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

The aim of this study was to determine the effect of dexamethasone (DEX) on renal ischemia/reperfusion injury (IRI). C57BL/6 mice were randomly divided into Sham group, IRI group and DEX group. The mice in IRI and DEX groups subjected to renal ischemia for 60 min, were treated with saline or DEX (4 mg/kg, i.p.) 60 min prior to I/R. After 24 h of reperfusion, the renal function, renal pathological changes, activation of extracellular signal-regulated kinase (ERK) and glucocorticoid receptor (GR), and the levels of iNOS and eNOS were detected. The results showed DEX significantly decreased the damage to renal function and pathological changes after renal IRI. Pre-treatment with DEX reduced ERK activation and down-regulated the level of iNOS, whereas up-regulated the level of eNOS after renal IRI. DEX could further promote the activation of GR. These findings indicated GR activation confers preconditioning-like protection against acute IRI partially by up-regulating the ratio of eNOS/iNOS.
Animals ; Dexamethasone ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Glucocorticoids ; pharmacology ; Male ; Mice ; Nitric Oxide Synthase Type II ; biosynthesis ; Nitric Oxide Synthase Type III ; biosynthesis ; Receptors, Glucocorticoid ; agonists ; Reperfusion Injury ; enzymology ; pathology ; Up-Regulation ; drug effects

To explore the effects of bilirubin on alveolar macrophages (AM) and expression of iNOS and NO in them in emphysema model, the rats were pretreated with bilirubin before exposed to smoke. AM were isolated from bronchoalveolar lavage fluid (BALF) and cultured. Pathological microscopic examination of AM and immunohistochemical analysis of iNOS were performed. Nitric oxide (NO) content in the samples was determined by nitrate reductase technique. The results showed both alveoli and alveolar septum appeared normal in size and shape in normal group. AM showed kidney-shaped nucleus and were rich in Golgi complexes and primary lysosomes in the cytoplasm. The inner membrane of mitochondrion was continuous. Most cristae of the mitochondria were intact. In model group, the alveoli were expanded, ruptured and bullaes were formed. Both the population and sizes of AM increased significantly. Secondary lysosomes were rich in the cytoplasm. Deformation and pyknosis of the nucleus, swelling of the mitochondrions and rupture of the inner mitochondrial membrane could also be seen. At high magnification, most of the mitochondrial cristae were broken, or completely lost at certain points. In bilirubin group, alveoli partly expanded and the population of AM also increased, with morphological changes being slighter than that in model group. Both NO contents and expression of iNOS in model group were higher than those in normal group (P<0.05). In bilirubin group the two indice were lower than those in model group (P<0.05). Our findings suggested that high expression of iNOS and high NO content in AM accelerate the development of emphysema associated with smoking in rats. Bilirubin may exert protective effects on AM and retards the development of emphysema in rats.
Animals ; Antioxidants ; pharmacology ; Bilirubin ; pharmacology ; Cells, Cultured ; Emphysema ; metabolism ; pathology ; Female ; Macrophages, Alveolar ; drug effects ; metabolism ; pathology ; Male ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; biosynthesis ; Nitric Oxide Synthase Type II ; Rats ; Rats, Wistar

OBJECTIVETo evaluate the effect of endogenous nitric oxide (NO) on the ability of 5-fluourouracil (5-FU) to induce apoptosis in the liver carcinoma Bel7402 cell line, and to observe the anti-tumor mechanism and effective adjuvant of 5-FU.

METHODSCells were cultured under routine conditions with Dulbecco's modified Eagle's medium (DMEM) without L-Arginine (L-Arg). We observed the expression of inducible nitric oxide synthase (iNOS) and apoptosis of cells induced by 5-FU with L-Arg added to the medium. The production of nitric oxide was determined by the cell expression of iNOS detected by immunohistochemical staining, and by the concentrations of nitrite and nitrate in the supernatant.

RESULTS5-fluourouracil significantly increased the iNOS expression to 0.1687 +/- 0.01968 (P < 0.05, vs control group), and the concentration of nitric oxide to 213 +/- 30.2 micromol/L (P < 0.05, vs control group). The apoptotic cell rate increased significantly to 17.85 +/- 0.78%, while the necrotic cell rate decreased to 3 2.99 +/- 0.83% (P < 0.05,compared with the 5-FU group). N(omega)-nitro-L-Arginine methyl ester (L-NAME), the antagonist of L-Arg, can block the apoptotic effects of endogenous nitric oxide.

CONCLUSIONS5-FU had a synergistic effects with L-Arg by increasing the production of endogenous nitric oxide. Endogenous nitric oxide plays an important role in the process where 5-FU induces apoptosis in liver carcinoma cells. L-Arg may be a good adjuvant for chemotherapy with 5-FU.

Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Arginine ; pharmacology ; Fluorouracil ; pharmacology ; Humans ; Immunohistochemistry ; Liver Neoplasms ; pathology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; analysis ; Nitric Oxide Synthase Type II ; Tumor Cells, Cultured

The role of nitric oxide (NO) in the ocular surface remains unknown. We investigated the conditions leading to an increase of NO generation in tear and the main sources of NO in ocular surface tissue. We evaluated the dual action (cell survival or cell death) of NO depending on its amount. We measured the concentration of nitrite plus nitrate in the tears of ocular surface diseases and examined the main source of nitric oxide synthase (NOS). When cultured human corneal fibroblast were treated with NO producing donor with or without serum, the viabilities of cells was studied. We found that the main sources of NO in ocular surface tissue were corneal epithelium, fibroblast, endothelium, and inflammatory cells. Three forms of NOS (eNOS, bNOS, and iNOS) were expressed in experimentally induced inflammation. In the fibroblast culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblast cells caused by serum deprivation in a dose dependent manner up to 500 micrometer SNAP, but a higher dose decreased cell viability. This study suggested that NO might act as a doubleedged sword in ocular surface diseases depending on the degree of inflammation related with NO concentration.
Animals ; Apoptosis/drug effects/physiology ; Aqueous Humor/metabolism ; Blood Proteins/pharmacology ; Cell Survival/drug effects/physiology ; Cells, Cultured ; Epithelium, Corneal/*cytology/*enzymology ; Fibroblasts/cytology/enzymology ; Humans ; Nitric Oxide/biosynthesis/*physiology ; Nitric Oxide Donors/pharmacology ; Nitric Oxide Synthase/metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Penicillamine/*analogs & derivatives/pharmacology ; Peroxynitrous Acid/biosynthesis ; Rabbits ; Tears/metabolism ; Uveitis/metabolism

OBJECTIVETo evaluate the effects of taurine in diet on the expression of inducible nitric oxide synthase (iNOS) in rat lung induced by silica.

METHODSWistar rats were established by direct tracheal instillation of silica into rat lungs exposed surgically, and the animals of taurine-treated group were silica-instilled with a diet containing taurine. The taurine concentration of serum was analyzed by means of HPLC. The expression of iNOS protein in paraffin-embedded lung sections with Streptavidin/peroxidase (SP) immunohistochemistry on tissue microarray was measured by Image-Pro Plus.

RESULTSThe concentration of taurine in serum of taurine-treated group was significantly higher than those in saline-treated and silica-treated groups (P < 0.05). The activities of total NOS and iNOS in BALF supernatant and iNOS positive area percentage of rat lung in silica-treated group were at the peak on 14th day, which were 1.84 U/ml, 1.12 U/ml and 5.42% more respectively than those in saline-treated group (P < 0.05). There were no significant differences between taurine-treated group and silica-treated group in total NOS and iNOS activities of BALF supernatant, and iNOS positive area of the lung (P > 0.05).

CONCLUSIONTreatment with taurine hardly influences on the increase in expression of nitric oxide synthase in rat lung induced by silica dust.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Female ; Lung ; drug effects ; enzymology ; Male ; Nitric Oxide Synthase Type II ; biosynthesis ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Taurine ; blood ; pharmacology

OBJECTIVETo observe effect of porcine relaxin(pRLX) on NO production of human microvascular endothelial cells(HMVECs) and discuss its possible mechanism.

METHODSiNOS and cNOS expression of HMVECs with or without pRLX were detected using western blotting. NO production of HMVECs with pRLX at different concentration or different time were determined by method of Griess. NO production of pRLX of HMVECs plus Non-selective NOS inhibitor NG-monomethyl-L-arginine(L-NMMA), selective iNOS inhibitor aminoguanidine(AG) or nuclear factors-kappaB (NF-kappaB) inhibitor pyrrolidine dithiocarbamate(PDTC) were also analysed.

RESULTSpRLX promoted iNOS protein expression of HMVECs, but not cNOS protein expression. NO production of HMVECs was promoted by pRLX on concentration-dependent pattern instead of time-dependent one. AG, L-NMMA and PDTC were showed to block the effect of pRLX on NO production of HMVECs.

CONCLUSIONpRLX promote iNOS expression and NO production of HMVECs.

Animals ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Lung ; blood supply ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; biosynthesis ; Relaxin ; pharmacology ; Swine ; Time Factors

OBJECTIVETo explore the influence of lanthanum chloride (LaCl3) on inducible nitric oxide synthase (iNOS) expression in RAW264.7 macrophages with lipopolysaccharide (LPS) induction, and to investigate its possible mechanisms.

