• The Journal of Korean Medicine
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• v.23 no.2
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• pp.19-27
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• 2002

Objective : This study was designed to investigate the effect of Hyeolbuchukeo-tang (HCT) on NO production and the molecular mechanism of NO production modulated by HCT in the primary VSMC (vascular smooth muscle cells). Method : Primary VSMC was established from aorta and cultured VSMC used in this study. NO production of VSMC was assayed by Griess reagent and the expression of iNOS gene was assayed by Western, RT-PCR. Result : $TNF-{\gamma}$ induced NO production, but $IFN-{\gamma}$ or HCT alone did not induce NO production in cultured VSMC. However, $IFN-{\gamma}$ or HCT potentiated NO production in $TNF-{\gamma}-treated$ VSMC in a time- and dose-dependent manner. $TNF-{\gamma}$ induced the iNOS gene expression corresponding to NO production in $TNF-{\gamma}-treated$ VSMC. HCT potentiated NO production in $TNF-{\gamma}-treated$ VSMC by about 20%, but HCT did not increase the level of iNOS mRNA in $TNF-{\gamma}-treated$ VSMC. HCT slightly increased the level of iNOS protein in $TNF-{\gamma}-treated$ VSMC. Calcium ionophore A23187 decreased NO production in $TNF-{\gamma}-treated$ VSMC, but HCT attenuated the effect of A23187. Conclusion : As NO is deeply involved in the development of arteriosclerosis and dilation of blood vessels, drugs or chemicals modulating NO production in VSMC could be used for preventing and treating arteriosclerosis. Considering the effect of HCT on the modulation of NO production in VSMC, MCT has a potential capacity for preventing and treating diseases of the circulation system including arteriosclerosis.

• The Journal of Korean Medicine
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• v.24 no.2
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• pp.166-178
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• 2003

Objectives : Nitric oxide (NO) plays an important role in normal and pathophysiological cells as a messenger molecule, neurotransmitter, microbiological agent, or dilator of blood vessels and arteriosclerosis, respectively. This study was undertaken to understand the mechanism of NO production and effect of Hyeolbuchukeo-tang (Xiefuzhuyu-tang) on NO production in cultured vascular smooth muscle cell (VSMC). Methods and Results : VSMC was isolated from aorta and cultured. Cultured primary cells were identified as VSMC with anti--smooth muscle actin antibody. A large amount of NO was produced in cultured VSMC treated with $IFN-{\gamma}$ plus TNF in a time- and dose-dependent manner. $TNF-{\alpha}$ was a more efficient stimulator than $IFN-{\gamma}$ in NO production of cultured VSMC. iNOS protein wasdetected within 3 hrs and it increased up to 12 hrs in a time-dependent manner. However, accumulated NO in cytokine-treated VSMC was not detected within 3 hrs. NO production in cytokine-treated VSMC showed the dose- and time-dependent manner, and increased up to 48 hrs. The activated VSMC produced a large amount of NO (about 60 uM). Hyeolbuchukeo-tang (Xiefuzhuyu-tang) alone did not induceNO production, but it potentiated the effect of $TNF-{\alpha}$ on NO production and increased NO production by about 20%. Hyeolbuchukeo-tang (Xiefuzhuyu-tang) did not affect the transcriptional activity of iNOS gene, but increased the accumulation of iNOS. These results indicate that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) could modulate the translational level of iNOS. PKC did not modulate NO production, but calcium ionophore A23187 decreased NO production. However, Hyeolbuchukeo-tang (Xiefuzhuyu-tang) elevated the decreased NO production in A23187-treated VSMC by modulating the stability of iNOS transcripts. Half-life of the synthesized transcripts appeared to have about 6 hrs. PDTC, an $NF-{\kappa}B$ inhibitor, blocked the accumulation of iNOS mRNA, indicating that $NF-{\kappa}B$ served as an important modulator in the transcriptional regulation of iNOS. As Hyeolbuchukeo-tang (Xiefuzhuyu-tang) potentiated the effect of the $TNF-{\alpha}$ on NO production but had no additional effect on PDTC-modulated NO production, it is suggested that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) enhances the $TNF-{\alpha}-mediated$ NO production of VSMC by modulating the iNOS activity and the stability of iNOS transcripts in activated VSMC having the elevated intracellular calcium ion. Conclusions : This study suggests that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) has a potential capacity for preventing and treating diseases of the circulation system, including arteriosclerosis.

• BMB Reports
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• v.41 no.1
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• pp.79-85
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• 2008

The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.

