• Archives of Pharmacal Research
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• v.27 no.2
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• pp.225-231
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• 2004

This study was designed to investigate the effect of Trolox, a hydrophilic analogue of vitamin E, on the alteration of vasoregulatory gene expression during hepatic ischemia and reperfusion (I/R). Rats were subjected to 60 min of hepatic ischemia in vivo. The rats were treated intravenously with Trolox (2.5 mg/kg) or the vehicle as a control 5 min before reperfusion. Liver samples were obtained 5 h after reperfusion for a RT-PCR analysis on the mRNA for the genes of interest. These mRNA peptides are endothelin-1 (ET -1), potent vasoconstrictor peptide, its receptor $ET_A$ and $ET_B$, vasodilator endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and cyclooxygenase-2 (COX-2). It was seen that serum alanine aminotransferase and lipid peroxi-dation levels were markedly increased after I/R and Trolox significantly suppressed this increase. In contrast, the glutathione concentration decreased in the I/R group, and this decrease was inhibited by Trolox. ET-1 mRNA expression was increased by I/R, an increase which was prevented by Trolox. The mRNA levels for $ET_A$ receptor was significantly decreased, whereas ET$_{B}$ receptor transcript increased in the I/R group. The increase in $ET_A$ was prevented by Trolox. The mRNA levels for iNOS and HO-1 significantly increased in the I/R group and Trolox attenuated this increase. There were no significant differences in eNOS mRNA expression among any of the experimental groups. The mRNA levels for COX-2 and TNF-$\alpha$ significantly increased in I/R group and Trolox also attenuated this increase. Our findings suggest that I/R induces an imbalanced hepatic vasoregulatory gene expression and Trolox ameliorates this change through its free radical scavenging activity.y.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.111-117
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• 2004

Hepatic microcirculatory failure is a major component of reperfusion injury in the liver. Recent data provided some evidence that endothelium-derived vasoconstrictors and vasodilators may be functionally important to the control of the total hepatic blood flow under these conditions of circulatory failure. Since Kupffer cells provide signals that regulate the hepatic response in ischemia/reperfusion (I/R), the aim of this study was to investigate the role of Kupffer cells in the I/R-induced imbalance of vasoregulatory gene expression. Rats were subjected to 60 min hepatic ischemia, followed by 5 h of reperfusion. The Kupffer cells were inactivated by gadolinium chloride ($GdCl_3$, 7.5 mg/kg body weight, intravenously) 1 day prior to ischemia. Liver samples were obtained 5 hrs after reperfusion for RT-PCR analysis of the mRNA for genes of interest: endothelin-1 (ET-1), its receptors $ET_A and ET_B$, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). ET-1 mRNA expression was increased by I/R. mRNA levels for $ET_A$ receptors showed no change, whereas $ET_B$ receptor transcripts increased in the I/R group. The increases in ET-1 and $ET_B$ mRNA were not prevented by the $GdCI_3$ pretreatment. The mRNA levels for iNOS and eNOS significantly increased within the I/R group with no significant difference between the I/R group and the $GdCl_3$-treated I/R group. HO-1 mRNA expression significantly increased in the I/R group and this increase was attenuated by $GdCI_3$. In conclusion, we have demonstrated that an imbalance in hepatic vasoregulatory gene expression occurs during I/R. Our findings suggest that the activation of Kupffer cells is not required for I/R-induced hepatic microvascular dysfunction.

• Biomedical Science Letters
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• v.14 no.2
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• pp.127-130
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• 2008

To investigate if there are tandem repeats in iNOS gene in Korean genome we applied PCR amplification followed by DNA sequencing. Tandem repeats we were looking at were (AAAT)n in the promoter region. Totally, 65 people were subjected for this experiment. Twenty of them were patients with metabolic disease. Only $(AAAT)_4$ was found in all of these Korean samples. This result was somewhat different trom the data for Caucasians and other Asian people. So, we assume this is specific VNTR (variable number of tandem repeat) in Korean which can be used for the purpose of diagnosis and for the differentiation of ethnic groups.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.5
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• pp.333-337
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• 2012

Gene expression of neuronal nitric oxide synthase (nNOS) changes in the hypothalamic paraventricular nucleus (PVN) depending on feeding conditions, which is decreased during food deprivation and restored by refeeding, and phosphorylated cAMP response element binding protein (pCREB) was suggested to play a role in its regulation. This study was conducted to examine if the fasting-induced down-regulation of the PVN-nNOS expression is restored by activation of cAMP-dependent protein kinase A (cAMP/PKA) pathway. Freely moving rats received intracerebroventricular (icv) injection of cAMP/PKA activator Sp-cAMP (40 nmol) or vehicle (sterilized saline) following 48 h of food deprivation. One hour after drug injections, rats were transcardially perfused with 4% paraformaldehyde, and the PVN tissues were processed for nNOS or pCREB immunohistochemistry. Sp-cAMP significantly increased not only nNOS but also pCREB immunoreactivities in the PVN of food deprived rats. Fastinginduced down-regulation of the PVN-nNOS was restored by 1 h after the icv Sp-cAMP. Results suggest that cAMP/PKA pathway may mediate the regulation of the PVN-nNOS expression depending on different feeding conditions.

• Natural Product Sciences
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• v.20 no.4
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• pp.227-231
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• 2014

Cimicifuga heracleifolia extract (CHE) was investigated for its effects on the release of nitric oxide (NO) and at the level of inducible nitric oxide synthase (iNOS) gene expression in mouse macrophages. We found that C. heracleifolia elicited a dose-dependent increase in NO production and the level of iNOS mRNA. Since, iNOS transcription has been shown to be under the control of the transcription factor $NF-{\kappa}B$, the effects of CHE on $NF-{\kappa}B$ activation were examined. Transient expression assays with $NF-{\kappa}B$ binding sites linked to the luciferase gene revealed that the increased level of iNOS mRNA, induced by CHE, was mediated by the $NF-{\kappa}B$ transcription factor complex. By using DNA fragments containing the $NF-{\kappa}B$ binding sequence, CHE was shown to activate the protein/DNA binding of $NF-{\kappa}B$ to its cognate site, as measured by electrophoretic mobility shift assay. These results demonstrate that C. heracleifolia stimulates NO production and is able to up-regulate iNOS expression through $NF-{\kappa}B$ transactivation.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2153-2159
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• 2015

Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation — only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

• Toxicological Research
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• v.23 no.2
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• pp.123-126
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• 2007

We isolated a lignan, kobusin from Geranium thunbergii and studied its effect on the expression of inducible nitric oxide synthase (iNOS) gene in a monocyte/macrophage cell line, RAW264.7 cells. Kobusin inhibited lipopolysaccharide (LPS)-stimulated NO production and the expression of iNOS in a concentration-dependent manner. To identify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of kobusin on both the luciferase reporter activity using $NF-{\kappa}B$ minimal promoter and the nuclear translocation of p65. Kobusin suppressed the reporter gene activity and the LPS-induced movement of p65 in to nucleus. $NF-{\kappa}B$ activation is controlled by the phosphorylation and subsequent degradation of $I-{\kappa}B{\alpha}$, and in the present study, we found that $I-{\kappa}B{\alpha}$ phosphorylation was also inhibited by kobusin. Our findings indicate that kobusin may provide a developmental basis for an agent against inflammatory diseases.

• Biomolecules & Therapeutics
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• v.22 no.4
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• pp.282-287
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• 2014

We show that silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), inhibits cytokine mixture (CM: TNF-${\alpha}$, IFN-${\gamma}$, and IL-$1{\beta}$)-induced production of nitric oxide (NO) in the pancreatic beta cell line MIN6N8a. Immunostaining and Western blot analysis showed that silymarin inhibits iNOS gene expression. RT-PCR showed that silymarin inhibits iNOS gene expression in a dose-dependent manner. We also showed that silymarin inhibits extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) phosphorylation. A MEK1 inhibitor abrogated CM-induced nitrite production, similar to silymarin. Treatment of MIN6N8a cells with silymarin also inhibited CM-stimulated activation of NF-${\kappa}B$, which is important for iNOS transcription. Collectively, we demonstrate that silymarin inhibits NO production in pancreatic beta cells, and silymarin may represent a useful anti-diabetic agent.

• Biomolecules & Therapeutics
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• v.28 no.6
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• pp.549-560
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• 2020

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser¹³³ level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser¹⁹⁸¹ and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser¹⁹⁸¹, p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

• The Journal of Korean Obstetrics and Gynecology
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• v.23 no.3
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• pp.78-90
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• 2010

Purpose: This study was performed to evaluate the effect of Clematidis Radix extract(CB) on osteoclast differentiation and gene expression. The osteocastogenesis and gene expression were determined in RANKL-induced RAW 264.7 cell. Methods: RANKL-induced RAW 264.7 cell with Clematidis Radix extract was stained by TRAP which is expressive marker of osteoclast. The gene expression of RANK, $TNF{\alpha}$, IL-6, iNOS and Cathepsin, those are factors related to bone resorption, was estimated by using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: Clematidis Radix extract decreased the number of TRAP-positive multi nuclei cell, and decreased the gene expression of RANK, $TNF{\alpha}$, IL-6, iNOS and Cathepsin K in RANKL-induced RAW 264.7 cell. Conclusion: It is concluded that Clematidis Radix extract might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression.