This study was designed to determine the inhibitory effect of astragaloside Ⅳ(AS-Ⅳ), a principal bioactive component extracted from the Chinese medicinal Astragali Radix, on the inflammatory response of vascular endothelial cells induced by angiotensin Ⅱ(Ang Ⅱ), the most major pathogenic factor for cardiovascular diseases, and to clarify the role of calcium(Ca~(2+))/phosphatidylinosi-tol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO) pathway in the process. To be specific, human umbilical vein endothelial cells(HUVECs) were cultured in the presence of AS-Ⅳ with or without the specific inhibitor of NO synthase(NG-monomethyl-L-arginine, L-NMMA), inhibitor of PI3K/Akt signaling pathway(LY294002), or Ca~(2+)-chelating agent(ethylene glycol tetraacetic acid, EGTA) prior to Ang Ⅱ stimulation. The inhibitory effect of AS-Ⅳ on Ang Ⅱ-induced inflammatory response and the involved mechanism was determined with enzyme-linked immunosorbent assay(ELISA), cell-based ELISA assay, Western blot, and monocyte adhesion assay which determined the fluorescently labeled human monocytic cell line(THP-1) adhered to Ang Ⅱ-stimulated endothelial cells. AS-Ⅳ increased the production of NO by HUVECs in a dose-and time-dependent manner(P<0.05) and raised the level of phosphorylated eNOS(P<0.05). The above AS-Ⅳ-induced changes were abolished by pretreatment with L-NMMA, LY294002, or EGTA. Compared with the control group, Ang Ⅱ obviously enhanced the production and release of cytokines(tumor necrosis factor-α, interleukin-6), chemokines(monocyte chemoattractant protein-1) and adhesion molecules(intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), and the number of monocytes adhered to HUVECs(P<0.05), which were accompanied by the enhanced levels of phosphorylated inhibitor of nuclear factor-κBα protein and activities of nuclear factor-κB(NF-κB)(P<0.05). This study also demonstrated that Ang Ⅱ-induced inflammatory response was inhibited by pretreatment with AS-Ⅳ(P<0.05). In addition, the inhibitory effect of AS-Ⅳ was abrogated by pretreatment with L-NMMA, LY294002, or EGTA(P<0.05). This study provides a direct link between AS-Ⅳ and Ca~(2+)/PI3K/Akt/eNOS/NO pathway in AS-Ⅳ-mediated anti-inflammatory actions in endothelial cells exposed to Ang Ⅱ. The results indicate that AS-Ⅳ attenuates endothelial cell-mediated inflammatory response induced by Ang Ⅱ via the activation of Ca~(2+)/PI3K/Akt/eNOS/NO signaling pathway.
Humans ; Angiotensin II/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; omega-N-Methylarginine/pharmacology* ; Egtazic Acid/pharmacology* ; Human Umbilical Vein Endothelial Cells ; NF-kappa B/metabolism* ; Nitric Oxide/metabolism* ; Cells, Cultured

The aim of this study was to explore the mechanism of the nervous system lesions induced by formaldehyde (FA). Male Balb/c mice were exposed to gaseous formaldehyde for 7 days (8 h/d) with three different concentrations (0, 0.5 and 3.0 mg/m(3)). A group of animals injected with the nitric oxide synthase inhibitor L-NMMA (0.01 mL/g) was also set and exposed to 3.0 mg/m(3) FA. The concentrations of cAMP, cGMP, NO and the activity of NOS in cerebral cortex, hippocampus and brain stem were determined by corresponding assay kits. The results showed that, compared with the control (0 mg/m(3) FA) group, the cAMP contents in cerebral cortex and brain stem were significantly increased in 0.5 mg/m(3) FA group (P < 0.05), but decreased in 3.0 mg/m(3) FA group (P < 0.05); The concentration of cAMP in hippocampus was significantly decreased in 3.0 mg/m(3) FA group (P < 0.05). In comparison with the control group, L-NMMA group showed unchanged cAMP contents and NOS activities in different brain regions, but showed increased cGMP contents in hippocampus and NO contents in cerebral cortex (P < 0.05). In addition, compared with 3.0 mg/m(3) FA group, L-NMMA group showed increased contents of cAMP and reduced NOS activities in different brain regions, as well as significantly decreased cGMP contents in cerebral cortex and brain stem and NO content in brain stem. These results suggest that the toxicity of FA on mouse nervous system is related to NO/cGMP and cAMP signaling pathways.
Animals ; Brain Stem ; chemistry ; drug effects ; Cerebral Cortex ; chemistry ; drug effects ; Cyclic AMP ; chemistry ; Cyclic GMP ; chemistry ; Formaldehyde ; toxicity ; Hippocampus ; chemistry ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; chemistry ; Nitric Oxide Synthase ; antagonists & inhibitors ; omega-N-Methylarginine ; pharmacology

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 (Tbeta4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tbeta4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tbeta4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(beta-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tbeta4 expression with Tbeta4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tbeta4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tbeta4 plasmids for Tbeta4 overexpression. Taken together, these results suggest that Tbeta4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.
Actins ; Anoxia ; Nitric Oxide Synthase ; Nitric Oxide* ; omega-N-Methylarginine ; Plasmids ; Response Elements ; RNA ; Thymosin* ; Transfection ; Uterine Cervical Neoplasms* ; Vascular Endothelial Growth Factor A*

PURPOSE: The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. MATERIALS AND METHODS: Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650Omega, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [3H]-thymidine, and [35S]-sulfate incorporation. To detect phenotypical expression, reverse transcription-polymerase chain reactions (RT-PCR) were performed for aggrecan, collagen type I, and type II mRNA expression. To assess action mechanism of EMF, IVD cells were exposed to EMF with NG-Monomethyl-L-arginine (NMMA) and acetylsalicylic acid (ASA). RESULTS: There was no cytotoxicity in IVD cells with the EMF group in MTT assay. Cellular proliferation was observed in the EMF group (p < 0.05). There was no difference in newly synthesized proteoglycan normalized by DNA synthesis between the EMF group and the control. Cultures with EMF showed no significant change in the expression of aggrecan, type I, and type II collagen mRNA compared to the control group. Cultures with NMMA (blocker of nitric oxide) or ASA (blocker of prostaglandin E2) exposed to EMF demonstrated decreased DNA synthesis compared to control cultures without NMMA or ASA (p < 0.05). CONCLUSION: EMF stimulated DNA synthesis in human IVD cells while no significant effect on proteoglycan synthesis and chondrogenic phenotype expressions. DNA synthesis was partially mediated by nitric oxide and prostaglandin E2. EMF can be utilized to stimulate proliferation of IVD cells, which may provide efficient cell amplification in cell therapy to degenerative disc disease.
Adult ; Aspirin/pharmacology ; Cell Proliferation/*radiation effects ; Collagen/metabolism ; Dinoprostone/metabolism ; *Electromagnetic Fields ; Enzyme Inhibitors/pharmacology ; Female ; Humans ; Intervertebral Disk/*pathology/radiation effects ; Male ; Middle Aged ; Nitric Oxide/metabolism ; Tetrazolium Salts/pharmacology ; Thiazoles/pharmacology ; omega-N-Methylarginine/pharmacology

Effects of quercetin, a kind of flavonoids, on the vasodilating actions were investigated. Among the mechanisms for quercetin-induced vasodilatation in rat aorta, the involvement with the Ca2+ activated K+ (KCa) channel was examined. Pretreatment with NE (5 micrometer) or KCl (60 mM) was carried out and then, the modulation by quercetin of the constriction was examined using rat aorta ring strips (3 mm) at 36.5degrees C. Quercetin (0.1 to 100 micrometer) relaxed the NE-induced vasoconstrictions in a concentration-dependent manner. NO synthesis (NOS) inhibitor, NG-monomethyl-L-arginine acetate (L-NMMA), at 100 micrometer reduced the quercetin (100 micrometer)-induced vasodilatation from 97.8+/-3.7% (n=10) to 78.0+/-11.6% (n=5, p<0.05). Another NOS inhibitor, L-NG-nitro arginine methyl ester (L-NAME), at 100 micrometer also had the similar effect. In the presence of both 100 micrometer L-NMMA and 10 micrometer indomethacin, the quercetin-induced vasodilatation was further attenuated by 100 micrometer tetraethylammonium (TEA, a KCa channel inhibitor). Also TEA decreased the quercetin-induced vasodilatation in endothelium-denuded rat aorta. Used other KCa channel inhibitors, the quercetin-induced vasodilatation was attenuated by 0.3 micrometer apamin (a SK channel inhibitor), but not by 30 nM charybdotoxin (a BK and IK channel inhibitor). Quercetin caused a concentration-dependent vasodilatation, due to the endothelium-dependent and -independent actions. Also quercetin contributes to the vasodilatation selectively with SK channel on smooth muscle.
Animals ; Aorta ; Apamin ; Arginine ; Charybdotoxin ; Constriction ; Endothelium ; Flavonoids ; Indomethacin ; Muscle, Smooth ; omega-N-Methylarginine ; Potassium Channels, Calcium-Activated ; Quercetin ; Rats ; Tea ; Tetraethylammonium ; Vasoconstriction ; Vasodilation

OBJECTIVETo detect the nitric oxide (NO) production and energy metabolism of the interleukin (IL)-1beta-treated residual hepatocytes from rats after partial hepatectomy.