• The Journal of Internal Korean Medicine
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• v.20 no.1
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• pp.73-82
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• 1999

Nitric oxide (NO) is now recognized as a mediator of several biological and immunological functions, but unlike classical neurotransmitters. NO simply diffuse of the postsynaptic cells and around affecting cells. Taraxacum officinale (Compositae) has been used for maintenance of vitality, and they still occupy an important place in the traditional Korean medicine. We have examined that the effect of Taraxacum officinale water extract on NO synthesis in microglial cells of murine's brain, using the Griess method. And this study was evident that Taraxacum officinale did not induce NO production without recombinant interferon gamma ($rIFN-{\gamma}$), whereas Taraxacum officinale (10-1000 g/ml) with $rIFN-{\gamma}$ effectively produced NO in microglial cells of brain. As result. NO production in microglial cells increased most significantly in dose of 100 g/ml of the Taraxacum officinale and the production of NO was dependent on the dose of Taraxacum officinale, NG-monomethyl-L-arginine, competitive inhibitor of NO synthase, reduced the NO production by Taraxacum officinale stimulation with $rIFN-{\gamma}$ in microglial cells of murine. The effect of Taraxacum officinale was mainly dependent on Taraxacum officinale-induced tumor necrosis factor- secretion. Conclusively, this study suggested that Taraxacum officinale stimulate NO production at microglial cells in brain, which may be an important factor for mediating immune and neuroendocrinologic regulation in nervous system.

• Biomedical Science Letters
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• v.24 no.1
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• pp.50-54
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• 2018

N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU), direct alkylating chemical mutagens and carcinogens, are shown to be the upregulators of cellular $NF-{\kappa}B$, regulating various genes that mediate tumorigenesis and carcinogenesis. Nitric oxide (NO), a toxic reactive radical gas, has been known to induce programmed cell death or apoptosis in various cells. Therefore, the assessment of NO production was examined to elucidate the possible contribution of NO release to the chemical carcinogenic potency of NMU and NEU in human skin cells. NMU and NEU did not alter the NO production, but they caused a significant downregulation of the NO generation on lipopolysaccharide (LPS)-induced NO production at concentrations ranging from $2{\sim}5{\mu}M$. The degree of downregulation of NO by NMU and NEU decreased up to 15% and 20%, respectively, compared to the control. These results demonstrate that the LPS-inducible keratinocytes NO synthase is involved in modulating carcinogenic potency by NMU and NEU, and the regulation of the cellular $NF-{\kappa}B$ activity by NMU and NEU is negatively correlated with the level of LPS-induced NO production in human skin cells. The findings of this study suggest the hypothesis that NMU and NEU-induced carcinogenesis may be associated with the downregulation of NO production, and the inducible NO may play an important role in NMU and NEU-induced carcinogenicity in human epidermal keratinocytes.

• The Journal of Korean Medicine
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• v.24 no.2
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• pp.138-147
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• 2003

Objectives : In this study, we investigated the mechanism by which Chelidonium majus (CM) regulates nitric oxide (NO) production. Methods : Using mouse peritoneal macrophages, the mechanism by which CM regulates NO or tumor necrosis $factor-{\alpha}(TNF-{\alpha})$ production was examined. NO release was measured by the Griess method. $TNF-{\alpha}$ production was measured by the ELISA method. The protein extracts were prepared and samples were analyzed for the inducible NOS(iNOS) expression and nuclear factor kappa $B(NF-{\kappa}B)$ activation by Western blotting. Results : When CM was used in combination with recombinant $interferon-{\gamma}{\;}(rIFN-{\gamma})$, there was a marked cooperative induction of NO production. CM had an effect on NO production by itself. The expression of the iNOS gene was increased in $rIFN-{\gamma}$ plus CM-stimulated peritoneal macrophages and almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of $NF-{\kappa}B$. The $NF-{\kappa}B$ activation was increased in rIFN-{\gamma} plus CM-induced peritoneal macrophages. The increased production of NO from $rIFN-{\gamma}$ plus CM-stimulated peritoneal rnacrophages was decreased by the treatment with $N^{G}-monomethyl-{_L}-arginine{\;}(N^{G}MMA){\;}N^{\alpha}-Tosyl-Phe$ chloromethyl ketone (TPCK) , and was almost completely inhibited by pre-treatment with PDTC. Furthermore, treatment with CM alone or rIFN-{\gamma} plus CM in peritoneal macrophages caused a significant increase in $TNF-{\alpha}$ production. PDTC decreased CM-induced $TNF-{\alpha}$ production significantly. After CM treatment in HT-29 or AGS cells, cell viability decreased. Conclusions : These findings demonstrate that CM increases the production of NO and $TNF-{\alpha}{\;}by{\;}rIFN-{\gamma}-primed$ macrophages and suggest that NF-B plays a critical role in mediating these effects of CM.

• Preventive Nutrition and Food Science
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• v.11 no.4
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• pp.273-277
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• 2006

Recently, Rhei Rhizoma extracts (RRE) have begun to receive more attention as potential biological response modifiers. In the present study, we studied the antiproliferative effect of RRE on tumor cells and the effect of RRE on macrophage function. A variety of tumor cells and macrophages were treated with RRE at various concentrations. The effect of RRE on cell proliferation was measured by MTT assay and the effect of RRE on the production of nitric oxide (NO) was determined in the macrophage-like cell lines Raw264.7, C6 and peritoneal macrophages (pMQ). RRE inhibited the growth of tumor cells (e.g., B16, HOS). However, the effects of RRE on the production of NO varied with macrophage types. RRE had no effect on C6 cell growth and slightly increased the growth of Raw264.7 cells. In addition, treatment of normal pMQ with RRE enhanced NO production in a concentration-dependent manner, whereas RRE suppressed NO production at $50\;{\mu}g/mL$ in both Raw264.7 and C6 cells. However, RRE suppressed NO production in LPS/IFN-$\gamma$-stimulated C6 cells. Overall, these results suggest that RRE elicits an antiproliferative property and differentially modulates NO production in various macrophages, and have a potential for therapeutic application.

• The Journal of Korean Medicine
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• v.21 no.3
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• pp.20-30
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• 2000

Objective : This experiment was performed in order to study the effect of an aqueous extract of Salviae radix root(SRRAE) on NO production and NOS gene induction from macrophages Methods : To investigate dose-dependent effects of SRRAE for NO release on the $rIFN-{\gamma}-treated$ macrophages, the cells were incubated for 6 hrs in a medium containing $rIFN-{\gamma}$ (5 U/ml), stimulated with SRRAE and incubated in a CO2 incubator. The cells were treated with 5 U/ml $rIFN-{\gamma}$ plus 100 g/ml of SRRAE, Then, the cells were incubated with various concentrations of NGMMA at $37^{\circ}C$ for 48 hrs, Results : SRRAE had no effect on NO production by itself, whereas recombinant $interferon-{\gamma}(rIFN-{\gamma})$ alone showed modest activity, When SRRAE was used in combination with $rIFN-{\gamma}$, there was a marked cooperative induction of NO production in a dose-dependent manner. The optimal effect of SRRAE on NO production was shown at 6hrs after treatment with $rIFN-{\gamma}$. The SRRAE-induced production of NO was inhibited by NG-monomethyl- L-arginine(NGMMA) and arginase. $rIFN-{\gamma}$ in combination with SRRAE showed a marked increase of the expression of the inducible NOS(iNOS) gene. In addition, the effect of SRRAE was mainly dependent on the SRRAE-induced tumor necrosis $factor-{\alpha}(TNF-{\alpha})$ secretion. Conclusions : SRRAE induces NO production from macrophages as a result of SRRAE-induced $TNF-{\alpha}$ secretion. SRRAE may provide a second signal for synergistic induction of NO production in macrophages already induced to express iNOS gene by $rIFN-{\gamma}$.

• Korean Journal of Microbiology
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• v.9 no.1
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• pp.32-38
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• 1971

In solid culture of Endomycopsis fibuliger No.55, Eudomuopsis javanensis No.112 and Candida tropicalis No.340, the conditions of enzyme (protease, anylase and cellulase) production and the influence of addition of $(NH_4)_2;SO_4$ were examined, and the results obtained were as follows. 10 Wheat bran medium is found to be the best on the enzyme production in case of simple material. The optimum conditions ; are water content added 100 to 120%, temperature 25 to $80^{\circ}C$ and incubation times 2 to 3 days. 2) The cellulase production was scarely produced in the case of Endomyopsis fibuliger No.55, as well as, the amylase production was scarely producted in the case of Endomycopsis javanensis No.112 and Candida tropicalis No.340. 3) The enzyme production was remarkably increased when 5% of$(NH_4)_2;SO_4$ as inorganic nitrogen sources was admixed to wheat bran. 4) When 5% of $(NH_4)_2;SO_4$ was admixed to medium, the ratio of protein increase was 10.2 to 17.7% in wheat bran medium and 10.6 to 17.9% in sweet potato cake medium.

• Biomolecules & Therapeutics
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• v.2 no.2
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• pp.131-135
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• 1994

The effect of ginsenoside (GS) from Panax ginseng on basal and nitro-L-arginine suppressed nitric oxide (NO) production was studied in rat kidney. NO production was determined by conversion to [$^{14C}$]=L-citrulline from [$^{14C}$]-L-arginine both in whole kidney and three renal segments; glomerulus, cortex excluding glomerulus (cortex-) and medulla. Nitro-L-arginine (total dose of 30 mg/kg/3 days, i.p.) significantly reduced NO production in whole kidney, which was prevented by GS pretreatment (30 mg/kg/3 days, i.p.). Relative high dose of GS (120 mg/kg/4 days, i.p..) selectively increased NO production in glomerulus and cortex-. Protein content, on wet weight basis, in cortex- and glomerular DNA content were significantly reduced by GS. Our results confirm the existence of constitutive nitric oxide synthase in kidney and it seems that target nephron segment for volume expansion due to GS'NO-mediated vasodilation and for NO production stimulated by GS is cortex including glomerulus.lus.