• Journal of Trauma and Injury
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• v.21 no.2
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• pp.120-127
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• 2008

Purpose: Many studies on the time course of inducible nitric oxide synthase (iNOS) gene expression have been performed in the LPS (Lipopolysaccharide)-induced endotoxemic model, but there have been few experimental approaches to continuous peritonitis-induced sepsis model. We conducted this study to establish basic data for future sepsis-related research by investigating the time course of iNOS gene expression and the relationship with the production of inflammatory mediators in the early sepsis model induced by cecal ligation and puncture (CLP). Methods: Male Sprague-Dawley rats were operated on by sing the CLP method to induce of peritonitis; and then, they were sacrificed and samples of blood and lung tissues were obtained at various times (1,2,3,6,9 and 12 h after CLP). We observed the expression of iNOS mRNA from lung tissues and measured the synthesis of nitric oxide, $IL-1{\beta}$, and $TNF-{\alpha}$ from the blood. Results: iNOS mRNA began to be expressed at 3 h and was maintained untill 12 h after CLP. The nitric oxide concentration was increased significantly at 6 h, reached its peak level at 9 h, and maintained a plateau untill 12 h after CLP. $TNF-{\alpha}$ began to be detected at 3 h, increased gradually, and decreased steeply from 9 h after CLP. $IL-1{\beta}$ showed its peak level at 6 h after CLP, and tended to decrease without significance. Conclusion: We observed that the iNOS gene was expressed later in peritonitis-induced sepsis than in LPS-induced sepsis. Nitric oxide and key inflammatory mediators were also expressed later in peritonitis-induced sepsis than in LPS-induced sepsis.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.7
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• pp.2929-2937
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• 2015

Endothelial nitric oxide synthase (eNOS or NOS3) produces nitric oxide and genetic polymorphisms of NOS3 gene play significant roles in various processes of carcinogenesis. The results from published studies on the association between NOS3 G894T and NOS3 intron 4 (4a/b) polymorphisms and cancer risk are conflicting and inconclusive. However, i n order to assess this relationship more precisely, a meta-analysis was performed with PubMed (Medline), EMBASE and Google web searches until February 2014 to select all published case-control and cohort studies. Genotype distribution data were collected to calculate the pooled odd ratios (ORs) and 95% confidence intervals (CIs) to evaluate the strength of association. A total of 10,546 cancer cases and 10,550 controls were included from twenty four case-control studies for the NOS3 G894T polymorphism. The results indicated no significant association with cancer risk as observed in allelic (T vs G: OR=1.024, 95%CI=0.954 to 1.099, p=0.508), homozygous (TT vs GG: OR=1.137, 95%CI=0.944 to 1.370, p=0.176), heterozygous (GT vs GG: OR=0.993, 95%CI=0.932 to 1.059, p=0.835), recessive (TT vs GG+GT: OR=1.100, 95%CI=0.936 to 1.293, p=0.249) and dominant (TT+GT vs GG: OR=1.012, 95%CI=0.927 to 1.105, p=0.789) genetic models. Similarly, a total of 3,449 cancer cases and 3,691 controls were recruited from fourteen case-control studies for NOS3 4a/b polymorphism. Pooled results indicated no significant association under allelic (A vs B: OR=0.981, 95%CI=0.725 to 1.329, p=0.902), homozygous (AA vs BB: OR=1.166, 95%CI=0.524 to 2.593, p=0.707), heterozygous (BA vs BB: OR=1.129, 95%CI=0.896 to 1.422, p=0.305), dominant (AA+BA vs BB: OR=1.046, 95%CI=0.779 to 1.405, p=0.763) and recessive (AA vs BB+BA: OR=1.196, 95%CI=0.587 to 2.439, p=0.622) genetic contrast models. This meta-analysis suggests that G894T and 4a/b polymorphisms of NOS3 gene are not associated with increased or decreased risk of overall cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.5
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• pp.283-287
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• 2003

While nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is beneficial for host survival, it is also detrimental to the host. Thus, regulation of iNOS gene expression may be an effective therapeutic strategy for the prevention of unwanted reactions at various pathologic conditions. During the screening process for the possible iNOS regulators, we observed that esculetin is a potent inhibitor of cytokine-induced iNOS expression. The treatment of 3T3-L1 adipocytes with the tumor necrosis factor-${\alpha}$ (TNF) induced iNOS expression, leading to enhanced NO production. TNF-induced NO production was inhibited by esculetin in a dose-dependent manner. Esculetin inhibited the TNF-induced NO production at the transcriptional level through suppression of iNOS mRNA and subsequent iNOS protein expression. These results suggest esculetin, a component of natural products, as a naturally occurring, nontoxic means to attenuate iNOS expression and NO-mediated cytotoxicity.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Molecular & Cellular Toxicology
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• v.5 no.4
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• pp.354-363
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• 2009

Cytotoxic Nitric oxide (NO) overproduced by inducible NO Synthase (iNOS or NOS2), which was induced in inflammatory reactions and immune responses directly or indirectly affects the functions as host defense and can cause normal tissue damage. Microarray analysis was performed to identify gene profiles of both NO-dependent and -independent transcripts in RAW 264.7 macrophages that use selective NOS2 inhibitors aminoguanidine ($100\;{\mu}M$) and L-canavanine (1 mM). A total of 3,297 genes were identified that were up- or down-regulated significantly over 2-fold in lipopolysaccharide (LPS)-treated macrophages. NO-dependency was determined in the expressed total gene profiles and also within inflammatory conditions-related functional categories. Out of all the gene profiles, 1711 genes affected NO-dependently and -independently in 567 genes. In the categories of inflammatory conditions, transcripts of 16 genes (Pomp, C8a, Ifih1, Irak1, Txnrd1, Ptafr, Scube1, Cd8a, Gpx4, Ltb, Fasl, Igk-V21-9, Vac14, Mbl1, C1r and Tlr6) and 29 geneas (IL-1beta, Mpa2l, IFN activated genes and Chemokine ligands) affected NO-dependently and -independently, respectively. This NO dependency can be applied to inflammatory reaction-related functional classifications, such as cell migration, chemotaxis, cytokine, Jak/STAT signaling pathway, and MAPK signaling pathway. Our results suggest that LPS-induced gene transcripts in inflammation or infection can be classified into physiological and toxic effects by their dependency on the NOS2-mediated NO release.

• Journal of Photoscience
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• v.9 no.2
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• pp.205-208
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• 2002

Nitric oxide (NO) is a newly described transmitter involved with cell to cell communication that is generated in biologic tissues by specific types of nitric oxide synthase (NOS), which metabolize L-arginine and molecular oxygen to citrulline and nitric oxide. In the skin. NO has been reported to play an important role in such diseases as psoriasis, atopic dermatitis, and contact dermatitis, as well as act as an important modulator in UVB-induced erythema. Ultraviolet B irradiation to the skin evokes an increase in NO production in the epidermis through two pathways; induction of inducible NOS, mediated by inflammatory cytokines, and elevation of constitutive neuronal NOS activity. In a cell culture system, it has been demonstrated that NO functions as a melanogen after being produced in keratinocytes in response to UVB-irradiation. NO-stimulated melanogenesis in melanocytes is mediated by the cGMP/PKG pathway. In this study, up-regulation of tyrosinase gene expression by NO-stimulation and the involvement of NO in UVB-induced pigmentation were examined. In NO-induced melanogenesis, protein synthesis and tyrosinase activity increased along with an up-regulation of tyrosinase gene expression. In an animal model, UVB-induced pigmentation in skin was suppressed by sequential daily treatments with a specific inhibitor of NOS. Thus, NO plays an important role in UVB-induced pigmentation, where its function as a melanogen is considered to be one of the mechanisms. Together with its role in the development of erythema, NO contributes to the total protective response of skin against UVB-irradiation.

• Advances in nano research
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• v.12 no.5
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• pp.501-514
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• 2022

This study aimed to investigate the effects and potential mechanisms of Chikusetsusaponin V (CsV) on endothelial nitric oxide synthase (eNOS) and vascular endothelial cell functions. Different concentrations of CsV were added to animal models, bovine aorta endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) cultured in vitro. qPCR, Western blotting (WB), and B ultrasound were performed to explore the effects of CsV on mouse endothelial cell functions, vascular stiffness and cellular eNOS mRNA, protein expression and NO release. Bioinformatics analysis, network pharmacology, molecular docking and protein mass spectrometry analysis were conducted to jointly predict the upstream transcription factors of eNOS. Furthermore, pulldown and ChIP and dual luciferase assays were employed for subsequent verification. At the presence or absence of CsV stimulation, either overexpression or knockdown of purine rich element binding protein A (PURA) was conducted, and PCR assay was employed to detect PURA and eNOS mRNA expressions, Western blot was used to detect PURA and eNOS protein expressions, cell NO release and serum NO levels. Tube formation experiment was conducted to detect the tube forming capability of HUVECs cells. The animal vasodilation function test detected the vasodilation functions. Ultrasonic detection was performed to determine the mouse aortic arch pulse wave velocity to identify aortic stiffness. CsV stimulus on bovine aortic cells revealed that CsV could upregulate eNOS protein levels in vascular endothelial cells in a concentration and time dependent manner. The expression levels of eNOS mRNA and phosphorylation sites Ser1177, Ser633 and Thr495 increased significantly after CsV stimulation. Meanwhile, CsV could also enhance the tube forming capability of HUVECs cells. Following the mice were gavaged using CsV, the eNOS protein level of mouse aortic endothelial cells was upregulated in a concentration- and time-dependent manner, and serum NO release and vasodilation ability were simultaneously elevated whereas arterial stiffness was alleviated. The pulldown, ChIP and dual luciferase assays demonstrated that PURA could bind to the eNOS promoter and facilitate the transcription of eNOS. Under the conditions of presence or absence of CsV stimulation, overexpression or knockdown of PURA indicated that the effect of CsV on vascular endothelial function and eNOS was weakened following PURA gene silence, whereas overexpression of PURA gene could enhance the effect of CsV upregulating eNOS expression. CsV could promote NO release from endothelial cells by upregulating the expression of PURA/eNOS pathway, improve endothelial cell functions, enhance vasodilation capability, and alleviate vessel stiffness. The present study plays a role in offering a theoretical basis for the development and application of CsV in vascular function improvement, and it also provides a more comprehensive understanding of the pharmacodynamics of CsV.

• IMMUNE NETWORK
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• v.5 no.1
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• pp.45-49
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• 2005

Background: Inflammation in the brain has known to be associated with the development of a various neurological diseases. The hallmark of neuro-inflammation is the activation of microglia, brain macrophage. Pro-inflammatory compounds including nitric oxide (NO) are the main cause of neuro-degenerative disease such as Alzheimer's disease (AD) which is resulted in cell death. Among those pro-inflammatory compounds, NO contributes to the cell death by directly or indirectly. Methods: In the study, we examined whether ursodeoxycholic acid (UDCA), a non-toxic hydrophilic bile acid, inhibits the NO production by a direct method using Griess reagent and by RT-PCR in the gene expression of inducible nitric oxide synthase (iNOS). In signal transduction, we also examined the NF-${\kappa}B$ (p65/p50), IKK, and I ${\kappa}B$, which are associated with the expression of iNOS gene using western blots. Results: In the present study, we found that UDCA effectively inhibited NO production in BV-2 microglial cell, and NF-${\kappa}B$ activation was reduced by suppressing IKK gene expression and by increasing the I${\kappa}B$ in cytosol comparing those to the positive control LPS. Conclusion: Taken together, these data suggested that UDCA may playa crucial role in inhibiting the NO production and the results imply that UDCA suppresses a cue signal of the microglial activation via stimulators, such as ${\beta}$-amyloid peptides which are known to stimulate microglia in AD pathogenesis.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.6
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• pp.363-369
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• 2011

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor $N^6$-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependent manner. Pretreatment of 10 mg/kg/day L-NIL significantly suppressed the foot pad swelling by MSU. Plasma level of nitric oxide (NO) metabolites and gene expression and protein level of iNOS in feet were increased by MSU, which was suppressed by L-NIL pretreatment. Similar pattern of change was observed in nitrotyrosine level. MSU increased the gene expression of tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-$1{\beta}$ and L-NIL pretreatment suppressed MSU-induced cytokines expression. The mRNA levels of superoxide dismutase and glutathione peroxidase1 were increased by MSU and L-NIL pretreatment normalized the gene expression. Phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was increased by MSU, which was suppressed by L-NIL pretreatment. The mRNA levels of iNOS, TNF-${\alpha}$, and IL-$1{\beta}$ were increased by MSU in human dermal fibroblasts, C2C12 myoblasts, and human fetal osteoblasts in vitro, which was attenuated by L-NIL in a dose dependent manner. This study shows that L-NIL inhibits MSU-induced inflammation and edema in mice feet suggesting that iNOS might be involved in MSU-induced inflammation.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.6
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• pp.1514-1518
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• 2003

Angelica sinensis radix, Danggui, is a traditional oriental medication, which has been used to modulate immune response. We report here that aqueous extract of Angelica sinensis radix (ASR) can induces NO production, and inhibit LPS-induced NO production in dose-dependent manner in RAW 264.7 macrophage cells. ASR also induces iNOS mRNA and iNOS protein expression, and exhibit inhibitory effect on iNOS mRNA and protein expression in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophage cells. Cytokines involved in the regulation of inflammatory reaction and immune response may play a role in the pathogenesis. ASR induces. pro-inflammatory cytokine gene expression (IL-1α, IL-1β and IL-6 gene) in a dose-dependent manner, and inhibits the expressions of these cytokines in LPS-stimulated RAW 264.7 macrophage cells. These data indicate that (1) ASR may be a potential therapeutic modulator of NO synthesis in various pathological conditions, and (2) the immunomodulatory effects of ASR may be, in part, associated with the inducing or suppression of pro-inflammatory cytokine gene expressions.