• Journal of Korean Neurosurgical Society
• /
• 제29권7호
• /
• pp.877-885
• /
• 2000

Objective : The NOS inhibitors exhibit antinociceptive activity in rat model of neuropathic pain. NOS activity increases in the dorsal root ganglia(DRG) in neurop-athic pain. However, NOS activity decreases in the dorsal horn of spinal cord in the nerve injury models of neuropathic pain. To investigate whether the mechanism of decrease of NOS expression in the dorsal horn is related to a secondary effect resulting from increased NO production and likewise in the spinal DRG in the spinal nerve ligation model of neuropathic pain. Methods : We conducted behavioral tests for neuropathic pain, and nNOS immunohistochemistry and NADPH-diaphorase histochemistry after tight ligation of the 5th lumbar(L5) and 6th lumbar(L6) spinal nerves and L5 dorsal rhizotomy. Results : Typical neuropathic pain behaviors occurred 7 days after post-ligation in the neuropathic surgery group, but neuropathic pain behaviors in the dorsal rhizotomy group were absent or weak 7 days after post-operation. There was a decrease in the number of nNOS immunoreactive dorsal horn neurons on the both side(especially ipsilateral side) 7 days after post-ligation. The number of nNOS immunoreactive neurons in both side of the dorsal horn was not decreased 7 days after L5 dorsal rhizotomy. Conclusion : These data indicate that the changes in the injured DRG is essential for development and maintenance of neuropathic pain, and mechanism of decrease of nNOS expression in the dorsal horn is a secondary effect against the changes in the DRG including increased NO production in the spinal nerve ligation model of neuropathic pain.

• 한국자원식물학회지
• /
• 제25권3호
• /
• pp.317-322
• /
• 2012

In this study we investigated the effects of supplementation with fucoidan from brown alga on the function of natural-killer (NK) cells to evaluate the possibility as an immunomodulator in ovariectomized (OVX) rats. A total of 18 female Wistar rats (six weeks) were used this study and 12 rats were OVX, and the rest of rats were sham-operated. The sham and one OVX group were fed standard diet, and the remaining OVX group received fucoidan (0.05% supplemented diet). After 12 weeks of supplementation, rats were sacrificed to assess the tumoricidal activity of the NK cells and the NO-iNOS regulation from splenocytes. The mass of body and the immune organs such as spleen and thymus were also studied. In OVX rats, body and thymus weights increased, however fucoidan supplementation did not change the body mass and organs weight compared to OVX group. Fucoidan supplementation increased NK cell activity and reduced NO-iNOS production in OVX rats. Ex vivo treatment of fucoidan increased NK cell activity in splenocytes from shame and OVX rats. Ex vivo, we confirmed that fucoidan partially reduced the NK cell activity in the presence of iNOS inhibitors in OVX-splenocytes. These results indicate fucoidan supplementation has a NK cell tumoricidal activity, which are regulated by the iNOS production in OVX rats. This suggests that fucoidan is useful for potential therapeutic strategies as a nutrient in regulating the NK cells in postmenopausal osteoporosis patients.

• Journal of Acupuncture Research
• /
• 제36권2호
• /
• pp.80-87
• /
• 2019

Background: This study investigated the anti-inflammatory effects of bee venom (BV) through the inhibition of nuclear factor kappa beta ($NF-{\kappa}B$) expression in macrophages and keratinocytes. Methods: Cell viability assays were performed to investigate the cytotoxicity of BV in activated macrophages [lipopolysaccharide (LPS)] and keratinocytes [interferon-gamma/tumor necrosis factor-alpha ($IFN-{\gamma}/TNF-{\alpha}$)]. A luciferase assay was performed to investigate the cellular expression of $NF-{\kappa}B$ in relation to BV dose. The expression of $NF-{\kappa}B$ inhibitors ($p-I{\kappa}B{\alpha}$, $I{\kappa}B{\alpha}$, and p50 and p65) were determined by Western Blot analysis, and the electromobility shift assay. A nitrite quantification assay was performed to investigate the effect of BV, and $NF-{\kappa}B$ inhibitor on nitric oxide (NO) production in macrophages. In addition, Western Blot analysis was performed to investigate the effect of BV on the expression of mitogen-activated protein kinases (MAPK) in activated macrophages and keratinocytes. Results: BV was not cytotoxic to activated macrophages and keratinocytes. Transcriptional activity of $NF-{\kappa}B$, and p50, p65, and $p-I{\kappa}B{\alpha}$ expression was reduced by treatment with BV in activated macrophages and keratinocytes. Treatment with BV and an $NF-{\kappa}B$ inhibitor, reduced the production of NO by activated macrophages, and also reduced $NF-{\kappa}B$ transcriptional activity in activated keratinocytes (compared with either BV, or $NF-{\kappa}B$ inhibitor treatment). Furthermore, BV decreased p38, p-p38, JNK, and p-JNK expression in LPS-activated macrophages and $IFN-{\gamma}/TNF-{\alpha}$-activated keratinocytes. Conclusion: BV blocked the signaling pathway of $NF-{\kappa}B$, which plays an important role in the inflammatory response in macrophages and keratinocytes. These findings provided the possibility of BV in the treatment of atopic dermatitis.

