26.11±3.55)% and (37.15±3.58)%, P < 0.01]. ② Changes of amino acid content: At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group ( P < 0.05-0.01). However, contents in L-NA group were similar to those in ischemic group (P > 0.05). At 12 hours after ischemia, the contents of aspartate [(0.21±0.06), (0.36±0.05), (0.29±0.12) mg/g] and glutamate [(0.55±0.06), (0.78±0.10), (0.52±0.10) mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [(0.49±0.17), (0.63±0.03), (0.51±0.15) mg/g; (0.98±0.30), (1.15±0.15), (0.93±0.15) mg/g, P < 0.05-0.01]. Glycine in hippocampus was (0.40±0.07) mg/g, which was higher than that in ischemic group [(0.21±0.07) mg/g, P < 0.05]. GABA in striatum, hippocampus and cortex was (0.93±0.10), (0.62±0.12) and (0.81±0.10) mg/g, respectively, which was higher than that in ischemic group [(0.60±0.08), (0.37±0.17), (0.59±0.10) mg/g, P < 0.05-0.01]. CONCLUSION: It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA.展开更多
Pancreatic cancer is a common tumor of the digestive system.At present,the pathogenesis is still unclear,but current research on inducible nitric oxide synthase in relation to the pathogenesis of pancreatic malignant ...Pancreatic cancer is a common tumor of the digestive system.At present,the pathogenesis is still unclear,but current research on inducible nitric oxide synthase in relation to the pathogenesis of pancreatic malignant tumors is particularly extensive.Therefore,this article focuses on the research progress on inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.展开更多
Background:Overexpression of inducible nitric oxide synthase(iNOS)has been reported in diabetic retinopathy(DR).The kinin B1 receptor(B1R)is also overexpressed in DR,and can stimulate iNOS via Gαi/ERK/MAPK pathway.We...Background:Overexpression of inducible nitric oxide synthase(iNOS)has been reported in diabetic retinopathy(DR).The kinin B1 receptor(B1R)is also overexpressed in DR,and can stimulate iNOS via Gαi/ERK/MAPK pathway.We previously showed that the topical administration of a B1R antagonist,LF22-0542,significantly reduces leukocyte infiltration,increased vascular permeability and overexpression of several inflammatory mediators,including iNOS in DR.Thus,the aim of this study was to determine whether the pro-inflammatory effects of B1R are attributed to oxidative stress caused by the activation of iNOS pathway in order to identify new therapeutic targets for the treatment of DR.iNOS and B1R being absent in the normal retina,their inhibition is unlikely to result in undesirable side effects.The approach will be no invasive by eye application of drops.Methods:Diabetes was induced in male Wistar rats(200-230 g)by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg b.w).One week later,rats were randomly divided into four groups(N=5)and treated for one week as follows:Gr 1:control rats treated with the selective iNOS inhibitor(1,400 W,0.06μM twice a day by eye-drops×7 days),Gr 2,STZ-diabetic rats treated with 1,400 W,Gr 3:control rats received a selective B1R agonist[Sar(D-Phe8)-des-Arg9-BK,100μg twice a week]by intravitreal injections(itrv)and treated with 1,400 W,Gr 4:STZ-diabetic rats+B1R agonist+1,400 W.At the end of treatment and two weeks post-STZ,three series of experiments were carried out to measure vascular permeability(by Evans blue dye method)and the expression of vasoactive and inflammatory mediators,including iNOS,VEGF-A,VEGF-R2,IL-1β,Cox-2,TNF-α,bradykinin 1 and 2 receptors and carboxypeptidase M/kininase 1(by Western Blotting and qRT-PCR).The nitrosative stress(nitrosylation of proteins)was also assessed by Western Blotting.One-way Anova test with Bonferroni post hoc was used for statistical analysis.Results:STZ-diabetic rats showed a significant increase in retinal vascular permeability(22.8μg/g Evans blue dye per g of fresh retinas,P=0.016)compared with control rats and control treated rats(17.2 and 16.8μg/g respectively).The injections of B1R agonist amplified the increase of vascular permeability which was normalized by the 1,400 W.The overexpression of inflammatory markers was also normalized by the 1,400 W in STZ-diabetic rats received or not the B1R agonist.Conclusions:These results support a contribution of iNOS in the deleterious effects of B1R in this model of diabetic retinopathy.Hence,iNOS inhibition by ocular application of 1,400 W may represent a promising and non-invasive therapeutic approach in the treatment of diabetic retinopathy.展开更多
Pancreatic cancer is a common tumor of the digestive system,at present,the pathogenesis is still unclear,but in the current research on the pathogenesis of pancreatic malignant tumors,the research on inducible nitric ...Pancreatic cancer is a common tumor of the digestive system,at present,the pathogenesis is still unclear,but in the current research on the pathogenesis of pancreatic malignant tumors,the research on inducible nitric oxide synthase is particularly extensive.Therefore,this article focuses on the research progress of inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.This is a review.展开更多
AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus.METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histo...AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus.METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique.RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus. The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62 -± 38 cells/mm2(antrum), 43 ± 32 cells/mm2(body), and 32 ± 28 cells/mm2 (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels.CONCLUSION: Nitric oxide might he involved in the modulation of motility, secretion and blood ciroulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach.展开更多
基金supported by the Natural Nature Science Foundation of China,Nos.82030071,81874004the Science and Technology Major Project of Changsha,No.kh2103008(all to JZH).
文摘Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.
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 Natural Science Foundation of Shandong Province(No.ZR2020QC214)the Young Experts of Taishan Scholars(No.tsqn201909130)+3 种基金the Science and Technology Support Plan for Youth Innovation of Colleges and Universities in Shandong Province(No.2019KJF003)the‘First Class Fishery Discipline’Programme in Shandong Provincea special talent programme‘One Thing One Decision(YishiYiyi)’Programme in Shandong Province,Chinathe Breeding Plan of Shandong Provincial Qingchuang Research Team(2019)。
文摘Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.
文摘BACKGROUND Although the associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)induces more rapid liver regeneration than portal vein embolization,the mechanism remains unclear.AIM To assess the influence of inflammatory cytokines and endothelial nitric oxide synthase(eNOS)activation on liver regeneration in ALPPS.METHODS The future liver remnant/body weight(FLR/BW)ratio,hepatocyte proliferation,inflammatory cytokine expression,and activation of the Akt-eNOS pathway were evaluated in rat ALPPS and portal vein ligation(PVL)models.Hepatocyte proliferation was assessed based on Ki-67 expression,which was confirmed using immunohistochemistry.The serum concentrations of inflammatory cytokines were measured using enzyme linked immune-solvent assays.The Akt-eNOS pathway was assessed using western blotting.To explore the role of inflammatory cytokines and NO,Kupffer cell inhibitor gadolinium chloride(GdCl3),NOS inhibitor N-nitro-arginine methyl ester(L-NAME),and NO enhancer molsidomine were administered intraperitoneally.RESULTS The ALPPS group showed significant FLR regeneration(FLR/BW:1.60%±0.08%,P<0.05)compared with that observed in the PVL group(1.33%±0.11%)48 h after surgery.In the ALPPS group,serum interleukin-6 expression was suppressed using GdCl3 to the same extent as that in the PVL group.However,the FLR/BW ratio and Ki-67 labeling index were significantly higher in the ALPPS group administered GdCl3(1.72%±0.19%,P<0.05;22.25%±1.30%,P<0.05)than in the PVL group(1.33%±0.11%and 12.78%±1.55%,respectively).Phospho-Akt Ser473 and phospho-eNOS Ser1177 levels were enhanced in the ALPPS group compared with those in the PVL group.There was no difference between the ALPPS group treated with L-NAME and the PVL group in the FLR/BW ratio and Ki-67 labeling index.In the PVL group treated with molsidomine,the FLR/BW ratio and Ki-67 labeling index increased to the same level as in the ALPPS group.CONCLUSION Early induction of inflammatory cytokines may not be pivotal for accelerated FLR regeneration after ALPPS,whereas Akt-eNOS pathway activation may contribute to accelerated regeneration of the FLR.
文摘AIM To observe the nitric oxide synthase (NOS) distribution in the esophageal mucosa andhemodynamic changes in cirrhotic rats.METHODS NOS distribution in the loweresophagus of rats with carbon tetrachlorideinduced cirrhosis was assessed by using NADPHdiaphorase (NADPH-d ) histochemical method.Concentration of NO in serum were measured byfluorometric assay. Mean arterial pressure(MAP), cardiac output (CO), cardiac index (CI),splanchnic vascular resistance (SVR ), andsplanchnic blood flow (SBF ) were alsodetermined using 5’CO-labeled microspheretechnique.RESULTS Intensity of NOS staining in theesophageal epithelium of cirrhotic rats wassignificantly stronger than that in controls.There was a NOS--positive staining area in theendothelia of esophageal submucosal vessels ofcirrhotic rats, but the NOS staining was negativein normal rats. NO concentration of serum incirrhotic rats were significantly higher incomparison with that of controls. Cirrhotic ratshad significantly lower MAP, SVR and higherSBF than those of the controls.CONCLUSION SPlanchnic hyperdynamiccirculatory state was observed in rats withcirrhosis. The endogenous NO may play animportant role in development of esophagealvarices and in changes of hemodynamics incirrhosis.
基金supported by the National Natural Science Foundation of China,No. 81400989 (to WCY)。
文摘Studies have shown that hyperglycemia aggravates brain damage by affecting vascular endothelial function. However, the precise mechanism remains unclear. Male Sprague-Dawley rat models of diabetes were established by a high-fat diet combined with an intraperitoneal injection of streptozotocin. Rat models of traumatic brain injury were established using the fluid percussion method. Compared with traumatic brain injury rats without diabetic, diabetic rats with traumatic brain injury exhibited more severe brain injury, manifested as increased brain water content and blood-brain barrier permeability, the upregulation of heme oxygenase-1, myeloperoxidase, and Bax, the downregulation of occludin, zona-occludens 1, and Bcl-2 in the penumbra, and reduced modified neurological severity scores. The intraperitoneal injection of a nitric oxide synthase inhibitor N(5)-(1-iminoethyl)-L-ornithine(10 mg/kg) 15 minutes before brain injury aggravated the injury. These findings suggested that nitric oxide synthase plays an important role in the maintenance of cerebral microcirculation, including anti-inflammatory, anti-oxidative stress, and anti-apoptotic activities in diabetic rats with traumatic brain injury. The experimental protocols were approved by the Institutional Animal Care Committee of Harbin Medical University, China(approval No. ky2017-126) on March 6, 2017.
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the Natural Sci-ence Foundation of HenanProvince, No. 984021100 agrant from Key Subject Fund ofXinxiang Medical College
文摘BACKGROUD: Ethanol can influence neural development and the ability of learning and memory, but its mechanism of the neural toxicity is not clear till now. Endogenous nitric oxide (NO) as a gaseous messenger is proved to play an important role in the formation of synaptic plasticity, transference of neuronal information and the neural development, but excessive nitro oxide can result in neurotoxicity. OBJECTIVE: To observe the effects of acute alcoholism on the learning and memory ability and the content of neuronal nitric oxide synthase (nNOS) in brain tissue of rats. DESIGN: A randomized controlled animal experiment. SETTING: Department of Physiology, Xinxiang Medical College. MATERIALS: Eighteen male clean-degree SD rats of 18-22 weeks were raised adaptively for 2 days, and then randomly divided into control group (n = 8) and experimental group (n = 10). The nNOS immunohistochemical reagent was provided by Beijing Zhongshan Golden Bridge Biotechnology Co.,Ltd. Y-maze was produced by Suixi Zhenghua Apparatus Plant. METHODS: The experiment was carried out in the laboratory of the Department of Physiology, Xinxiang Medical College from June to October in 2005. ① Rats in the experimental group were intraperitoneally injected with ethanol (2.5 g/kg) which was dissolved in normal saline (20%). The loss of righting reflex and ataxia within 5 minutes indicated the successful model. Whereas rats in the control group were given saline of the same volume. ② Examinations of learning and memory ability: The Y-maze tests for learning and memory ability were performed at 6 hours after the models establishment. The rats were put into the Y-maze separately. The test was performed in a quiet and dark room. There was a lamp at the end of each of three pathways in Y-maze and the base of maze had electric net. All the lamps of the three pathways were turned on for 3 minutes and then turned off. One lamp was turned on randomly, and the other two delayed automatically. In 5 seconds after alternation, pulsating electric current presented in the base of unsafe area to stimulate rat’s feet to run to the safe area. The lighting lasted for 15 seconds as one test. Running from unsafe area to safe area at one time in 10 seconds was justified as successful. Such test was repeated for 10 times for each rat and the successful frequency was recorded. The qualified standard of maze test was that the rat arrived in the safe area 9 times during 10 experiments. The number of trainings for the qualified standard was used to represent the result of spatial learning. ③ Determination of the content of nNOS in brain tissue: After the Y-maze test, the rats were anaesthetized, and blood was let from the incision on right auricle, transcardially perfused via the left ventricle with about 200 mL saline, then fixed by perfusion of 40 g/L paraformaldehyde. Hippocampal CA1 region, corpus striatum and cerebellum were taken to prepare serial freezing coronal sections. The nNOS contents in the brain regions were determined with the immunohistochemical methods to reflect the changes of nitric oxide in brain tissue. MAIN OUTCOME MEASURES: The changes of learning and memory ability and the changes of the nNOS contents in the brain tissue of rats with acute alcoholism were observed. RESULTS: One rat in the experimental group was excluded due to its slow reaction to electric stimulation in the Y-maze test, and the other 17 rats were involved in the analysis of results. ① The training times to reach qualifying standards of Y-maze in the experimental group was more than that in the control group [(34.33 ±13.04), (27.50±8.79) times, P < 0.05]. ② Forms and numbers of nNOS positive neurons in brain tissue: It could be observed under light microscope that in the hippocampal CA1 region, there were fewer nNOS positive neurons, which were lightly stained, and the processes were not clear enough; But the numbers of the positive neurons which were deeply stained as buffy were obviously increased in the experimental group, the cell body and cytoplasm of process were evenly stained, but the nucleus was not stained. The nNOS positive neurons in corpus striatum had similar forms and size in the experimental group and control group. The form of the nNOS positive neurons in cerebellum were similar between the two groups. The numbers of nNOS positive neurons in hippocampal CA1 region and corpus striatum in the experimental group [(18.22±7.47), (11.38±5.00) cells/high power field] were obviously higher than those in the control group [(10.15±4.24), (6.15±3.69) cells/high power field. The number of nNOS positive neurons in cerebellum had no significant difference between the two groups [(49.56±18.84), (44.43±15.42) cells/high power field, P > 0.05]. CONCLUSION: Acute alcoholism may impair learning and memory ability, and nitric oxide may be involved in mediating the neurotoxic role of ethanol.
文摘Purpose: The purpose of this study was to develop a method to quantitatively assess the effect of nitric oxide synthase (NOS) inhibition on tumor vascular activity using dynamic contrast-enhanced computed tomography (DCE-CT) and to investigate its usefulness using animal experiments. Mate-rials and Methods: The DCE-CT studies were performed in anesthetized Fisher rats bearing tumors using a 4-row multi-slice CT. The scanning started 4 s before a bolus injection of iodinated contrast agent (CA) (150 mgI/kg) from the tail vein using an automatic injector and lasted 60 s at 1-s in-tervals. The contrast enhancement (CE) images were generated by subtracting the CT images before and after the administration of CA. First, the DCE-CT studies were performed before and 15, 30, and 45 min after administration of N-nitro-L-arginine (L-NNA) (1, 3, and 10 mg/kg) or vehicle, and the relative CE values were calculated by normalizing the CE image at each time point by that obtained from the first DCE-CT study. Second, we investigated the case when L-arginine (L-ARG) (200 mg/kg) and L-NNA (1, 3, and 10 mg/kg) were administered after the first and second DCE-CT studies, respectively. Third, we investigated the case when L-NNA (1, 3, and 10 mg/kg) and L-ARG (200 mg/kg) were administered after the first and second DCE-CT studies, respectively. Finally, we investigated the case when L-NNA (1, 3, and 10 mg/kg) and L-ARG (200 mg/kg) were administered simultaneously after the first DCE-CT study. Results: The relative CE value significantly decreased after L-NNA administration in a dose-dependent manner (p-values = 0.0074 and <0.0001 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 15 min, 0.0003 and <0.0001 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 30 min, and 0.0367 and 0.0004 for 0 vs. 3 mg/kg and 0 vs. 10 mg/kg, respectively, at 45 min). When L-ARG was administered prior to the administration of 1 mg/kg L-NNA, the relative CE value at 45 min was significantly higher than that at 15 min. When L-ARG was administered after L-NNA administration, there was no significant difference between the relative CE values at 15 min and 45 min. These results suggest that when using L-NNA in combination with L-ARG, their effect on tumor vascular activity differs depending on the order of their administration. When L-NNA and L-ARG were administered simultaneously, there was a tendency for the relative CE value to be higher than that when only L-NNA was administered, at all injected doses of L-NNA. Conclusion: Our method using DCE-CT is useful for monitoring the effect of NOS inhibition on tumor vascular activity and for determining the optimal injected dose and timing of NOS inhibitors for anticancer therapy.
