OBJECTIVETo study the effects and mechanisms of Zuoguiyin (ZGY) on the ovarian nitric oxide (NO) production in peri-menopausal rats.

METHODSThe peri-menopausal model rats were respectively administered with low (13.78 g/kg), middle (20.67 g/kg), and high (31.00 g/kg) dose ZGY, and nilestriol for 8 weeks. Normal saline was given by gastrogavage to rats in the model group and the young control group (as the control group). The ovarian NO content and the activity of total nitric oxide synthase (NOS) were detected using nitrate reductase method and chemical colorimetry respectively. The mRNA and protein expressions of inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) were detected using RT-PCR and immunohistochemical assay.

RESULTS(1) Compared with that in the control group, the ovarian NO content and the activity of total NOS in peri-menopausal rats were significantly lower (P < 0.01). Middle and high dose ZGY could obviously up-regulate them (P < 0.01, P < 0.05). (2) The three kinds of NOS expression levels in perimenopausal rats were obviously lower when compared with those of the control group (P < 0.01). Middle dose ZGY could significantly promote all the three kinds of NOS expression levels of pre-senile rats (P < 0.01). High dose ZGY could up-regulate the expressions of iNOS and eNOS, while low dose ZGY could only enhance the iNOS expression (P < 0.01).

CONCLUSIONSThe down-regulated expressions of eNOS, iNOS, and nNOS in local ovaries resulted in decreased NOS activity and NO production, which were closely correlated with damaged microcirculatory vascular functions of ovaries in peri-menopausal rats. ZGY could protect rats' ovarian microcirculation by up-regulating the expressions of eNOS, iNOS, and nNOS, and enhancing the ovarian NOS activity and NO production.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Ovary ; drug effects ; metabolism ; Perimenopause ; Rats

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

Animals ; Female ; Isocyanates ; toxicity ; Male ; Malondialdehyde ; metabolism ; Mice ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; Superoxide Dismutase ; metabolism ; Testis ; drug effects ; metabolism

OBJECTIVE: To observe the effect of thrombin on the cytotoxicity of astrocytes injured by hypoxia/reoxygenation(H/R) and to explore its relationship with inducible nitric oxide synthase (iNOS). METHODS: Primary astrocytes were cultured in DMEM with 10% approximately 15% calf serum and divided into 6 groups: a control group, a Tm control group, an H/R group, a Tm+H/R group, a hirudin (HR) control group, and a Tm+HR+ H/R group. The cell damage and viability were detected by the 3-(4, 5-di-methyl-thazol-2-yl)-2, 5 diphenyl-tetrazol-iumbromide (MTT) conversion method. The NO level in the cultured cell supernatant was assayed by Griess reagent. The flow cytometry was performed to evaluate the apoptosis rate of astrocytes. The iNOS mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemistry was used to observe the expression of iNOS protein. RESULTS: The cell viability injured by H/R was lower than that of the control group, the NO production and apoptosis rate in the cell of H/R group were higher than those of the control group. Incubation of H/R cell with 10kU/L Tm enhanced the cytotoxicity of H/R stimulation compared with the cells injured by H/R. Hirudin can reverse the effect of thrombin. RT-PCR and immunocytochemistry analysis demonstrated that the levels of iNOS mRNA and iNOS protein increased in the cells treated by H/R. Tm enhanced the expression of iNOS mRNA and iNOS protein in the cells treated by H/R. Hirudin blocked the effect of Tm. CONCLUSION Increasing the level of iNOS and enhancing the production of NO may be the mechanism of thrombin cytotoxicity in astrocytes injured by H/R.
Animals ; Apoptosis ; drug effects ; Astrocytes ; cytology ; drug effects ; Cell Hypoxia ; Cells, Cultured ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Sprague-Dawley ; Thrombin ; pharmacology

OBJECTIVETo detect the protective effect of ligustrazine hydrochloride on homocysteine-injured ECV304 cells.

METHODIn the in vitro study, human umbilical vein endothelial cells were selected as objects, with homocysteine as the molding agent, to judge the injury degree by monitoring NOS and NO contents. Based on that, the best homocysteine concentration in ECV304 cells, the best reaction time could be determined, and an endothelial cell injury model was established. After adding ligustrazine hydrochloride, NOS and NO contents in injured endothelial cells were determined to observe the protective effect of ligustrazine hydrochloride.

RESULTIt was proved that the optimal concentration of homocysteine on injured ECV304 cell was 1 mmol x L(-1), the best reaction time was 48 h. An injured endothelial cell model was established. At the same time, positive drug nitroglycerin and ligustrazine hydrochloride displayed a protection effect on injured ECV304 cells, NOS and NO formation were significantly increased compared with the model group.