• 생명과학회지
• /
• 제23권6호
• /
• pp.736-742
• /
• 2013

Achyranthoside E dimethyl ester (AEDE)는 Achyranthes japonica에서 분리한 oleanolic acid glycoside이다. 본 연구에서는 대식세포에서 lipopolysaccharide (LPS)로 인한 nitric oxide (NO)의 생성에 미치는 AEDE의 효과를 관찰하고 그 작용 기전을 연구하였다. AEDE는 NO 생성과 inducible NO synthase (iNOS) 발현을 억제하였으며 세포에 독성을 유도하지 않았다. 또한 AEDE는 heme oxygenase-1 (HO-1)의 발현을 유도하였으며, HO-1 siRNA를 처리했을 때 AEDE가 iNOS의 발현을 억제하지 못하였다. AEDE는 HO-1의 발현에 관여하는 전사인자인 nuclear factor E2-related factor 2 (Nrf2)를 핵으로 이동시켰다. 한편 AEDE에 의한 HO-1의 발현은 phosphatidylinositol 3-kinase (PI-3K) 및 extracellular signal regulated kinase (ERK1/2) 억제제에 의해 감소되었으며, AEDE가 Akt와ERK1/2의 인산화를 유도하였다. 이상의 결과를 종합해보면, AEDE는 대식세포에서 PI-3K/Akt/ERK-Nrf2 신호전달과정을 통해 HO-1의 발현을 유도함으로써 NO와 같은 염증매개물질의 생성을 억제한다는 것을 알 수 있다. 이러한 연구결과는 AEDE가 항염증제로 사용될 수 있음을 시사한다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제5권4호
• /
• pp.343-351
• /
• 2001

This study was undertaken to investigate the mechanism of lipopolysaccharide (LPS) and nitric oxide (NO) as a regulator of vascular smooth muscle cell (VSMC) proliferation. VSMC was primarily cultured from rat aorta and confirmed by the immunocytochemistry with anti-smooth muscle myosin antibody. The number of viable VSMCs were counted, and lactate dehydrogenase (LDH) activity was measured to assess the degree of cell death. Concentrations of nitrite in the culture medium were measured as an indicator of NO production. LPS was introduced into the medium to induce the inducible nitric oxide synthase (iNOS) in VSMC, and Western blot for iNOS protein and RT-PCR for iNOS mRNA were performed to confirm the presence of iNOS. Inhibitors of iNOS and soluble guanylate cyclase (sGC), sodium nitroprusside (SNP) and L-arginine were employed to observe the action of LPS on the iNOS-NO-cGMP signalling pathway. LPS and SNP decreased number of VSMCs and increased the nitrite concentration in the culture medium, but there was no significant change in LDH activity. A cell permeable cGMP derivative, 8-Bromo-cGMP, decreased the number of VSMCs with no significant change in LDH activity. L-arginine, an NO substrate, alone tended to reduce cell count without affecting nitrite concentration or LDH level. Aminoguanidine, an iNOS specific inhibitor, inhibited LPS-induced reduction of cell numbers and reduced the nitrite concentration in the culture medium. LY 83583, a guanylate cyclase inhibitor, suppressed the inhibitory actions of LPS and SNP on VSMC proliferation. LPS increased amounts of iNOS protein and iNOS mRNA in a concentration-dependent manner. These results suggest that LPS inhibits the VSMC proliferation via production of NO by inducing iNOS gene expression. The cGMP which is produced by subsequent activation of guanylate cyclase would be a major mediator in the inhibitory action of iNOS-NO signalling on VSMC proliferation.