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 IgG 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Objective To investigate the distribution of nitric oxide synthase (NOS), nerve growth factor receptor (NGFR) and interstitial cells of Cajal (ICCs) in Hirschsprung's disease (HD). Methods The distribution of NOS, NGFR and ICCs was studied by using NADPH diaphorase histochemistry, immunohistochemistry with a monoclonal antibody to human NGFR and the specific polyclonal antibody against c-kit in 8 normal controls and 10 cases of HD. Results NOS and NGFR were abundantly present in the myenteric plexus and in the nerve fibers of musculature. ICCs were intensively distributed in the surface of circular musculature and around the myenteric plexus to form a network in normal control colon. In contrast, NOS and NGFR were scarce or absent in the myenteric plexus and in the nerve fibers of musculature, while the hypertrophic nerve trunks were NGFR positive, ICCs were scarcely distributed and the network was disrupted in the aganglionic colon in HD. Conclusion These findings suggest the involvement of NOS, NGFR and ICCs in the pathophysiology of HD.
基金supported by the Army on the Subject of China (10ZYZ219)
文摘Background: To investigate the effects of unsafe decompression on rat pulmonary endothelial function and its relevant mechanisms.Methods: Sixty male Sprague-Dawley(SD) rats were randomly divided into a control group(n=30) and a decompression sickness(DCS) group(n=30). The DCS model was established by placing the rats in the DCS group in a pressurized cabin where they were exposed to a 600 k Pa compressed air environment for 60 min, and the pressure was then reduced by 100 k Pa/min until it reached atmospheric pressure. After the surviving rats in the DCS group and the rats in the control group were anesthetized, their pulmonary arteries were stripped to test the in vitro pulmonary artery endothelium-dependent vasodilation capacity. Western blotting was used to measure the expression and dissociation of endothelial nitric oxide synthase(e NOS) in pulmonary artery tissues and all protein nitration levels in pulmonary artery tissues; reactive oxygen species(ROS) formation was measured via in vitro pulmonary artery superoxide anion probe dihydroethidium(DHE) staining.Results: After experiencing unsafe decompression, 10 of the 30 rats in the DCS group died. The pulmonary artery endothelium-dependent vasodilation capacity in the surviving rats decreased significantly(P<0.05). The difference in e NOS expression between the DCS group and the control group was statistically insignificant(P>0.05), but the ratio of e NOS monomer/dimer in the DCS group was significantly higher than that in the control group(P<0.05). All protein tyrosine nitration levels in the pulmonary artery tissues of the DCS group were significantly higher than those of the control group(P<0.05). The results of DHE staining showed that the amount of ROS formation in the pulmonary arteries of the DCS group was significantly higher than that of the control group(P<0.05).Conclusion: Unsafe decompression during a simulated submarine escape process can lead to e NOS dimer uncoupling in the pulmonary artery endothelium. The dissociated e NOS monomer cannot synthesize nitric oxide(NO) and thus affect the endothelium-dependent vasodilation capacity. The e NOS monomer can promote peroxynitrite(ONOO–) synthesis, leading to an increase in protein tyrosine nitration levels in pulmonary artery tissues and causing disorder in cell cycle regulation. The e NOS monomer can also cause an increase in the formation of ROS and thus mediate peroxidation damage.
文摘BACKGROUND: Melatonin is a kind of hormones derived from pineal gland. Recent researches demonstrate that melatonin is characterized by anti-oxidation, anti-senility and destroying free radicals. While, effect and pathogenesis of pineal gland on learning ability should be further studied. OBJECTIVE: To investigate the effects of pinealectomy on learning abiliy, distribution of cholinesterase and expression of neuronal nitric oxide synthase (nNOS) in cerebral cortex of rats and probe into the effect of melatonin on learning ability, central cholinergic system and nNOS expression. DESIGN: Randomized grouping design and animal study. SETTING: Department of Neurology, the 187 Hospital of Chinese PLA. MATERIALS: A total of 12 male SD rats, of normal learning ability testing with Y-tape maze, of clean grade, weighing 190-210 g, aged 6 weeks, were selected in this study. METHODS: The experiment was carried out in the Department of Neurology, Zhujiang Hospital from July 1997 to June 2000. All SD rats were divided into experimental group (n =6, pinealectomy) and control group (n =6, sham operation). Seven days later, rats in both two groups were continuously fed for 33 days. ① Learning ability test: The learning ability of rats was tested by trisection Y-type maze and figured as attempting times. ② Expression of acetylcholinesterase (AchE) was detected by enzyme histochemistry and nNOS was measured by SABC method. ③ Quantitative analysis of AchE fibers: AchE fibers density in unit area (surface density) was surveyed with Leica Diaplan microscope and Leica Quantimet 500+ image analytic apparatus and quantitative parameter was set up for AchE fibers covering density (μm2) per 374 693.656 μm2, moreover, the AchE fibers density was measured in Ⅱ-Ⅳ layers of motor and somatosensory cortex (showing three layers per field of vision at one time), in radiative, lacunaria and molecular layers of CA1, CA2 and CA3 areas, and in lamina multiforms of dentate gyrus. Three tissue slices were picked up randomly in the same part of each rat, together six tissue slices for nNOS expression and four near view (× 400) were selected in the parts of right neocortex, medial septal nucleus-diagonal band nucleus (SM-DB), corpus striatus and hippocampus to count nNOS-positive cells. MAIN OUTCOME MEASURES: Learning ability; distribution and quantitative analysis of AchE fibers; expression of nNOS in various cerebral areas. RESULTS: The twelve rats were all involved in the final analysis. ① Learning ability test: The learning abilities before operation in the experimental group [(14.67±4.97) times] were consistent with those in the control group [(14.33±4.32) times, P > 0.05], the learning abilities in the experimental group at 40 days after pinealectomy [(28.67±2.42) times] were obviously more than those before pinealectomy and those in the control group after operation [(13.83±8.33) times, P < 0.01]. ② Results of AchE-positive fibers density in cerebral cortex of rats: The AChE-positive fibers densities in motor and somatosensory cortex, CA1, CA2 and CA3 areas of hippocampus and in lamina multiforms of dentate gyrus in the experimental group were obviously lower than those in the control group [experimental group: (15 244±1 339), (14 764±1 391), (12 991±970), (15 077±1 020), (19 546±1 489), (19 337±1 378) μm2; control group: (21 001±1 021), (17 930±2 225), (17 260±1 342), (18 911±1 048), (24 108±1 671), (22 917±1 909) μm2, P < 0.01]. ③ Expression of nNOS in various cerebral areas: nNOS-positive cells in cerebral cortex of rats of the experimental group were more, furthermore the ones in somatosensory cortex were slightly more in motor cortex and the number (5.90±0.68) was more than that in the control group (3.68±0.39,P < 0.05). The nNOS-positive cells in SM-DB (16.21±2.03) were markedly more than those in the control group (9.32±1.05,P < 0.01). The nNOS-positive cells in hippocampus (4.27±0.75) and in corpus striatus (9.35±2.58) were not different with those in the control group (3.94±0.53, 8.96±2.31, P > 0.05). CONCLUSION: Decrease of melatonin due to pinealectomy of rats can result in learning disorder, which may be related to trauma of cholinergic neuron in cerebral cortex which were caused by nitric oxide neurotoxicity arose from the overexpression of nNOS in cerebral neocortex and SM-DB.
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.