CONCLUSIONLigustrazine hydrochloride has a protective effect on homocysteine-injured ECV304 cells. The model established in this study can be used to screen anti-myocardial ischemia drugs targeting at an endothelial cell protective agent.

Cytoprotection ; drug effects ; Homocysteine ; adverse effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; biosynthesis ; Pyrazines ; pharmacology

OBJECTIVETo investigate the effects of glycosides of tripterygium wilfordii (GTW), methyltestosterone and Zhuanggushenjin capsule on nitric oxide synthase (NOS) in rat testes.

METHODSForty-five rats were equally divided into 5 groups, and respectively given GTW [10 mg/(kg x d)], methyltestosterone [2 mg/(kg x d)], Zhuanggushenjin capsule [0.3 g/(kg x d)], distilled water plus Tween 80 (control I), and distilled water alone (control II) for 4 weeks. At the end of the 5th week, the immunochemical ABC method was used to observe the effects of the three drugs on the NOS positive Leydig cells of the rats.

RESULTSCompared with control II, the GTW group had a significant decrease in the numbers of nNOS and eNOS positive Leydig cells, the methyltestosterone group showed an increase in the number of nNOS but a decrease in that of eNOS positive Leydig cells, and the Zhuanggushenjin group had an increase in the numbers of both nNOS and eNOS positive Leydig cells.

CONCLUSIONGTW can reduce NO production by inhibiting eNOS and nNOS, and hence influence the spermatogenic process. Zhuanggushenjin capsule plays an important role in improving male sexual function by enhancing nNOS and eNOS expression and NO synthesis.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Leydig Cells ; drug effects ; enzymology ; Male ; Methyltestosterone ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type I ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis ; drug effects ; Tripterygium

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

The aim of this study was to investigate the effect of urotensin II (U II) on the nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. The endothelial nitric oxide synthase (eNOS) mRNA expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction. The activity of nitric oxide synthase (NOS) and NO content in cardiomyocytes were measured. The current results showed that U inhibited eNOS mRNA expression, the NOS activity and the NO production of cardiomyocytes. U II (0.1 micromol/L) inhibited the NOS activity and the NO production in cardiomyocytes in a time-dependent manner. These results suggest that the cardiovascular effect of U II might be partially associated with NO production in cultured neonatal rat cardiomyocytes.
Animals ; Animals, Newborn ; Cells, Cultured ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Urotensins ; pharmacology

OBJECTIVEN-Acetylcysteine (NAC) is a sulfhydryl donor molecule with antioxidant and antiinflammatory effects. A major role has been described for inducible nitric oxide (NO) synthase in several inflammatory liver diseases. NAC attenuates NO generation following lipopolysaccharide injection in rats. The purpose of this study was to investigate the effect of NAC against lipopolysaccharide injury in hepatocytes of neonatal mice and the molecular mechanisms by which NAC influences inflammatory responses of the hepatocytes.

METHODSThe liver of neonatal mouse was digested by collagenase to dissociate the hepatocytes. The hepatocytes were cultured and isolated. After 7 days of culture the normal hepatocytes were divided into two groups: LPS group and NAC group. In LPS group, 10 microg/ml LPS was added into the culture medium. In NAC group, 5 mmol/L NAC was added into the culture medium firstly, 10 microg/ml LPS was added after 1 h of culture. There were 12 mice in each group. The cell supernatants and the hepatocytes were collected at 0, 6 and 12 hours after adding LPS. The cell supernatants were taken to measure the alanine aminotransferase (ALT) level and nitric oxide (NO) production by the biochemical methods. The cells were taken to analyze the gene expression of induced nitric oxide synthase (iNOS) by the RT-PCR.

RESULTSIn LPS group, the levels of ALT, NO and iNOS mRNA increased significantly at the time points 6 h and 12 h compared with the time point 0, (P < 0.01). Compared with the LPS group, the levels of ALT, NO and iNOS mRNA of NAC group were lower at the time points 6 h and 12 h (P < 0.01).

CONCLUSIONSNAC may play a protective role in the hepatocytes injury caused by LPS in the neonatal mice. The protective mechanism works partially through the inhibition of iNOS activation by LPS.

Acetylcysteine ; pharmacology ; Alanine Transaminase ; metabolism ; Animals ; Animals, Newborn ; Anti-Inflammatory Agents ; pharmacology ; Cells, Cultured ; Hepatocytes ; drug effects ; Lipopolysaccharides ; Mice ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism

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