• 생명과학회지
• /
• 제25권9호
• /
• pp.976-983
• /
• 2015

Achyranthoside C dimethyl ester (ACDE)는 우슬에서 분리한 oleanolic acid glycoside이다. 본 연구에서는 RAW264.7 대식세포에서 ACDE의 항염증 효과를 관찰하고 그 작용 기전을 연구하였다. ACDE는 세포에 독성을 유도하지 않으면서 heme oxygenase-1 (HO-1)의 발현을 유도하였다. ACDE 는 HO-1의 발현에 관여하는 전사인자인 nuclear factor E2-related factor 2 (Nrf2)를 핵으로 이동시켰다. 또한 ACDE에 의한 HO-1의 발현은 phosphatidylinositol 3-kinase (PI-3K) 및 mitogen activated protein kinases (MAPK) 억제제에 의해 감소되었으며, ACDE가 Akt, c-Jun kinase (JNK), extracellular signal regulated kinase (ERK), p38 kinase의 인산화를 유도하였다. 한편 ACDE는 lipopolysaccharide (LPS)로 인한 nitric oxide (NO)의 생성과 inducible NO synthase (iNOS) 발현을 억제하였으며 HO-1 siRNA를 처리했을 때 ACDE가 iNOS의 발현을 억제하지 못하였다. 이상의 결과를 종합해보면, ACDE는 대식세포에서 PI3K/Akt 및 MAPK와 Nrf2 신호전달과정을 통해 HO-1의 발현을 유도함으로써 NO와 같은 염증매개물질의 생성을 억제한다는 것을 알 수 있다. 이러한 연구결과는 ACDE가 항염증제로 사용될 수 있음을 시사한다.

• Journal of Preventive Medicine and Public Health
• /
• 제32권4호
• /
• pp.459-466
• /
• 1999

Objectives:eurotoxicity is mediated by nitric oxide(NO) in the rat primary neuronal cultures and assess the effect of $Mn^{2+}$ on the N-methyl-D aspartate(NMDA) receptors. Methods: We have used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay to examine the effect of cytotoxicity of $MnCl_2$ in neuronal cells , NO production was determined by measuring nirites, a stable oxidation product of NO. The neurons in the rat that contains neuronal nitric oxide synthase(nNOS) were examined by immunofluorescence and confocal microscopy. The effects of $Mn^{2+}$ on the NMDA receptors was assesed by the whole cell voltage clamp technique. Results: We showed that the NO release and NOS expression was increased with 500uM $MnCl_2$ treatment and an NOS inhibitors, $N^G-nitro-L-arginine$, prevented neurotoxicity elicited by manganese. In the electrophysiological study, $Mn^{2+}$ does not block or activate the NMDA receptors and not pass through the NMDA receptors in a neurons of basal ganglia. Conclusions: It is concluded that manganese neurotoxicity in basal ganglia was partially mediated by nitric oxide in the cell culture model.

• Biomolecules & Therapeutics
• /
• 제8권2호
• /
• pp.140-146
• /
• 2000

Recently, we reported that immunostimulation of primary rat cortical astrocyte caused stimulation of glucose deprivation induced apoptotic cell death. To enhance the understanding of the mechanism of the potentiated cell death of clucose-deprived astrocyte by immunostimulation, we investigated the effect of immunostimulation on the glucose deprivation induced cell death of rat C6 glioma cells. Co-treatment of C6 glioma cells with lipopolysaccharide (LPS, $1\;{\mu}\textrm{g}/ml$) and interferon ${\gamma}(IFN{\gamma},\;100U/ml)$ is serum free condition caused marked elevationo f nitric oxide production ($>50\;{\mu}M$). In this condition, glucose deprivation caused significant release of lactate dehdrogenase (LDH) from C6 glioma cells while control cells did not show LDH release. To investigate whether elevated level of nitric oxide is responsible for the enhanced LDH release in glucose-deprived condition, C6 glioma cells were treated with 3-morphorinosydnonimine (SIN-1) and it was observed that SIN-1 caused increase in LDH release from glucose-deprived C6 glioma cells. Treatment of C6 glioma cells with $25\;{\mu}M$ of pyrrolidinedithiocarbamate (PDTC) which inhibit Nuclear factor kB (NF-kB) activation, caused complete inhibition of nitric oxide production. Treatment of C6 glioma cells with NO synthase inhibitors, $N^{G}$-nitro-L-arginine (NNA) or L-$N{\omega}$-nitro-L-arginine methyl ester (L-NAME), caused inhibition of nitric oxide production and also glucose deprivation induced cell death of cytokine-stimulated C6 glioma cells. In addition, diaminohydroxypyrimidine (DAHP, 5 mM) which inhibits the synthesis of tetrahydrobiopterine (BH4), one of essential cofactors for iNOS activity, caused complete inhibition of NO production from immunostimulated C6 glioma cells. The results from the present study suggest that immunostimulation causes potentiation of glucose deprivation induced death of C6 glioma cells which is mediated at least in part by the increased production of nitric oxide. The vulnerability of immunostimulated C6 glioma cells to hypoglycemic insults may implicate that the elevated level of cytokines in various ischemic and neurodegenerative diseases may play a role in their pathogenesis.