文摘BACKGROUND: Some researches showed that motoneurons in spinal cord anterior horn wound die following brachial plexus injury, but the concrete mechanism of motoneurons death remains unclear. OBJECTIVE: To observe the expression of nitric oxide synthase (NOS) and survival of C7 motoneurons in spinal cord of rats after selective brachial plexus injury. DESIGN: A randomized controlled animal experiment. SETTING:Department of Anatomy, Sun Yet-sen Medical College, Sun Yet-sen University. MATERIALS: Totally 35 adult healthy male Sprague-Dawley rats with the body mass of 200-300 g were provided by Experimental Animal Center, Sun Yet-sen Medical College, Sun Yat-sen University. The rats were divided into control group (n =5) and experimental group(n =30) by random number table method, and the experimental group was divided into three injury subgroups: anterior root avulsion group, dorsal root transection group and spinal cord hemisection group, 10 rats in each group. There were horse anti-neuronal NOS (nNOS) polycolonal antibody (Sigma company) and nicotina mideadeninedinucleotide phosphate (NADPH-d) (Sigma Company). METHODS: The experiment was performed at Department of Anatomy, Sun Yet-sen Medical College, Sun Yet-sen University between September 2004 and April 2005. ①After anesthetizing the rats, the spinous process of second thoracic vertebra as a marker, the vertebra was exposed from C5 to T1 and the lamina of vertebra was unclenched, and spinal dura mater was carved to expose the spinal nerve dorsal roots of C5-T1. The right ventral root of C7 was avulsed, and the residual root was removed in anterior root avulsion group. The right ventral root of C7 was avulsed and the right dorsal roots of brachial plexus (C5-T1) were cut off in dorsal root transection group. In spinal cord hemisection group, the hemisection between the C5 and C6 spinal segment on right side and avulsion of right ventral root of C7 were made. In the control group, the vertebra from C5 to T1 was unclenched and the skin of wound was sutured. ②Three weeks after operation, behavior of rats was observed. The rats were killed after anesthesia. The C7 segment of spinal cord was removed and treated with NADPH-d staining, neutral red counterstaining and NOS immunohistochemistry staining to detect the expression of NOS. MAIN OUTCOME MEASURES: The expression of NOS and survival of C7 motoneurons in spinal cord of rats 3 weeks after operation. RESULTS: Among the 35 included rats, 3 rats died 2 weeks following operation, so totally 32 rats were involved in the result analysis. ①NADPH-d positive neurons of in anterior horn of C7 in the three groups: The NADPH-d positive neurons could be found in anterior horn of C7 in the three groups. The percentage of that in anterior root avulsion group to that of non-injury side of spinal cord was(20.98±2.65)%, (29.43±6.81)% in dorsal root transection group and (31.74±6.80)% in spinal cord hemisection group. There was significant difference among the three injury groups(F =5.135,P =0.016). There was significant difference in anterior root avulsion group with dorsal root transection group and spinal cord hemisection group (t =2.562,3.167,P < 0.05). There was no significant difference between the dorsal root transection group and spinal cord hemisection group (P =0.534). ②survival rate of motoneurons in anterior horn of C7: There were dead motoneurons in the three injury groups, the percentages of surviving motoneurons to that of non-injured side of spinal cord were (69.22±4.04)%,(62.01±3.82)% and (56.74±6.86)%, respectively. There were significant differences among the three groups (F =9.508,P =0.002). The anterior root avulsion group was significantly different from the other two groups(t =2.764,4.587,P < 0.05). There was no significant difference between the dorsal root transection group and spinal cord hemisection group(P =0.073). CONCLUSION: The selective brachial plexus injury can induce the up-regulation of NOS expression in motorneurons of spinal cord anterior horn and block descending pathway of cortex to cause the more significant up-regulation of NOS and low survival rate in motoneurons. It indicates that descending pathway of cortex can inhibit the NOS expression in motorneurons of spinal cord anterior horn, and the high NOS expression might induce the death of motorneurons in spinal cord anterior horn.
基金the Natural Sci-ence Foundation of HebeiProvince, No. C2005000840
文摘BACKGROUND: Nitric oxide synthase (NOS) inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids (EAA) during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine (L-NA) reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear.OBJECTIVE: By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid (GABA) in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism. DESIGN: A randomized and controlled experiment.SETTING: Department of Pharmacology, Hebei Academy of Medical Sciences.MATERIALS: A total of 42 male healthy SD rats (grade Ⅱ, weighting 250-300 g) were provided by the Experimental Animal Center of Hebei Province (Certification: 04036). Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Sigma Chemicals Co, St Louis, MO, USA. HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC.METHODS: The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups: sham-operated group (n = 6), ischemic group (n = 18), L-NA group (n = 18). The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared, but the filament was not inserted. Each group was further divided into 3 subgroups (n = 6 for each): drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion (MCAO) respectively. L-NA (20 mg/kg, ip) was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain (IV%) in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett’s test.MAIN OUTCOME MEASURES: ① The volume of cerebral infarction; ② The contents of aspartate, glutamate, glycine and GABA in brain tissue after cerebral ischemia.RESULTS: All 42 rats were involved in the final analysis. ① Infarcted volume: Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was (20.13±3.59)% and (23.12±5.84)% in L-NA group, which was not similar to that in ischemic group [(22.10±3.98)%, (25.38±5.37)%, P > 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [(26.11±3.55)% and (37.15±3.58)%, P < 0.01]. ② Changes of amino acid content: At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group ( P < 0.05-0.01). However, contents in L-NA group were similar to those in ischemic group (P > 0.05). At 12 hours after ischemia, the contents of aspartate [(0.21±0.06), (0.36±0.05), (0.29±0.12) mg/g] and glutamate [(0.55±0.06), (0.78±0.10), (0.52±0.10) mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [(0.49±0.17), (0.63±0.03), (0.51±0.15) mg/g; (0.98±0.30), (1.15±0.15), (0.93±0.15) mg/g, P < 0.05-0.01]. Glycine in hippocampus was (0.40±0.07) mg/g, which was higher than that in ischemic group [(0.21±0.07) mg/g, P < 0.05]. GABA in striatum, hippocampus and cortex was (0.93±0.10), (0.62±0.12) and (0.81±0.10) mg/g, respectively, which was higher than that in ischemic group [(0.60±0.08), (0.37±0.17), (0.59±0.10) mg/g, P < 0.05-0.01]. CONCLUSION: It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA.
文摘Pancreatic cancer is a common tumor of the digestive system.At present,the pathogenesis is still unclear,but current research on inducible nitric oxide synthase in relation to the pathogenesis of pancreatic malignant tumors is particularly extensive.Therefore,this article focuses on the research progress on inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.
文摘Background:Overexpression of inducible nitric oxide synthase(iNOS)has been reported in diabetic retinopathy(DR).The kinin B1 receptor(B1R)is also overexpressed in DR,and can stimulate iNOS via Gαi/ERK/MAPK pathway.We previously showed that the topical administration of a B1R antagonist,LF22-0542,significantly reduces leukocyte infiltration,increased vascular permeability and overexpression of several inflammatory mediators,including iNOS in DR.Thus,the aim of this study was to determine whether the pro-inflammatory effects of B1R are attributed to oxidative stress caused by the activation of iNOS pathway in order to identify new therapeutic targets for the treatment of DR.iNOS and B1R being absent in the normal retina,their inhibition is unlikely to result in undesirable side effects.The approach will be no invasive by eye application of drops.Methods:Diabetes was induced in male Wistar rats(200-230 g)by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg b.w).One week later,rats were randomly divided into four groups(N=5)and treated for one week as follows:Gr 1:control rats treated with the selective iNOS inhibitor(1,400 W,0.06μM twice a day by eye-drops×7 days),Gr 2,STZ-diabetic rats treated with 1,400 W,Gr 3:control rats received a selective B1R agonist[Sar(D-Phe8)-des-Arg9-BK,100μg twice a week]by intravitreal injections(itrv)and treated with 1,400 W,Gr 4:STZ-diabetic rats+B1R agonist+1,400 W.At the end of treatment and two weeks post-STZ,three series of experiments were carried out to measure vascular permeability(by Evans blue dye method)and the expression of vasoactive and inflammatory mediators,including iNOS,VEGF-A,VEGF-R2,IL-1β,Cox-2,TNF-α,bradykinin 1 and 2 receptors and carboxypeptidase M/kininase 1(by Western Blotting and qRT-PCR).The nitrosative stress(nitrosylation of proteins)was also assessed by Western Blotting.One-way Anova test with Bonferroni post hoc was used for statistical analysis.Results:STZ-diabetic rats showed a significant increase in retinal vascular permeability(22.8μg/g Evans blue dye per g of fresh retinas,P=0.016)compared with control rats and control treated rats(17.2 and 16.8μg/g respectively).The injections of B1R agonist amplified the increase of vascular permeability which was normalized by the 1,400 W.The overexpression of inflammatory markers was also normalized by the 1,400 W in STZ-diabetic rats received or not the B1R agonist.Conclusions:These results support a contribution of iNOS in the deleterious effects of B1R in this model of diabetic retinopathy.Hence,iNOS inhibition by ocular application of 1,400 W may represent a promising and non-invasive therapeutic approach in the treatment of diabetic retinopathy.
文摘Pancreatic cancer is a common tumor of the digestive system,at present,the pathogenesis is still unclear,but in the current research on the pathogenesis of pancreatic malignant tumors,the research on inducible nitric oxide synthase is particularly extensive.Therefore,this article focuses on the research progress of inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.This is a review.
文摘AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus.METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique.RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus. The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62 -± 38 cells/mm2(antrum), 43 ± 32 cells/mm2(body), and 32 ± 28 cells/mm2 (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels.CONCLUSION: Nitric oxide might he involved in the modulation of motility, secretion and blood ciroulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach.