METHODSThe RAW264.7 macrophages were randomly divided into four groups: i. e, control group (without treatment), LaCl3 group (with treatment of 2.5 micromol/L of LaCl3 for 24 hrs), LaCl3 + LPS group (with treatment of 2.5 micromol/L LaCl3 for 24h), and LPS group (with treatment of 1 mg/L LPS for 24 hrs). The iNOS protein expression was measured by immunofluorescence and Western blot. iNOS gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). NO production in culture supernatant was assayed by nitrate reductase method.

RESULTSImmunofluorescence analysis showed that iNOS was located mainly in the cytoplasm. RAW264.7 cells with overexpression of iNOS accounted for 44.4%, which was obviously higher than that in LaCl3 + LPS group (11.8%, P < 0.05). There was a faint signal of FITC-labeled green tint in control group or LaCl3 group. The iNOS mRNA and protein expression, and the NO content in LPS group were significantly higher than those in control, LaCl3, and LaCl3 + LPS groups (P < 0.05).

CONCLUSIONLaCl3 can suppress LPS-induced iNOS overexpression at mRNA and protein level and reduce NO production, indicating that LaCl3 can antagonize the excessive activation of iNOS induced by LPS.

Animals ; Cell Line ; Lanthanum ; pharmacology ; Lipopolysaccharides ; toxicity ; Macrophages ; drug effects ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

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

OBJECTIVE: To investigate the effect of cardiomyopeptidin for injection on energy metabolism in isolated hearts of young rats after ischemia-reperfusion injury. METHODS: Fifty young healthy SD rats(aged 20 +/- 3 days and weighing 50-70 g) were randomly divided into 5 groups: a normal control group (NC group, n = 10 ): the isolated hearts were stable for 20 min, and then 150 min continuous perfusion; a normal + cardiomyopeptidin group (NCMP group, n = 10): the same as the normal control group, but K-H buffer solution was added with 50 mg/L cardiomyopeptidin, and 3 ischemia-reperfusion injury model groups, including a model control group (n = 10): the isolated rat hearts were perfused with K-H buffer and then arrested with cardioplegic solution; a CMP1 group (n = 10): the ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin; CMP2 group (n=10): K-H buffer and ST.Thomas'II cardioplegic solution was added with 50 mg/L and 100 mg/L cardiomyopeptidin respectively. The cardiac functional indexes were monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow. In the 3 ischemia-reperfusion injury model groups, myocardial ultrastructure was observed through transmission electron microscopy; the creatine kinase isoenzyme (CK-MB) concentration was measured in the fluid outflow of coronary; the content of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, total ATPase, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS) and aldosereductase were measured in the myocardium tissue; the relative expression levels of iNOS, eNOS, and Akr1b4 mRNA in the myocardial tissue were also detected by real-time fluorescence quantitative PCR. RESULTS: In the NC group, after prolonged perfusion, the cardiac function of isolated hearts had no significant change. Cardiomyopeptidin for injection had no significant effect on normal isolated hearts. Compared with the model control group, the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin decreased much less. Cardiac myofibrillar fragmentation and mitochondrial swelling were observed in the control group, while in the CMP groups, the myocardial structure was nearly complete, and only mild mitochondria swelling and degeneration could be seen. After the reperfusion, the content of CK-MB was increased in the control group. Compared with the model control group, the CK-MB content was lower in the CMP1 and CMP2 groups. There was a slight decline in the contents of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, and Total ATPase in the CMP1 and CMP2 groups, and an increase in SOD activity (P < 0.01 or P < 0.05). The concentration of NO and MDA produced after the ischemia-reperfusion injury was much lower in the CMP1 and CMP2 groups. The activity of iNOS and aldosereductase was inhibited, the expression levels of iNOS, and Akr1b4 mRNA were significantly down-regulated in the CMP1 and CMP2 groups. These changes were more prominent in the CMP2 group (P < 0.01 or P < 0.05). The eNOS mRNA levels in the CMP2 group was up-regulated (P < 0.05). CONCLUSION Cardiomyopeptidin for injection may improve the energy metabolism, improve coronary blood flow and cardiac function after the reperfusion, thus protecting immature myocardial against ischemia-reperfusion injury in young rats. Administration of it in both K-H buffer and ST.Thomas'II cardioplegic solution is better than adding it in cardioplegic solution alone. The mechanism may be associated with the inhibition the mRNA expression of iNOS and Akr1b4 in cardiomyocytes, the inhibition activity of iNOS and aldosereductase, and the decrease of NO production.
Aldehyde Reductase ; genetics ; metabolism ; Animals ; Energy Metabolism ; drug effects ; Female ; In Vitro Techniques ; Male ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; metabolism ; ultrastructure ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peptides ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley