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.Effect of urotensin II on iNOS expression in human umbilical vein endothelial cells.
Chinese Journal of Applied Physiology 2006;22(3):288-291
AIMTo observe the direct effect of urotensin II (U II) on the release of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) mRNA in human umbilical vein endothelial cells (HUVEC).
METHODSHUVEC were cultured with different concentrations of U II (10(-9)-10(-7) mol/L) for 24 hours. Then the supernatant was collected to detect the level of NO and the activity of iNOS, the expression of iNOS mRNA of HUVEC was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).
RESULTSIn comparison with controls, the level of NO, the activity of iNOS and the iNOS mRNA expression increased significantly (P < 0.05).
CONCLUSIONU II may up-regulate the expression of iNOS mRNA and increase NO generation in HUVEC, it suggests that U II may relax blood vessel by activating iNOS/NO pathway.
Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; RNA, Messenger ; genetics ; Urotensins ; pharmacology
3.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
4.Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats.
Wu-Jiang LIU ; Zhong-Cheng XIN ; Hua XIN ; Yi-Ming YUAN ; Long TIAN ; Ying-Lu GUO
Asian Journal of Andrology 2005;7(4):381-388
AIMTo investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS) isoforms in castrated rats.
METHODSThirty-two adult male Wistar rats were randomly divided into one sham-operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal saline (groups A and B) or oral icariin (1 mg/[kg.day] for group C and 5 mg/[kg.day] for group D) for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP) during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle (PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in corpus cavernosum (CC) were also evaluated.
RESULTSICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A (P 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B (P 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant (P 0.05) in groups C and D compared with those in group B.
CONCLUSIONOral treatment with icariin ( 98.6 % purity) for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.
3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; metabolism ; Animals ; Blood Pressure ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Drugs, Chinese Herbal ; pharmacology ; Erectile Dysfunction ; drug therapy ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Male ; Muscle, Smooth ; drug effects ; physiology ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Orchiectomy ; Penile Erection ; drug effects ; Penis ; drug effects ; enzymology ; Pressure ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Testosterone ; blood
5.Effects of 2, 3, 4', 5-tetrahydroxystilbene -2-O-beta-D glucoside on content of nitric oxide synthase and expression of nitric oxide synthase in artery vessels of experimental atherosclerotic rats.
Yan SHEN ; Chun-Hua WANG ; Yu-Qin WANG ; Feng LI ; Wei ZHANG
China Journal of Chinese Materia Medica 2008;33(8):919-923
OBJECTIVETo investigate the effects of TSG on the content of nitric oxide synthase and the expression of endothelium nitric oxide synthase in artery vessels of experimental atherosclerotic rats.
METHODThe atherosclerosis model of rat was made by feeding high grease food and injecting Vit D3. Sixty male rats were randomly divided into six groups: normal control; model control; TSG high dose; TSG middle dose; TSG low dose; Simvastatin. After 12 weeks, several aorta were randomly tested, and the model made was successful when we found plaque. And after six weeks of treatment, the levels of NOS in serum were measured with a biochemical method. The biochemical method was adopted to detect the content of nitric oxide synthase and half-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect eNOS and iNOS gene expression in artery vessels.
RESULTData of the study demonstrated that compared with model group, the activity of NOS and the gene expression of eNOS were increased remarkably, and however the gene expression of iNOS was reduced markedly in simvastatin group and TSG 60, 120 mg x kg(-1) x d(-1) group.
CONCLUSIONTSG can enhance the expression of eNOS gene and reduce the expression of iNOS gene in aorta vessels of experimental atherosclerotic rats, which may be one of the anti-atherosclerosis mechanisms of TSG.
Animals ; Arteries ; drug effects ; metabolism ; pathology ; Atherosclerosis ; drug therapy ; enzymology ; pathology ; Gene Expression Regulation, Enzymologic ; drug effects ; Glucosides ; pharmacology ; therapeutic use ; Male ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Stilbenes ; pharmacology ; therapeutic use
6.Inducible nitric oxide synthase is involved in the oxidation stress induced by HIV-1 gp120 in human retina pigment epithelial cells.
Qiu-rong YU ; Zhen-ping ZHANG ; Hui ZHANG ; Hao-tian LIN ; Xiu-mei LI ; Ling BAI ; Wei-bin CAI
Chinese Medical Journal 2008;121(24):2578-2583
BACKGROUNDThe human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 has been implicated in the development of AIDS-associated retinopathy. The present study tested the hypothesis that gp120 may induce oxidative stress including up regulation of inducible nitric oxide synthase (iNOS) and production of malondialdehyde (MDA) and nitric oxide (NO) to mediate retinopathy in retinal pigment epithelial (RPE) cells.
