1.Effects of insulin-like growth factor II on regulating nitric oxide synthase gene expression in mouse osteoblast-like MC3T3-E1 cells.
Wei-lian SUN ; Li-li CHEN ; Jie YAN ; Zhong-sheng YU
Chinese Journal of Stomatology 2004;39(3):201-204
OBJECTIVETo study the effects of insulin-like growth factor II (IGF-II) on regulating the levels of nitric oxide (NO) and the mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells.
METHODSMouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different intervals of time, MTT colorimetry was used for examining the cell proliferation. Nitrate reductase method was applied for detecting the NO concentrations in cell culture supernatants and RT-PCR employed for determining the levels of cellular iNOS and eNOS mRNAs.
RESULTSAfter the MC3T3-E1 cells were treated with IGF-II at the dosages of 1 microg/L for 72 h, 10 and 100 microg/L for 24, 48 and 72 h respectively, all the MTT values increased markedly (P < 0.05 or P < 0.01). After the cells were treated for 48 and 72 h at the dosage of 100 microg/L IGF-II respectively, the levels of NO in the supernatants of cell cultures and cellular iNOS mRNA decreased significantly (P < 0.01). However, the levels of eNOS mRNA in the cells treated with any of the IGF-II dosages for the different times were stable (P > 0.05).
CONCLUSIONSIGF-II at the dosages of 1 approximately 100 microg/L showed the effects on promoting proliferation, which as probably due to the maintenance of low NO levels. Inducible NOS gene expression at the level of transcription was down regulated in the MC3T3-E1 cell treated with higher dosage of IGF-II (100 microg/L) but eNOS mRNA was not, which might be one of the mechanisms for the maintenance of low NO levels.
3T3 Cells ; Animals ; Insulin-Like Growth Factor II ; pharmacology ; Mice ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Osteoblasts ; cytology ; drug effects ; enzymology ; RNA, Messenger ; biosynthesis
2.Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases.
Xiang-Feng ZHANG ; Shuang LIU ; Yu-Jie ZHOU ; Guang-Fa ZHU ; Hussein D FODA
Chinese Medical Journal 2010;123(7):929-935
BACKGROUNDExposure of adult mice to more than 95% O(2) produces a lethal injury by 72 hours. Nitric oxide synthase (NOS) is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS.
METHODSOne hundred and forty-four osteopontin knock-out (KO) mice and their matched wild type background control (WT) were exposed in sealed cages > 95% oxygen or room air for 24- 72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase (eNOS) and iNOS mRNA in lung tissues at 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry (IHC) was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues.
RESULTSOPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85 +/- 0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31 +/- 0.92 in the WT group (P < 0.05). iNOS mRNA (48 hours: 1.04 +/- 0.08 vs. 0.63 +/- 0.09, P < 0.01; 72 hours: 0.89 +/- 0.08 vs. 0.72 +/- 0.09, P < 0.05) and eNOS mRNA (48 hours: 0.62 +/- 0.08 vs. 0.43 +/- 0.09, P < 0.05; 72 hours: 0.67 +/- 0.08 vs. 0.45 +/- 0.09, P < 0.05) expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS (20.54 +/- 3.18 vs. 12.52 +/- 2.46, P < 0.05) and eNOS (19.83 +/- 5.64 vs. 9.45 +/- 3.82, P < 0.05) in lung tissues of OPN KO mice at 72 hours of hyperoxia.
CONCLUSIONOPN can protect against hyperoxia-induced lung injury by inhibiting NOS.
Animals ; Hyperoxia ; genetics ; physiopathology ; Immunohistochemistry ; Lung ; metabolism ; Lung Injury ; etiology ; genetics ; metabolism ; Mice ; Mice, Knockout ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; Nitric Oxide Synthase Type III ; genetics ; Osteopontin ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction
3.The relationship between resting heart rate and single nucleotide polymorphism at 3 sites of nitric oxide synthase gene in the Chinese Han nationality population.
Hou-xun MA ; Yong-hong NIU ; Zhang-yong LI ; Zheng-xiang XIE
Chinese Journal of Medical Genetics 2006;23(6):670-672
OBJECTIVETo study the relationship between resting heart rate and single nucleotide polymorphisms (SNP) at 3 sites of nitric oxide synthase (NOS) gene including NOS3 -922A/G, NOS3 894G/T and NOS2 -1173C/T SNPs.
METHODSGenomic DNA was gained from 211 Chinese Han nationality population. The SNPs of NOS3 -922A/G, NOS3 894G/T and NOS2 -1173C/T were genotyped by allele-specific primer-polymerase chain reaction (ASP-PCR) technique.
RESULTSThe distribution frequencies of GG, GT and TT genotypes of NOS3 894G/T and AA, AG and GG genotypes of NOS3 -922A/G and CC, CT and TT genotypes of NOS2 -1173C/T were consistent with Hardy-Weinberg equilibrium (P> 0.05). The resting heart rate of Chinese Han nationality population with AA genotypes was higher than that with GG genotype of NOS3 -922A/G (P< 0.01). The resting heart rate of the sub-population with GG genotype was higher than that with TT genotype of NOS3 894G/T (P< 0.05). There were no difference among the resting heart rates of the sub-populations with the allele genotypes of NOS2 -1173C/T.
CONCLUSIONThe resting heart rate of Chinese Han nationality population with mutated genotype GG of NOS3 -922A/G and with mutated genotype TT of NOS3 894G/T were lower than those with wild genotype of NOS3 -922A/G and NOS3 894G/T. The finding suggests that resting heart rate is associated with SNP of NOS3 -922A/G and NOS3 894G/T.
Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; China ; Female ; Gene Frequency ; Genotype ; Heart Rate ; Humans ; Male ; Middle Aged ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type II ; genetics ; Nitric Oxide Synthase Type III ; genetics ; Polymorphism, Single Nucleotide
4.Gene expression of two kinds of constitutive nitric oxide synthase in injured spinal cord tissue.
Chenglong LIU ; Anmin JIN ; Chusong ZHOU ; Shaoxiong MIN
Chinese Journal of Traumatology 2002;5(1):3-6
OBJECTIVETo investigate the gene expression of two kinds of constitutive nitric oxide synthase (cNOS): neuronal NOS (nNOS) and endothelial NOS (eNOS) in injured spinal cord tissue.
METHODSThirty-six adult Sprague-Dawley rats were divided randomly into six groups: the normal group and the injury groups (2, 6, 12, 24, 48 h after injury, respectively). A compression injury model of the spinal cord wa s ma de and gene expression of nNOS and eNOS were examined by reverse transcription polymerase chain reaction (RT-PCR).
RESULTSThe gene expression of nNOS and eNOS was detected in the normal group and they were up-regulated quickly after injury, reaching the maximum at 6 h. There was no difference between gene expression of nNOS and eNO S in the normal group, but in each injury group the gene expression of eNOS was much higher than that of nNOS.
CONCLUSIONSExpression of constitutive NOS (cNOS) in spinal co rd tissue was up-regulated after injury mainly in the early stage. cNOS as a wh ole offers protection in spinal cord injury, but different cNOS may play different roles.
Animals ; Gene Expression ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type II ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord Injuries ; enzymology ; genetics ; Up-Regulation
5.Study on the Relationship between susceptibility of stomach neoplasm cancer and polymorphism of inducible nitric oxide synthase gene.
Jing SHEN ; Runtian WANG ; Liwei WANG ; Zhaoxi WANG ; Houxun XING ; Binyan WANG ; Maosen LI ; Zhaolai HUA ; Jianming WANG ; Chunhua GUO ; Xinru WANG ; Xiping XU
Chinese Journal of Epidemiology 2002;23(5):374-377
OBJECTIVETo study the relationship between polymorphism of inducible Nitric Oxide Synthase (iNOS) gene and the susceptibility of intestinal type stomach cancer and stomach cardia cancer in Chinese people.
METHODSA community-based case-control study was designed. Ninety-three intestinal type of stomach cancer and 50 stomach cardia cancer patients with endoscopy and pathology diagnosis were identified as cases. Two hundred and forty-six controls served as controls.
RESULTSC-->T polymorphism was found in exon 16 of iNOS gene, which changed the coding amino acid from serine to leucine, and formed a recognition site identified by Tsp 509 I restriction enzyme (we called it C-->T polymorphism). The T allele gene frequency in the control group was 13.21%. No statistically significant difference was found between C-->T polymorphism alone and the increased susceptibility to intestinal stomach cancer or stomach cardia cancer. A significant type 2 multiplicative interaction was found in increasing both the risk of intestinal stomach cancer and stomach cardia cancer when both C-->T polymorphism and tobacco smoking exposure existed. An additive interaction model, which showed statistically significant difference, was found to increase only the risk of stomach cardia cancer when CagA antibody shared negative but C-->T polymorphism occurred.
CONCLUSIONC-->T polymorphism of iNOS gene was considered as one of the possible susceptible genes, which specifically increased the risk of tobacco-related but CagA negative types of intestinal stomach cancer and stomach cardia cancer.
Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Bacterial Proteins ; immunology ; Genetic Predisposition to Disease ; Humans ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type II ; Polymorphism, Genetic ; Stomach Neoplasms ; genetics
6.Effect of spinal cord injury on iNOS and Bcl-2 gene expression in spermatogenic cell of the male rat.
Yong WEI ; Xiao-Bin ZHANG ; Jie ZHANG ; Fan CHENG
National Journal of Andrology 2003;9(1):28-54
OBJECTIVESTo investigate the cause of spermatogenic cells decreasing after spinal cord injury.
METHODSImmunohistochemical S-P method was used to detect the expression of iNOS and Bcl-2 protein in the testis of spinal cord injured and sham-operated adult rats in second week and fourth week after operation.
RESULTSSecond week and fourth week after operation, the expression of iNOS of operated rats increased significantly (P < 0.05). Fourth week after operation, the number of Bcl-2 positive cells in spinal cord injured group decreased significantly (P < 0.05).
CONCLUSIONSThe expression veition of iNOS and Bcl-2 is the cause that inducing the decrease of spermatogenic cells in testis of spinal cord injured rats.
Animals ; Male ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Nitric Oxide Synthase Type II ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism
7.Inducible nitric oxide synthase and brain hypoxic-ischemic brain damage.
Chinese Journal of Contemporary Pediatrics 2014;16(9):962-967
Brain hypoxia-ischemia has been considered as critical factors in many human central nervous system diseases, including stroke and neonatal hypoxic-ischemic encephalopathy. In brain hypoxia-ischemia processes, inducible NO synthase (iNOS) is induced to produce excessive nitric oxide (NO) which leads to cascade reactions of inflammation and neuronal death, deteriorating primary brain injury. Inhibiting iNOS expression has opened new perspectives in the treatment of brain hypoxia-ischemia because iNOS inhibitor has been shown as a potent therapeutic agent. This reviews focus on recent research achievements regarding the relationship between iNOS and ischemic-hypoxic brain damage and the perspective of using iNOS inhibitors as therapeutic strategies for brain ischemic-hypoxic brain damage.
Animals
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Humans
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Hypoxia-Ischemia, Brain
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drug therapy
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metabolism
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Nitric Oxide Synthase Type II
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antagonists & inhibitors
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genetics
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physiology
8.Nitric oxide synthase gene expression in injured spinal cord tissue.
Chenglong LIU ; Anmin JIN ; Chusong ZHOU ; Bin CHEN
Chinese Medical Journal 2002;115(5):740-742
OBJECTIVETo investigate gene expression of three nitric oxide synthase isozymes in injured spinal cord tissue.
METHODSThirty-six adult SD rats were randomly divided into six groups: a normal group and five injury groups, with six per each group. Animals in the injury groups were sacrificed at 2, 6, 12, 24, 48 h after injury. A compression injury model on the spinal cord was made according to Nystrom B et al and gene expression of the three NOS isozymes were examined by reverse transcription polymerase chain reaction (RT-PCR).
RESULTSGene expression of nNOS and eNOS were detectable in the normal group and were up-regulated quickly after injury, reaching a maximum at 6 h: (0.633 +/- 0.012) and (1.236 +/- 0.207). Gene expression of iNOS was detectable only in the injury groups and it was gradually up-regulated after injury, reaching a maximum at 24 h: (1.043 +/- 0.049).
CONCLUSIONInjury to the spinal cord leads to early up-regulation of cNOS and late up-regulation of iNOS. Different NOS isozymes may play different roles in secondary spinal cord injury.
Animals ; Female ; Gene Expression Regulation, Enzymologic ; Male ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; RNA ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord ; enzymology ; pathology ; Spinal Cord Injuries ; enzymology ; genetics
9.Effect of dexamethasone on nitric oxide synthase and Caspase-3 gene expressions in endotoxemia in neonate rat brain.
Hua WANG ; Yu-Bin WU ; Xiu-Hua DU
Biomedical and Environmental Sciences 2005;18(3):181-186
OBJECTIVETo investigate the gene and protein expressions of three isoforms of nitric oxide synthase (NOS) and gene expression of Caspase-3, and effect of dexamethasone on them in neonatal rats with lipopolysaccharide (LPS)-induced endotoxemic brain damage.
METHODSExpressions of the three isoforms of NOS and caspase-3 mRNA in the brain were investigated by RT-PCR in postnatal 7-day Wistar rats with acute endotoxemia by intraperitoneal administration of LPS. Regional distributions of NOSs were examined by immunohistochemical technique.
RESULTSnNOS and Caspase-3 mRNA were obviously detected. eNOS mRNA was faintly expressed, but iNOS mRNA was undetectable in the control rat brain. The expressions of NOS mRNA of three isoforms were weak 2 h after LPS (5 mg/mg) delivery, peaked at 6 h, and thereafter, reduced gradually up to 24 h. The expression intensity was in the order of nNOS> iNOS> eNOS. Widespread nNOS, scattered eNOS distribution and negative iNOS were identified in the control rat brain and all isoforms of NOS could be induced by LPS which reached the apex at 24 h in the order of nNOS> iNOS> eNOS as detected by immunostaining. Although Caspase-3 mRNA could be found in all groups, DNA fragmentation was only seen at 6 h and 24 h. The expressions of NOS and Caspase-3 mRNA were inhibited in the rat brain when dexamethasone was administrated.
CONCLUSIONLPS-induced NO production induces apoptosis of neurons through mechanism involving the Caspase-3 activation, which may play an important role in the pathogenesis of brain damage during endotoxemia, and neuro-protective effects of dexamethasone may be partially realized by inhibiting the expression of NOS mRNA.
Animals ; Animals, Newborn ; Apoptosis ; Brain ; drug effects ; enzymology ; Caspase 3 ; Caspases ; genetics ; metabolism ; Dexamethasone ; pharmacology ; Disease Models, Animal ; Endotoxemia ; chemically induced ; enzymology ; Female ; Lipopolysaccharides ; Male ; Nerve Tissue Proteins ; genetics ; metabolism ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction
10.Effects of Naoxintong on atherosclerosis and inducible nitric oxide synthase expression in atherosclerotic rabbit.
Xiao-Nan ZHONG ; Hong-Hao WANG ; Zheng-Qi LU ; Yong-Qiang DAI ; Jian-Hua HUANG ; Wei QIU ; Ya-Qing SHU ; Wen XU ; Chen CHENG ; Xue-Qiang HU
Chinese Medical Journal 2013;126(6):1166-1170
BACKGROUNDHigh levels of nitric oxide (NO) produced by inducible NO synthase (iNOS) have been associated with atherosclerosis processes. Naoxintong is a traditional Chinese medicine for treatment of cerebrovascular and cardiovascular disease. The aim of the present study was to detect and quantify changes of iNOS mRNA and NO levels in the vessel wall after the administration of Naoxintong in an atherosclerotic rabbit model.
METHODSForty New Zealand white rabbits were randomly divided into five groups (n = 8). Rabbits were fed a standard diet (group A), an atherogenic diet consisting of 79% standard feed + 1% cholesterol + 5% lard + 15% egg yolk powder (group B), an atherogenic diet with Naoxintong 0.25 mg×kg(-1)×d(-1) (group C), an atherogenic diet with Naoxintong 0.5 mg×kg(-1)×d(-1) (group D), or atherogenic diet with Naoxintong 1.0 mg×kg(-1)×d(-1) (group E) for 12 weeks.
RESULTSSupplemented administration of Naoxintong led to a down-regulation of cholesterol (CHOL) (P < 0.001) and low-density lipoprotein (LDL) (P < 0.001). The trend became more notable as the dose of Naoxintong increased; group C vs. group B (CHOL, P = 0.568; LDL-cholesterol (LDL-C), P = 0.119), group D vs. group B (CHOL, P = 0.264; LDL-C, P = 0.027), group E vs. group B (CHOL, P = 0.028; LDL-C, P = 0.002). Atherosclerotic lesions in aorta were reduced in Naoxintong groups (groups C, D, E) compared to group B. Group B had higher iNOS mRNA (P = 0.001) and NO level (P < 0.001) than group A. Compared with the atherogenic diet fed-rabbits, Naoxintong supplements decreased the expression of iNOS mRNA (P < 0.001) and the NO level (P < 0.001) in the vessel wall. Groups given a higher Naoxintong dose exhibited greater benefits. iNOS mRNA and NO levels seemed to be reduced in group C, although the difference did not quite reach statistical significance (iNOS mRNA, P = 0.130; NO, P = 0.038). iNOS mRNA and NO levels significantly decreased in group D (iNOS mRNA, P = 0.019; NO, P = 0.018) and group E (iNOS mRNA, P = 0.004; NO, P < 0.001).
CONCLUSIONNaoxintong has beneficial effects on atherosclerosis treatment by reducing expression of iNOS mRNA and the NO level in the vessel wall.
Animals ; Atherosclerosis ; drug therapy ; enzymology ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Rabbits