METHODSForty rats were equally divided into partial hepatectomies (PH) group and control group. In the control group the rats were otherwise matched and underwent sham surgeries. The residual hepatocytes were separated by the collagenase perfusion method. The hepatocytes were cultured with cytokines such as IL-1beta. The production of NO in the two groups were measured with Griess reagent method, the production of inducible nitric oxide synthase (iNOS) protein detected with Western blot, the content of the nucleotide in the hepatocytes detected with high-performance liquid chromatography, and the content of the ketone body in the hepatocytes of the two groups determined with the enzymatic method. Afterwards the ketone body ratio (acetoacetate/beta-hydroxy butyrate, KBR) was calculated.

RESULTSThe production of NO in the PH group was twice as much as that in the Sham group. IL-1beta decreased the content of ATP and the KBR in the hepatocytes of both groups, and the decrease magni tude in the PH group was significantly larger than that in the Sham group. After the injection of L-arginine, the production of NO in the hepatocytes in the PH group increased, and the level of ATP and KBR decreased. N(G)-methyl-L-arginine (L-NMMA), the inhibitor of NO synthase, inhibited the production of NO and reversed the decrease of ATP and KBR.

CONCLUSIONAfter partial hepatectomy, increased NO production in the hepatocytes after the treatment of interleukin-1beta may disturb the function of mitochondria by inhibiting the synthesis of ATP.

Adenosine Triphosphate ; biosynthesis ; Animals ; Arginine ; pharmacology ; Cells, Cultured ; Hepatectomy ; Hepatocytes ; metabolism ; Interleukin-1beta ; pharmacology ; Ketone Bodies ; biosynthesis ; Nitric Oxide ; antagonists & inhibitors ; biosynthesis ; Nitric Oxide Synthase ; antagonists & inhibitors ; Rats ; omega-N-Methylarginine ; pharmacology

OBJECTIVETo investigate the differential expression of isocitrate lyase in Penicillium marneffei phagocytized by nonstimulated and stimulated murine macrophages, and explore the role of glyoxylate pathway in pathogenesis of Penicilliosis marneffei.

METHODSPenicillium marneffei conidia and Raw264.7 cells were incubated in 16 cultures, which were divided to 4 groups for treatment with N-monomethyl-L-arginine (LNMMA, CI group), murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) (T group), IFN-gamma plus LPS and LNMMA (TI group), or the same volume of culture medium (C group). The transcriptional levels of isocitrate lyase were detected using real-time RT-PCR, and its expression levels detected biochemically.

RESULTSThe transcriptional levels of isocitrate lyase in C, CI, T, TI groups were 1.00, 1.42, 33.09, and 74.88 (P<0.05), while the expression levels were 0.06, 0.07, 0.18, and 0.93, respectively (P<0.05). The content of nitric oxide in T group was significantly higher than that in the other groups (P<0.01), but the CFU of T group was the lowest (P<0.01).

CONCLUSIONReactive nitrogen intermediates induced by stimulated murine macrophages restrain the expression of isocitrate lyase of Penicillium marneffei and development of Penicillium marneffei, in which process the glyoxylate pathway may play an important role.

Animals ; Cell Line ; Fungal Proteins ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Fungal ; drug effects ; Host-Pathogen Interactions ; Interferon-gamma ; pharmacology ; Isocitrate Lyase ; genetics ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; immunology ; microbiology ; Mice ; Nitric Oxide ; immunology ; Penicillium ; genetics ; immunology ; physiology ; Phagocytosis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; omega-N-Methylarginine ; pharmacology

OBJECTIVETo dynamically observe the effect of Safflower Injection (SI) on mesenteric microvascular motion in vivo in rabbits, and to explore the effect of nitric oxide (NO) in the process to further investigate the action mechanism of activating blood to remove stasis of SI.

