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 investigate the antioxidant and anti-endotoxin effects of propofol on endothelial cells and the possible mechanisms.

METHODSCultured endothelial cells were treated with hydrogen peroxide (H(2)O(2)), propofol + H(2)O(2), lipopolysaccharide (LPS) and propofol + LPS, respectively. Endothelial cell damage was monitored for possible lactic dehydrogenase (LDH) release. The transcription and the protein expression levels of endothelial nitric oxide synthase (eNOS) were measured.

RESULTSLDH release was higher in groups treated with H(2)O(2) or LPS than in the control group. After pretreatment with propofol, the effects induced by H(2)O(2) were attenuated, but propofol did not decrease the LDH release induced by LPS. Both H(2)O(2) and LPS significantly increased the eNOS transcript levels and the increases were significantly attenuated after pretreatment with propofol. Both H(2)O(2) and LPS significantly increased the eNOS protein expression and the increase was attenuated after pretreatment with propofol.

CONCLUSIONPropofol could protect endothelial cells against oxidative stress by inhibiting eNOS transcription and protein expression, but could not antagonise endotoxin induced cell injuries.

Antioxidants ; pharmacology ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Endotoxins ; antagonists & inhibitors ; Free Radical Scavengers ; pharmacology ; Humans ; In Vitro Techniques ; Lipopolysaccharides ; pharmacology ; Nitric Oxide ; pharmacology ; Nitric Oxide Synthase ; biosynthesis ; Nitric Oxide Synthase Type III ; Propofol ; pharmacology

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 ; Brain ; metabolism ; Brain Injuries ; metabolism ; Enzyme Inhibitors ; pharmacology ; Guanidines ; pharmacology ; Neuroprotective Agents ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Rats

OBJECTIVETo investigate the vascular effect of acute and chronic treatment of interferon-alpha (IFN-alpha) in rat aortic rings.

METHODSIsolated thoracic aortic rings were mounted on the organ bath and the tension of the vessel was recorded.

RESULTSIFN-alpha(10, 100, 1,000 and 10,000 U/ml) caused concentration -dependent relaxation of endothelium-intact aorta rings preconstricted with phenylephrine (PE,10(-6)mol/L), to(90.1+/-0.91)%, (65.1+/-5.21)%, (39.5+/-8.22)% and (35.3+/-8.27)% of pre-drug control, respectively. Removal of the endothelium inhibited the relaxation by IFN-alpha. The vasorelaxant effect of IFN-alpha (100 U/ml ) was attenuated by pretreatment with L-NAME (10(-4)mol/L), methylene blue (10(-5)mol/L) or AMG (10(-4)mol/L), to (97.2+/-5.34)%, (95.1+/-6.25)% and (93.7+/-8.82)% of the control, respectively. Pretreatment with IFN-alpha (1,000,000 U/d, i.p.) for five days markedly inhibited the endothelium-dependent relaxation of the aortic rings to acetylcholine. But the endothelium-dependent relaxation to acetylcholine was not changed by pretreatment of IFN-alpha (10,000 U/ml) with the isolated aorta rings for 2 h.

CONCLUSIONThe vasorelaxation induced by IFN-alpha in rat aorta rings is endothelium-dependent and is possibly mediated by inducible nitric oxide synthase. Chronic treatment of IFN-alpha may impair the endothelium or NO-sGC pathway.

Acetylcholine ; pharmacology ; Animals ; Aorta, Thoracic ; drug effects ; physiology ; Endothelium, Vascular ; physiology ; Guanylate Cyclase ; physiology ; Interferon-alpha ; pharmacology ; Male ; Nitric Oxide ; physiology ; Nitric Oxide Synthase ; physiology ; Nitric Oxide Synthase Type II ; Phenylephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Vasodilation ; drug effects

BACKGROUNDThis study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.

METHODSMouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor kappaB (NF-kappaB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.

RESULTSM. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-kappaB activation, a possible mechanism for the induction of iNOS expression.

CONCLUSIONThis study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-kappaB.

Animals ; Bacterial Proteins ; pharmacology ; Cells, Cultured ; Enzyme Induction ; Lipoproteins ; pharmacology ; Membrane Proteins ; pharmacology ; Mice ; Mycoplasma penetrans ; chemistry ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; biosynthesis ; Nitric Oxide Synthase Type II ; RNA, Messenger ; analysis

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

In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expressions in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P > 0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal transduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.
Berberine/*pharmacology ; Connective Tissue/physiopathology ; Nitric Oxide Synthase/*biosynthesis ; Nitric Oxide Synthase/genetics ; Nitric Oxide Synthase Type I/biosynthesis ; Nitric Oxide Synthase Type I/genetics ; Nitric Oxide Synthase Type III/biosynthesis ; Nitric Oxide Synthase Type III/genetics ; Penile Erection/*physiology ; Penis/*metabolism ; Penis/physiology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics

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

AIMTo investigate the effects of L-arginine liposome on nitric oxide(NO) and nitric oxide synthase gene (NOS mRNA) in rats chronically exposed to hypoxia-hypercapnia.

METHODSFourty male SD rats were randomly divided into four groups (n=10): normal control group(NC), hypoxia-hypercapnia group (HH), hypoxia-hypercapnia + L-arginine group(HL) and hypoxia-hypercapnia + L-arginine liposome group (HP). Contents of NO in plasma were measured using colorimetric analysis. Expression of nitric oxide synthase gene were measured with situ hybridization.

RESULTS(1) The mean pulmonary artery pressure(mPAP) and weight ratio of right ventricle to left ventricle and septum(RV/LV + S) of HP group were obviously lower than those of HH group and HL group. (2) The NO contents in plasma of HP group were obviously higher than those of HH group and HL group (P < 0.01). (3) Situ hybridization showed the average value of integral light density(LD) of ecNOS mRNA in pulmonary arterioles was significantly higher in rats of HP group than that of HH group and HL group (P < 0.01). (4) Light microscopy showed WA/TA (vessel wall area/total area) and PAMT (media thickness ratio of pulmonary arterioles) were significantly lower in rats of HP group than those of HH group (P < 0.01).

CONCLUSIONL-arginine liposome could lower the mPAP and lighten the remodeling of pulmonary arterioles of the rats chronically exposed to hypoxia-hypercapnia than L-arginine does. It suggests that L-arginine liposome significantly promotes the membrane transportin of L-arginine.

Animals ; Arginine ; pharmacology ; Gene Expression ; Hypercapnia ; metabolism ; Hypoxia ; metabolism ; Liposomes ; pharmacology ; Male ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley