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

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

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

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

To examine the role and effect of nitric oxide synthase type II (NOS II) in cirrhotic rats, expression of NOS II mRNA was detected by real time RT-PCR. The enzymatic activity of nitric oxide synthase and the circulating levels of NO, systemic and portal hemodynamics and quantification of cirrhosis were measured. Chinese traditional medicine was used to treat cirrhotic rats and the effect of NO was evaluated. Double-blind method was used in experiment. Our results showed the concentration of NO and the enzymatic activity of NOS increased markedly at all stages of cirrhosis and iNOSmRNA was strongly expressed. Meanwhile, the portal-venous-pressure (PVP) and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the degree of hepatic fibrosis. Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA. Our results suggested that increased hepatic expression of NOS II is one of the important factors causing cirrhosis and portal hypertension. Tetrandrine can significantly ameliorate cirrhosis and portal hypertension.
Alkaloids ; pharmacology ; therapeutic use ; Animals ; Benzylisoquinolines ; pharmacology ; therapeutic use ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypertension, Portal ; drug therapy ; metabolism ; Liver ; enzymology ; pathology ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; Male ; Nitric Oxide ; antagonists & inhibitors ; blood ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Nitric Oxide Synthase Type II ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

The effect of extracellular beta-(1-->3), (1-->6)-glucan, produced by Paenibacillus polymyxa JB115, on nitric oxide (NO) production in RAW264.7 macrophages was investigated. beta-glucan induced the production of NO by RAW264.7 macrophages in a concentration- and time-dependent manner. Moreover, beta-glucan stimulation increased the mRNA expression of iNOS, COX-2 and IL-6 in RAW264.7 macrophages in a concentration-dependent manner.
Animals ; Bacillus/*metabolism ; Cell Line ; Cyclooxygenase 2/biosynthesis/genetics ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/*drug effects/enzymology/immunology ; Mice ; Nitric Oxide/*biosynthesis/immunology ; Nitric Oxide Synthase Type II/biosynthesis/genetics/metabolism ; RNA, Messenger/biosynthesis/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; beta-Glucans/metabolism/*pharmacology

OBJECTIVETo observe the effect of IPS-B2 on mouse peritoneal macrophages and the transcription of IL-1beta, IL-6, TNF-alpha and iNOS.

METHODELISA method and Griess method were used to detect the effect of mouse peritoneal macrophages produce cytokines IL-1beta, IL-6, TNF-alpha and cytotoxic effectors NO. The transcription of IL-1beta, IL-6, TNF-alpha and iNOS was detected by real-time RT-PCR method.

RESULTIPS-B2 could not promote mouse peritoneal macrophage production, but it could significantly improve the IL-1beta, IL-6, TNF-alpha content in mouse peritoneal macrophages culture supernatant, and increase the gene expression of IL-1beta, IL-6, TNF-alpha and iNOS.

CONCLUSIONIPS-B2 can enhance the ability of peritoneal macrophages to excrete bioactive substances and promote the transcription of bioactive substances to antitumor.

Agaricales ; chemistry ; Animals ; Gene Expression Regulation ; drug effects ; Interleukin-1beta ; biosynthesis ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Macrophages, Peritoneal ; drug effects ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Polymerase Chain Reaction ; Polysaccharides ; pharmacology ; RNA, Messenger ; biosynthesis ; Transcription, Genetic ; drug effects ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo investigate the effects of irbesartan for heart protection and on heart nitric oxide (NO) system in diabetic rats.

METHODSThirty adult male Wistar rats were randomly divided into three equal groups, namely control group, diabetes group and irbesartan group. Streptozotocin (STZ, 50 mg/kg) was injected to the abdomen to induce diabetes in the rats. After treatment for 12 weeks, the rats were sacrificed and the urine volume, body weight, ratio of heart to body weight, plasma glucose and glycosylated hemoglobin (HbA1c) were measured. NO levels in the serum and myocardium were determined. Inducible nitric oxide synthase (iNOS) expression was determined by immunohistochemistry, and iNOS mRNA detected by RT-PCR.

RESULTSUrine volume, ratio of heart to body weight, plasma glucose, HbA1C, NO levels in the urine, blood and myocardium in diabetic and irbesartan rats were significantly greater than those of normal controls (P<0.05). The ratio of heart to body weight and NO levels of urine, serum and heart tissue in rats of irbesartan group were significantly decreased as compared with those of diabetes rats (P<0.05). Myocardium iNOS mRNA and protein expression decreased significantly in irbesartan group, but not in diabetes group.

CONCLUSIONSThe abnormality in NO and iNOS mRNA expression might be related to diabetic cardiomyopathy. Irbesartan can decrease iNOS mRNA and protein expressions and reduce NO levels in STZ-induced diabetic rats.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Biphenyl Compounds ; pharmacology ; Diabetes Mellitus, Experimental ; enzymology ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Immunohistochemistry ; Male ; Myocardium ; enzymology ; metabolism ; Nitric Oxide ; blood ; metabolism ; urine ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazoles ; pharmacology

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.
Anti-Inflammatory Agents/immunology/pharmacology ; Cell Line, Tumor ; Cyclic GMP/analogs & derivatives/immunology/metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors/metabolism ; Humans ; Interleukin-1beta/*metabolism ; Male ; Nitric Oxide/*biosynthesis/genetics/immunology ; Nitric Oxide Synthase Type II/*biosynthesis/genetics/immunology ; Phosphodiesterase Inhibitors/immunology/*pharmacology ; Piperazines/immunology/*pharmacology ; Purines/immunology/pharmacology ; Signal Transduction/drug effects/genetics/immunology ; Sulfones/immunology/*pharmacology ; Synovial Membrane/enzymology/immunology

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappa B induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.
Animals ; Cell Line ; Cyclooxygenase 2/*metabolism ; Cysteamine/*analogs & derivatives ; Enzyme Activation ; Humans ; Lipopolysaccharides/*pharmacology ; Macrophages/*metabolism ; Mice ; NF-kappa B/metabolism ; Nitric Oxide/*biosynthesis ; Nitric Oxide Synthase Type II/metabolism ; PTEN Phosphohydrolase/*genetics ; Peptides/*genetics ; Recombinant Fusion Proteins/*biosynthesis/genetics ; Signal Transduction