METHODSTwenty healthy male albino rabbits were intraperitoneally injected with urethane for basic anesthesia and injected with alpha-chloralose via ear marginal venous to maintain anesthesia, spontaneously ventilated via tracheotomy tube, with the in-step record of breath and blood pressure. The vasomotion was induced by noradrenaline (NA) in vivo, then the changes of vasomotion after injecting SI and N(G)-monomethyl-L-arginine (L-NMMA, a NO synthase inhibitor) were measured respectively on a TV monitor using a TV camera mounted on the microscope, and the influence of L-NMMA on effect of SI was also observed.

RESULTSL-NMMA injection alone can inhibit the NA induced vasomotion in vasoconstriction state, while SI injection alone can inhibit it in vaso-dilation state. SI could abolish the effect of L-NMMA on vasomotion but L-NMMA did not influence the effect of SI on vasomotion. CONCLUSION SI can inhibit vasomotion in vaso-dilation status, but its mechanism is not mediated by endogenous NO.

Animals ; Carthamus tinctorius ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Injections ; Male ; Mesentery ; blood supply ; Microcirculation ; drug effects ; Rabbits ; Vasodilator Agents ; administration & dosage ; pharmacology ; omega-N-Methylarginine ; administration & dosage ; pharmacology

OBJECTIVETo investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms.

METHODSApoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting. Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1. Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction.

RESULTSGastric cancer cells were significantly apoptotic after treatment with N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01). The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253 phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P < 0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P < 0.01) and a significant increase in apoptosis (P < 0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation.

CONCLUSIONSL-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.

Antineoplastic Agents ; toxicity ; Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Enzyme Inhibitors ; toxicity ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; analysis ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Nitric Oxide ; antagonists & inhibitors ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Stomach Neoplasms ; enzymology ; metabolism ; pathology ; Transfection ; omega-N-Methylarginine ; toxicity ; rho-Associated Kinases

PURPOSE: This study was carried out to elucidate the effects of nitric oxide synthase(NOS) inhibitor, NG-monomethyl-L-arginine(L-NMMA) and nitric oxide precursor, L-arginine(L-Arg) on cerebral hemodynamics and energy metabolism during reoxygenation-reperfusion(RR) after hypoxia-ischemia(HI) in newborn piglets. METHODS: Twenty-eight newborn piglets were divided into 4 groups; Sham normal control(NC), experimental control(EC), L-NMMA(HI & RR with L-NMMA), and L-Arg(HI & RR with L-Arg) groups. HI was induced by occlusion of bilateral common carotid arteries and simultaneously breathing with 8 percent oxygen for 30 mins, and followed RR by release of carotid occlusion and normoxic ventilation for one hour. All groups were monitored with cerebral hemodynamics and cytochrome aa3 (Cyt aa3) using near infrared spectroscopy(NIRS). Na+, K(+)-ATPase activity, lipid peroxidation products, and tissue high energy phosphate levels were determined biochemically in the cerebral cortex. RESULTS: In experimental groups, mean arterial blood pressure, PaO2, and pH decreased, and base excess and blood lactate level increased after HI compared to NC group(P<0.05). These variables subsequently returned to baseline after RR except pH. There were no differences among the experimental groups. In NIRS, oxidized hemoglobin(HbO2) decreased and hemoglobin(Hb) increased during HI(P<0.05) but returned to base line immediately after RR; 40 min after RR, the HbO2 had decreased significantly compared to NC group(P<0.05). Changes of Cyt aa3 decreased significantly compared to NC after HI and recovered at the end of the experiment. Significantly reduced cerebral cortical cell membrane Na+, K(+)-ATPase activity and increased lipid peroxidation products(P<0.05) were not improved with L-NMMA or L-Arg. CONCLUSION: These findings suggest that NO is not involved in the mechanism of HI and RR brain damage during the early acute phase of RR.
Anoxia ; Arginine* ; Arterial Pressure ; Brain ; Carotid Artery, Common ; Cell Membrane ; Cerebral Cortex ; Electron Transport Complex IV ; Energy Metabolism* ; Hemodynamics* ; Humans ; Hydrogen-Ion Concentration ; Hypoxia-Ischemia, Brain* ; Infant, Newborn* ; Ischemia ; Lactic Acid ; Lipid Peroxidation ; Nitric Oxide ; omega-N-Methylarginine* ; Oxygen ; Perfusion ; Respiration ; Ventilation