METHODSHuman RPE cell line D407 was cultured and treated with gp120. HIV-1 gp120 protein induced lipid peroxidation product MDA. NO production and iNOS expression were examined in vitro by spectrophomtometry, real-time PCR, Western blotting, and confocal microscope.
RESULTSAddition of gp120 was able to induce RPE cells to produce NO and MDA in time- and dose-dependent manners (P < 0.05). Similarly, gp120 was also capable of up-regulating iNOS mRNA and protein in D407 cells in time- and dose-dependent manners.
CONCLUSIONSGp120 induces oxidative stress in D407 cell by stimulating MDA and NO production, which is mediated by up-regulating iNOS expression. Gp120 may mediate oxidation stress in AIDS-associated retinopathy.
Blotting, Western ; Cell Line ; Epithelial Cells ; drug effects ; metabolism ; HIV Envelope Protein gp120 ; pharmacology ; Humans ; Immunohistochemistry ; Microscopy, Confocal ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Polymerase Chain Reaction ; Retina ; cytology
7.Effects of insulin like growth factor-1 on cell viability and tissue factor in vascular endothelial cells.
Qi-Lin MA ; Tian-Lun YANG ; Ming SUN ; Yuan-Jian LI ; Can-E TANG ; Zhen-Yu PENG ; Shi-Lin HE ; Fang-Ping CHEN
Chinese Journal of Hematology 2007;28(9):605-608
OBJECTIVETo study the effects of insulin like growth factor-1 (IGF-1) on cell viability and tissue factor (TF) in angiotensin II (Ang II) induced vascular endothelial cells and to investigate its mechanisms.
METHODS10(-6) mol/L Ang II was added to human vascular endothelial cells (HUVECs) culture media alone or 30 min after pretreatment with IGF-1 (0.1 microg/ml , 0.5 microg/ml, 2.5 microg/ml). Cell viability and AngII type 1 receptor (AT1-R) mRNA were evaluated after 24 h incubation with AngII. At the optimum concentration of IGF-1 affecting cell viability, the time dependent manner for 12 - 48 h incubation with Ang II was evaluated. TF, NOS and NO were investigated after 24 h incubation with Ang II. In addition, NO synthase inhibitor Nomega-nitro-1-arginine methylester(L-NAME) was added 30 min before addition of IGF-1 and Ang II, and cell viability, TF, AT1-R mRNA, NOS and NO were evaluated after 24 h incubation.
RESULTS(1) Ang II induced a decrease in cell vitality, an upregulation of AT1-R mRNA, an increase in TF, and a decrease in the activity of NOS and content of NO. (2) Pretreatment with IGF-1 significantly inhibited the decreased cell viability and upregulation of AT1-R mRNA. IGF-1 at 0.5 microg/ml showed the most obvious effects. This effect of cell viability recovery was in a time dependent manner during 12 -48 h. (3) IGF-1 also inhibited the increased content of TF, the decreased activity of NOS and the decreased content of NO. (4) The beneficial effects of IGF-1 on cultured endothelial cells were completely abolished by L-NAME.
CONCLUSIONIGF-1 pretreatment could enhance the ang II injured cell viability and anti-thrombosis capacity, and the protective effects may be related to activation of NOS-NO signaling pathway which inhibited AT1-R.
Angiotensin II ; pharmacology ; Cell Survival ; drug effects ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; physiology ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism ; Thromboplastin ; metabolism
8.Effect of inducible nitric oxide synthase on tumour cells sensitivity to mitomycin C analogue 629 in vitro.
Acta Pharmaceutica Sinica 2006;41(8):712-715
AIMTo examine the effect of inducible nitric oxide synthase (iNOS) on tumour cells chemosensitivity to mitomycin C (MMC) analogue 5-aziridinyl-3-hydroxyl-1-methylindole-4,7-dione (629) in vitro, and elucidate the possible role of iNOS in the metabolism of 629.
METHODSHuman sarcoma cells (HT1080) and its iNOS gene transfected clones (iNOS9, iNOS12) were exposed to 629 at concentrations of 1 nmol x L(-1) - 100 micromol x L(-1). 3-[4, 5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay, agarose electrophoresis and flow cytometric analysis were used to determine cell sensitivity, deoxyribonucleic acid (DNA) damage and the change of cell cycle in above process, respectively. All experiments were performed both in air and under hypoxia parallelly.
RESULTS629 was more toxic than MMC, and enhanced cytotoxicity under hypoxia, which resulted in cell necrosis. Sixteen hours after treated with 629, HT1080 cells and related iNOS-transfected clone cells were obviously blocked in G2/M phase.
CONCLUSIONiNOS plays dual roles in 629 metabolism, enhancing or decreasing the cytoxicity of 629 depending on the intracellular oxygen pressure P(O2), which caused higher cytotoxicity to hypoxia cells of 629 with the increasing of iNOS activity.
Antibiotics, Antineoplastic ; pharmacology ; Aziridines ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; DNA Damage ; Fibrosarcoma ; metabolism ; pathology ; Flow Cytometry ; Humans ; Indoles ; pharmacology ; Mitomycin ; chemistry ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Transfection
9.Effects of extracts of cheezheng pain relieving plaster on nitric oxide and iNOS expression in macrophages induced by lipopolysaccharides.
Yang LIU ; Shan-Ying PENG ; Lin WANG ; Wen-Jie WANG
Acta Pharmaceutica Sinica 2009;44(8):863-867
This study is to explore the effects of extracts of Cheezheng pain relieving plaster (ECPRP) on nitric oxide (NO) production and expression of inducible nitric oxide synthase (iNOS) in macrophages induced by LPS and the mechanism involved. Nitric oxide level was measured with Griess reagent assay. Inducible nitric oxide synthase (iNOS) protein and NF-kappaBp65 fragment were detected with Western blotting. ECPRP (62.5 and 125 mgL(-1)) significantly inhibited the increase of nitric oxide level. Furthermore, ECPRP (62.5 and 125 mg x L(-1)) notably reduced the expression of iNOS mRNA and protein. ECPRP (62.5 and 125 mg x L(-1)) elevated the content of I-kappaB protein in cytoplasm, while decreased the content of NF-kappaBp65 protein in nucleus. These results suggest that ECPRP reduce nitric oxide level via down-regulation of NF-kappaB-iNOS-nitric oxide pathway, resulting in prevention of inflammation.
Animals
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Drugs, Chinese Herbal
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pharmacology
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Lipopolysaccharides
;
adverse effects
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Macrophages
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drug effects
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metabolism
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Male
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Mice
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Mice, Inbred C57BL
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Nitric Oxide
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metabolism
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Nitric Oxide Synthase Type II
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metabolism
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RNA, Messenger
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genetics
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Transcription Factor RelA
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metabolism
10.Effect of hypoxia on expression of iNOS mRNA in cultured rat astrocytes.
You-Ming FAN ; Yu-Qi GAO ; Guo-Bin ZHANG ; Wen-Xiang GAO ; Shu-Min XU
Chinese Journal of Applied Physiology 2003;19(1):12-15
AIMTo explore the effects of hypoxia on expression of inducible nitric oxide synthase (iNOS) mRNA in cultured rat astrocytes.
METHODSCultured rat astrocytes were randomly divided into 4 groups: glutamate group (G), hypoxic group (H), hypoxia + glutamate group (H + G) and the control (C). Cells of control group were exposed to normoxic (95% air, 5% CO2) condition, and cells of G and H + G were incubated with 100 micromol/L L-glutamate, cells of H and H + G exposed to hypoxic conditions (5% CO2, 95% N2) at 37 degrees C. Each group had five timepoints which included 0 h, 3 h, 6 h, 12 h, 24 h, respectively. Expression of mRNAs of iNOS were detected with reverse transcription polymerase chain reaction (RT-PCR).
RESULTSExpression of iNOS mRNA was not detectable in G and C, while it increased dramatically and continuously from 6 h to 24 h in H and G + H. Expression of iNOS mRNA was significantly higher in H than both in G and C at 6 h, 12 h and 24 h, and expression of iNOS mRNA was the highest of all groups in G + H.
CONCLUSIONHypoxia upregulates the expression of iNOS mRNA in cultured astrocytes. Glutamate does not induce the expression of iNOS mRNA but enhance the effect of hypoxia, which is maybe one of the adaptive mechanisms of hypoxia-induced cerebral dilation.
Animals ; Animals, Newborn ; Astrocytes ; drug effects ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Cerebral Cortex ; cytology ; Glutamic Acid ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats