• The Journal of Korean Medicine
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• v.29 no.1
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• pp.47-59
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• 2008

Objective : Anti-inflammatory effects of the extract of Lycii Fructus on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells were investigated. Method : In order to assess the cytotoxic effect of Lycii Fructus on the raw 264.7 macrophages 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was performed. Reverse transcription-polymerase chain reaction(RT-PCR) analysis of the mRNA levels of tumor necrosis factor-$\alpha$(TNF-$\alpha$) and inducible nitric oxide synthase(iNOS) was performed in order to provide an estimate of the relative level of expression of these genes. The protein level of the inhibitor of nuclear factor-${\kappa}B(I{\kappa}B)$ and nuclear factor-${\kappa}B$(NF-${\kappa}B$) activity was investigated by Western blot assay. NO production was investigated by NO detection. Result : Lycii Fructus suppressed NO production by inhibiting the LPS-induced expressions of iNOS and TNF-$^-\alpha$ mRNA and iNOS protein in RAW 264.7 macrophage cells. Also, Lycii Fructus suppressed activation of NF-${\kappa}B$ in the nucleus. Conclusion : These results show that the extract of Lycii Fructus has anti-inflammatory effect probably by suppressing iNOS expressions through the down-regulation of NF-${\kappa}B$ binding activity.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.4
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• pp.375-382
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• 1999

Growing evidence indicates that enhanced generation or actions of nitric oxide (NO) are implicated in the pathogenesis of hypertension in spontaneously hypertensive rats and diabetic nephropathy in streptozotocin (STZ)-induced diabetic rats. We investigated whether inducible nitric oxide synthase (iNOS) expression in STZ-induced diabetic rats is responsible for the alterations of vascular reactivity. Diabetic state was confirmed 28 days after injection of STZ (i.p) in rats by measuring blood glucose. In order to evaluate whether short term (4 weeks) diabetic state is related with altered vascular reactivity caused by iNOS expression, isometric tension experiments were performed. In addition, plasma nitrite/nitrate (NOx) levels and expression of iNOS in the lung and aorta of control and STZ-treated rats were compared by using Griess reagent and Western analysis, respectively. Results indicated that STZ-treated rats increased the maximal contractile response of the aorta to phenylephrine (PE), and high $K^+,$ while the sensitivity remained unaltered. Endothelium-dependent relaxation, but not SNP-mediated relaxation, was reduced in STZ-treated rats. Plasma nitrite/nitrates are significantly increased in STZ-treated rats compared to controls. The malondialdehyde (MDA) contents of liver, serum, and aorta of diabetic rats were also significantly increased. Furthermore, nitrotyrosine, a specific foot print of peroxynitrite, was significantly increased in endothelial cells and smooth muscle layers in STZ-induced diabetic aorta. Taken together, the present findings indicate that enhanced release of NO by iNOS along with increased lipid peroxidation in diabetic conditions may be responsible, at least in part, for the augmented contractility, possibly through the modification of endothelial integrity or ecNOS activity of endothelium in STZ-diabetic rat aorta.

• 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.

• The Journal of Internal Korean Medicine
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• v.34 no.1
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• pp.31-45
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• 2013

Objectives : Gokgisaeng (Korean mistletoe) is used for the treatment of inflammatory and cancer diseases in traditional Korean medicine and its major component lectins have been reported to induce nitric oxide (NO) in RAW 264.7 macrophages, and also induce apoptosis of various types of cancer cells, although its modulatory effects on cancer cell migration and macrophage activation is poorly understood. The aim of this study is to clarify molecular mechanisms of action responsible for the anti-inflammatory and antitumor migration potentials of Korean mistletoe extract (KME). Methods : We investigated the anti-inflammatory activity of KME on NO production and inducible nitric oxide synthase (iNOS) expression by lipopolysaccharide (LPS) in both RAW 264.7 macrophages and rat C6 glioma cells, and also evaluated inhibitory efficacy on glioma cell growth and migration. For assessment, XTT assay, nitrite assay, RT-PCR, scratch-wound and Boyden chamber assay, and western blot analysis were performed. Results : Previously reported, unlike the efficacy of Gokgisaeng lectin, KME inhibited NO production and iNOS expression, and suppressed pro-inflammatory mediators including IL-$1{\beta}$, IL-6, COX-2, iNOS in LPS-stimulated RAW 264.7 cells. Furthermore, KME suppressed tumor cell growth and migration, and it also inhibited LPS-induced NO release and iNOS activation by down-regulating expression of protein kinase C (PKC) and phosphorylation of ERK in C6 glioma cells. Conclusions : Our research findings provide evidence that KME can play a significant role in blocking pro-inflammatory reaction and malignant progression of tumors through the suppression of NO/iNOS by down-regulating of inflammatory signaling pathways, PKC/ERK.

• Fisheries and Aquatic Sciences
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• v.14 no.4
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• pp.275-282
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• 2011

Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been implicated in various inflammatory diseases. In this study, we investigated the anti-inflammatory activities of Chondria crassicaulis ethanolic extract (CCE) by measuring its effects on the expression of iNOS and COX-2 proteins in lipopolysaccharide (LPS)-treated RAW 264.7 murine macrophages. CCE significantly and dose-dependently inhibited the LPS-induced release of nitric oxide and prostaglandin $E_2$, and suppressed the expression of iNOS and COX-2 proteins in LPS-stimulated RAW 264.7 cells, without causing any cytotoxicity. It also inhibited the production of the pro-inflammatory cytokines such as interleukin (IL)-$1{\beta}$, IL-6, and tumor necrosis factor (TNF)-${\alpha}$ in LPS-stimulated RAW 264.7 cells. Moreover, treatment with CCE strongly suppressed nuclear factor-${\kappa}B$ (NF-${\kappa}B$) promoter-driven expression in LPS-treated RAW 264.7 cells. CCE treatment blocked nuclear translocation of the p65 subunit of NF-${\kappa}B$ by preventing proteolytic degradation of inhibitor of ${\kappa}B-{\alpha}$. These results indicate that CCE regulates iNOS and COX-2 expression through NF-${\kappa}B$-dependent transcriptional control, and identifies potential candidates for the treatment or prevention of inflammatory diseases.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.5
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• pp.273-280
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• 2004

Nitric oxide (NO) has been suggested to act as a mediator of cytokine-induced effects of turn over of bone. Activation of the inducible nitric oxide synthase (iNOS) by inflammation has been related with apoptotic cell death in osteoblast. YS 49, a synthetic isoquinoline alkaloid, inhibits NO production in macrophages activated with cytokines. In the present study, we investigated the molecular mechanism of YS 49 to inhibit iNOS expression in ROS 17/2.8 cells, which were activated with combined treatment of inflammatory cytokines $(TNF-{\alpha},\;IFN-{\gamma})$ and lipopolysaccharide (LPS). Results indicated that YS 49 concentration-dependently reduced iNOS mRNA and protein expression, as evidenced by Northern and Western blot analysis, respectively. The underlying mechanism by which YS 49 suppressed iNOS expression was not to affect iNOS mRNA stability but to inhibit activation and translocation of $NF-_kB$ by preventing the degradation of its inhibitory protein $I_kB_{\alpha}$. As expected, YS 49 prevented NO-induced apoptotic cell death by sodium nitroprusside. Taken together, it is concluded that YS 49 inhibits iNOS expression by interfering with degradation of phosphorylated inhibitory $_kB_{\alpha}\;(p-I_kB_{\alpha})$. These actions may be beneficial for the treatment of inflammation of the joint, such as rheumatoid arthritis.

• Toxicological Research
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• v.24 no.3
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• pp.169-174
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• 2008

This study examined the mechanisms underlying the effects of the vasorelaxation active substance(VAS), dimethyladenosine-5'-L-arabinose, and its partial purification fraction on nitric oxide synthase in improving erectile dysfunction with particular focus on the nitric oxide (NO)-cGMP pathways. Two rat models, 9-month-old SD rats and 11-month-old SD rats, were given VAS(40 mg/kg per day) for 4 days, The aqueous fraction of silworm male pupae extract; semi-purified VAS(100 mg/kg per day) for 10 days, respectively. The NOS activities of the following three enzymes were examined: neuronal NO synthase(nNOS), inducible NOS(iNOS), endothelial NOS(eNOS), vascular endothelial growth factor on endothelial cells(VEGF) and anti-inflammation effect of Tumor necrosis factor-$\alpha$. The results showed increases in the nitric oxide synthase activities. Western blotting of the tissue homogenate showed an increase in the nNOS level in the brain and tongue, and an increase in the endothelial NO synthase(eNOS) level in penis. However, there was little association with VEGF production in HUVEC endothelial cells and no relationship with TNF-$\alpha$ which showed low levels.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.2
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• pp.278-283
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• 2010

Inducible nitric oxide synthase (iNOS) are important inflammation enzyme and severe up-nitric oxide (NO) production by this enzyme has been intricate with pathogenesis of atopy dermatitis. The present study was designed in order to determine whether Lonicera japonica could inhibit atopy dermatitis through modulation of iNOS by NF-${\kappa}B$ suppression. We found that IKK mRNA and iNOS mRNA expression in RAW 264.7 macrophages stimulated with lipopolysaccharide dose-dependantly decreased by Lonicera japonica (0.4 - 1.0 mg/$m{\ell}$) and NO production decreased. The distribution of NF-${\kappa}B$ p65 and iNOS positive reacted cell in NC/Nga mice with atopy dermatitis were decreased by Lonicera japonica (45 mg/kg/day) and apoptosis were increased. These data likely indicate that Lonicera japonica may act as inflammatory regulator for atopy dermatitis through iNOS modulation by NF-${\kappa}B$B suppression and may be possible to develop useful agent for chemoprevention of NO intricate inflammatory diseases.

• Journal of Pharmacopuncture
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• v.5 no.2
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• pp.40-51
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• 2002

Objectives : The purpose of this study was to investigate the effect of Bee Venom on the lipopolysaccharide-induced expression phospholipase $A_2$, cyclooxygenase-2 and inducible nitrogen oxide synthase, and the generation of arachidonic acid, prostaglandin D2 and E2 in RAW 264.7 cells, a murine macrophage cell line. Methods : The expression of phospholipase $A_2$, cyclooxygenase and inducible nitrogen oxide synthase was determined by western blotting with corresponding antibodies, and the generation of arachidonic acid, prostaglandin $D_2$ and $E_2$ was assayed by ELISA method in RAW 264.7 cells. The non-toxic concentrations (0.1 to $5\;{\mu}g/ml$) of bee venom determined by MTT assay, were used in this study. Results : 1. Bee venom inhibited lipopolysaccharide-induced expression of phospholipase $A_2$ in a dose dependent manner after 48 hours treatment. 2. Bee venom inhibited lipopolysaccharide-induced expression of cyclooxygenase-2 in a dose dependent manner after 24 and 48 hours treatment. 3. Bee venom inhibited lipopolysaccharide-induced expression of inducible nitrogen oxidesynthase in a dose dependent manner after 48 hours treatment. 4. The generation of arachidonic acid, prostaglandin $D_2$ and $E_2$ was not much affected by the treatment of bee venom on the lipopolysaccharide-induced generation of arachidonic acid, prostaglandin $D_2$ and $E_2$ in RAW 264.7 cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.5
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• pp.1405-1410
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• 2005

Asthma is recognized today as an inflammatory disease of the lung characterized by acute non-specific airway hypersensitiveness in association with chronic pulmonary inflammation. Gamijihwang-tang(GJT), a fortified prescription of YMJHT, is applied for the treatments of chronic coughing and asthma, and post-delivery coughing and asthma in the gynecology. Also in the clinical practice, GJT is known to be very effective for controlling coughing and asthma as a cold sequoia. In this study, we investigated the effects of GJT on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) production, and on the level of inducible nitric oxide synthase (iNOS) and Cyclooxygenase-2 expression in murine macrophage RAW 264.7 cells. We found that GJT inhibited LPS-induced NO and $PGE_2$ production in a dose dependent manner. Furthermore, GJT inhibited the expression of LPS-induced iNOS and COX-2 protein and mRNA expression in RAW 264.7 macrophages. Treatment with GJT of RAW 264.7 cells transfected with a reporter construct indicated a reduced level of LPS-induced nuclear factor-KB (NF-kB) activity and effectively lowered NF-kB binding as measured by transient transfection assay. These results suggest that the main inhibitory mechanism of the GJT may be the reduction of iNOS and COX-2 gene expression through blocking of NF-kB activation.