• 생명과학회지
• /
• 제21권1호
• /
• pp.44-55
• /
• 2011

본 연구는 복분자의 수용성 추출물(RCE) 이 배양된 제대정맥내피세포에서 내피세포 NO 합성 효소(eNOS)의 발현과 활성에 미치는 효과를 연구하고, 이러한 RCE의 효과가 어떤 신호전달 과정을 거치는지를 밝히기 위한 것이다. 연구에 따르면 RCE가 제대정맥내피세포에서 NO의 생성을 증가시키는데 이는 iNOS 보다는 eNOS의 활성화에 의한 것임을 이들의 특이 억제제를 사용한 실험으로 확인할 수 있었다. 나아가 eNOS에 의한 NO 생성 증가는 이 효소의 활성 증가뿐만 아니라 mRNA 수준에서의 발현증가에도 기인함을 확인할 수 있었다. PKC-특이억제제인 RO-317549는 RCE에 의한 NO 생성의 증가에 별다른 영향을 주지 않았으나, 에스트로젠 수용체-특이 억제제인 Tamoxifen, ERK-특이 억제제인 PD98059와 PI3K/Akt-특이 억제제인 LY-294002는 제대정맥내피세포에서 RCE에 의해 증가된 NO 생성을 억제하였으며, 이는 두 저해제가 eNOS의 활성형인 pSer1177의 양을 감소시키며, 특히 PD98059는 비활성형인 pThr495의 양도 증가시키기 때문임을 알 수 있었다. 이상의 결과로써, 복분자 수추출물은 사람의 제대정맥내피세포에서 NO 생성을 증가시키며, 이는 eNOS의 발현을 증가시킬 뿐만 아니라, ERK와 PI3K/Akt의 신호전달과정을 거쳐서 eNOS의 활성도 증가시키기 때문임을 확인할 수 있었다.

• Journal of Ginseng Research
• /
• 제37권4호
• /
• pp.413-424
• /
• 2013

Korean Red Ginseng extract (KRGE) is a traditional herbal medicine utilized to prevent endothelium dysfunction in the cardiovascular system; however, its underlying mechanism has not been clearly elucidated. We here examined the pharmacological effect and molecular mechanism of KRGE on apoptosis of human umbilical vein endothelial cells (HUVECs) in a serum-deprived apoptosis model. KRGE protected HUVECs from serum-deprived apoptosis by inhibiting mitochondrial cytochrome c release and caspase-9/-3 activation. This protective effect was significantly higher than that of American ginseng extract. KRGE treatment increased antiapoptotic Bcl-2 and Bcl-$X_L$ protein expression and Akt-dependent Bad phosphorylation. Moreover, KRGE prevented serum deprivation-induced subcellular redistribution of these proteins between the mitochondrion and the cytosol, resulting in suppression of mitochondrial cytochrome c release. In addition, KRGE increased nitric oxide (NO) production via Akt-dependent activation of endothelial NO synthase (eNOS), as well as inhibited caspase-9/-3 activities. These increases were reversed by co-treatment of cells with inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) and pre-incubation of cell lysates in dithiothreitol, indicating KRGE induces NO-mediated caspase modification. Indeed, KRGE inhibited caspase-3 activity via S-nitrosylation. These findings suggest that KRGE prevents serum deprivation-induced HUVEC apoptosis via increased Bcl-2 and Bcl-$X_L$ protein expression, PI3K/Akt-dependent Bad phosphorylation, and eNOS/NO-mediated S-nitrosylation of caspases. The cytoprotective property of KRGE may be valuable for developing new pharmaceutical means that limit endothelial cell death induced during the pathogenesis of vascular diseases.