• 대한한의학회지
• /
• 제24권2호
• /
• pp.166-178
• /
• 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.

• 한방안이비인후피부과학회지
• /
• 제26권2호
• /
• pp.45-57
• /
• 2013

Objectives : Daucus carota sativa has been frequently used as food supplements in many of the Asian countries, and a nutritional medical drug in traditional medicine. This research investigated the effects of Daucus carota sativa extract (DCE) on acute phases of inflammation in Raw 264.7 cells treated with lipopolysaccharide (LPS) in terms of the inhibition of nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$) and pro-inflammatory cytokines production. Methods : NO, $PGE_2$, tumor necrosis factor (TNF)-${\alpha}$, interleukin-$1{\beta}$ and interleukin-6 contents were assayed by ELISA, and expressions of inflammation-related proteins such as inducible NO synthase (iNOS) were determined by immunoblot analyses. Results : DCE treatment attenuated the LPS ability to increase the productions of NO and $PGE_2$ as well as the protein level of iNOS in a concentration-dependent manner. Consistently, treatment of the cells with DCE suppressed the production of TNF-${\alpha}$, interleukin-$1{\beta}$ and interleukin-6. DCE also caused decreases of inhibitor of ${\kappa}B{\alpha}$ phosphorylation induced by LPS in the cells, which means DCE inhibition of NF-${\kappa}B$ activity. Furthermore, DCE blocked LPS-induced phosphorylation of p38 and SAPK/JNK. Conclusion : This study showing here may be of help to understand the action mechanism of DCE, and provide the information for the medical use of Daucus carota sativa for the inflammatory disease.

• 생명과학회지
• /
• 제32권11호
• /
• pp.899-907
• /
• 2022

Quercetin은 과일과 채소에 풍부한 플라보노이드 중의 하나로써, 항산화, 항염증, 항암, 항바이러스 활성 등 다양한 약리학적 활성을 가지고 있는 것으로 알려져 있다. 본 연구에서는 in vitro 모델에서 quercetin의 항염증 활성과 작용기전을 연구하였다. Quercetin은 LPS로 자극된 RAW264.7에서 세포 생존율에 영향 없이 NO 생산을 농도 의존적으로 저해하였고, iNOS와 COX-2 단백질의 발현을 억제하였다. 게다가, quercetin은 LPS로 유도된 p38, JNK, ERK의 인산화를 농도 의존적으로 저해하였고, NF-κB p65 단백질과 억제자인 IκBα 단백질의 인산화를 저해하였다. 이러한 결과는 quercetin의 항염증 활성이 MAPK 경로와 NF-κB를 조절함으로써 이루어진다는 것을 시사한다. Quercetin에 의해 4종류의 친 염증성 cytokine (CSF2, IL-1β, IL-6, TNF-α)의 발현 변화를 정량적 real-time PCR 방법으로 확인한 결과, 모든 cytokine 유전자의 발현이 감소됨을 확인하였다. 종합적으로, 본 연구결과는 플라보노이드 quercetin이 RAW264.7 세포에서 LPS로 유도된 염증반응을 MAPK 경로와 NF-κB경로를 통해 억제하고 친염증성 cytokine 유전자의 발현을 억제함으 로써 조절한다는 것을 제시한다.

• 생명과학회지
• /
• 제29권4호
• /
• pp.402-409
• /
• 2019

생삼을 쪄서 건조시킨 홍삼은 전통적으로 사용되고 있는 약재로서 면역, 내분비 및 중추신경계 작용을 증진시키며 염증을 억제하는 효과가 있는 것으로 알려져 있다. 본 연구에서는 그람 양성균의 세포벽 성분인 lipoteichoic acid (LTA)에 의한 염증반응에 홍삼추출액(RGE)이 항염증 효과를 가지는지 관찰하고 그 작용 기전을 연구하였다. BV-2 소교세포에서 RGE는 세포에 독성을 유도하지 않으면서 LTA로 인한 nitric oxide (NO)의 생성과 inducible nitric oxide synthase (iNOS) 발현을 억제하였으며, NF-kB p65의 핵으로의 이동과 IkB-a의 분해 또한 억제하였다. 한편, RGE는 농도의존적으로 heme oxygenase-1 (HO-1)의 발현을 유도하였으며, HO-1 siRNA를 처리했을 때는 RGE가 iNOS의 발현을 억제하지 못하였다. RGE는 HO-1의 발현에 관여하는 전사인자인 nuclear factor E2-related factor 2 (Nrf2)를 핵으로 이동을 촉진시켰다. 또한 RGE에 의한 HO-1의 발현은 phosphatidylinositol-3-kinase(PI-3K) 및 MAPK 억제제에 의해 감소되었으며, RGE가 Akt와 ERK, p38, JNK의 인산화를 유도하였다. 이상의 결과를 종합해보면, RGE는 PI-3K/Akt 및 ERK, p38, JNK 신호전달과정을 통해 HO-1의 발현을 유도함으로써 NO와 같은 염증매개물질의 생성을 억제한다는 것을 알 수 있다. 그러므로 홍삼추출액은 그람 양성균에 의한 신경염증과 염증관련 신경계 질환의 치료제로서 사용될 수 있을 것이라 사료된다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제36권6호
• /
• pp.481-489
• /
• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

• 생명과학회지
• /
• 제33권11호
• /
• pp.859-867
• /
• 2023

루페올은 5환성 트리테르펜의 일종으로 많은 질병에 치료 효과가 있는 것으로 보고되었으나, 인슐린 저항성에 미치는 영향은 명확하지 않다. 본 연구에서는 3T3-L1 지방세포에서 루페올의 IRS-1 인산화 억제능을 통해 인슐린 저항성 개선효과를 조사하였다. 3T3-L1 세포를 배양하고 TNF-α를 24시간 동안 처리하여 인슐린 저항성을 유도하였다. 서로 다른 농도의 루페올(15, 30 μM) 또는 100 nM의 rosiglitazone을 처리한 세포를 배양한 후, 용해된 세포를 이용하여 western blotting을 시행하였다. 실험결과 루페올은 지방세포에서 TNF-α에 의해 유발되는 인슐린 신호전달의 음성 조절자와 염증 활성화 단백질 kinase에 대한 개선 효과를 나타냈다. 인슐린 신호전달의 음성 조절자인 PTP-1B와 JNK의 활성 및 IKK와 염증활성화 단백질키나아제의 활성을 억제하였다. 또한, 루페올은 IRS-1의 serine 인산화는 하향 조절하고 tyrosine 인산화는 상향 조절하였다. 그 후, 하향 조절된 PI3K/AKT 경로가 활성화되고, GLUT 4의 세포막 전위가 자극되어, 결과적으로 인슐린 저항성이 유도된 3T3-L1 지방세포에서에서 세포내 포도당 흡수가 증가하였다. 본 연구결과, 루페올은 3T3-L1 지방세포에서 인슐린 신호전달 및 염증 활성화 단백질 kinsase들의 음성 조절인자를 억제하여, IRS-1의 serine 인산화를 하향 조절함으로써 TNF-α 유발 인슐린 저항성을 개선할 수 있을 것으로 사료된다.

• Animal Bioscience
• /
• 제37권2호
• /
• pp.303-314
• /
• 2024

Objective: Rutin, also called vitamin P, is a flavonoids from plants. Previous studies have indicated that rutin can alleviate the injury of tissues and cells by inhibiting oxidative stress and ameliorating inflammation. There is no report on the protective effects of rutin on goat rumen epithelial cells (GRECs) at present. Hence, we investigated whether rutin can alleviate lipopolysaccharide (LPS)-induced damage in GRECs. Methods: GRECs were cultured in basal medium or basal medium containing 1 ㎍/mL LPS, or 1 ㎍/mL LPS and 20 ㎍/mL rutin. Six replicates were performed for each group. After 3-h culture, the GRECs were harvested to detect the relevant parameters. Results: Rutin significantly enhanced the cell activity (p<0.05) and transepithelial electrical resistance (TEER) (p<0.01) and significantly reduced the apoptosis rate (p<0.05) of LPS-induced GRECs. Rutin significantly increased superoxide dismutase, glutathione peroxidase, and catalase activity (p<0.01) and significantly decreased lactate dehydrogenase activity and reactive oxygen species and malondialdehyde (MDA) levels in LPS-induced GRECs (p<0.01). The mRNA and protein levels of interleukin 6 (IL-6), IL-1β, and C-X-C motif chemokine ligand 8 (CXCL8) and the mRNA level of tumor necrosis factor-α (TNF-α) and chemokine C-C motif ligand 5 (CCL5) were significantly increased in LPS-induced GRECs (p<0.05 or p<0.01), while rutin supplementation significantly decreased the mRNA and protein levels of IL-6, TNF-α, and CXCL8 in LPS-induced GRECs (p<0.05 or p<0.01). The mRNA level of toll-like receptor 2 (TLR2), and the mRNA and protein levels of TLR4 and nuclear factor κB (NF-κB) was significantly improved in LPS-induced GRECs (p<0.05 or p<0.01), whereas rutin supplementation could significantly reduce the mRNA and protein levels of TLR4 (p<0.05 or p<0.01). In addition, rutin had a tendency of decreasing the protein levels of CXCL6, NF-κB, and inhibitor of nuclear factor kappa-B alpha (0.05

• 대한한방내과학회지
• /
• 제38권6호
• /
• pp.1021-1034
• /
• 2017

Objectives: The aim of this study was to investigate the effects of Gaejigayonggolmoryo-tang (GYT) on ulcerative colitis induced by dextran sodium sulfate (DSS) in mice. Methods: Colitis was induced by free drinking of 5% DSS in six-week-old male ICR mice. The experimental groups were the sample group, the control group, and the normal group. The sample group was treated with GYT for three days after being was given 5% DSS for five days. The control group was given water, instead of GYT, for three days after the five days of 5% DSS. The normal group was untreated (not given 5% DSS), for comparison purposes. Results: Cellular experiments showed that GYT inhibits the expression of the inflammatory enzymes COX-2 and iNOS, and the production of NO. Based on the primary cellular experiments, the effects of GYT on ulcerative colitis induced by DSS of mouse tissues were investigated. GYT reduced tissue damage and apoptosis by inhibiting the expression of the inflammatory enzymes $NF-{\kappa}B$ p65, COX-2, and iNOS. In the cellular experiment, GYT was more effective in inhibiting the expression of COX-2 than in inhibiting the expression of iNOS. GYT was evidently effective in tissues in inhibiting the expression of COX-2. Conclusions: Based on the results here, GYT may have therapeutic effects on ulcerative colitis induced by DSS. GYT is worthy of research and development as a COX-2 inhibitor and a potential drug for inflammatory bowel diseases from natural products. Further investigations for exact mechanisms will be needed.

• Applied Biological Chemistry
• /
• 제48권4호
• /
• pp.411-417
• /
• 2005

마우스 대식세포주인 RAW264.7 세포에서 곽향(Agastache rugosa)을 포함한 21종의 한약재에서 제조한 열수추출물의 NO생산에 대한 조절효과를 조사하였다. 모든 한약재 추출물은 LPS자극으로 생산된 NO에 대하여 뚜렷한 소거활성을 보이지 않았으나, LPS 무처리 조건에서 곽향이 RAW264.7 세포의 NO생산을 강력하게 유도하였다. $200{\mu}M$의 NOS2의 저해제인 $N^G-monomethyl-L-arginine(N^GMMA$)의 처리에 의하여 곽향이 유도하는 NO 생산은 유의적으로 감소되었다. 또한 $NF-{\kappa}B$ 저해제인 pyrrolidine dithiocarbamate(PDTC)의 처리로 NO 생산이 $100{\mu}M$에서 약 79%까지 감소하였다. 이상의 실험 결과는 곽향 열수추출물이 RAW264.7 세포의 NOS2 발현의 이차적인 세포 내 신호를 발생시킬 수 있으며, NO는 L-arginine 의존적 경로에 의하여 생성된다는 사실을 시사하였다.

• Journal of Microbiology and Biotechnology
• /
• 제24권8호
• /
• pp.1051-1058
• /
• 2014

Platelet-activating factor receptor (PAFR) plays an important role in bacterial infection and inflammation. We examined the effect of the bacterial cell wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) on PAFR expression in THP-1, a monocyte-like cell line. LPS and aLTA, but not pLTA, significantly increased PAFR expression, whereas priming with pLTA inhibited LPS-mediated or aLTA-mediated PAFR expression. Expression of Toll-like receptor (TLR) 2 and 4, and CD14 increased with LPS and aLTA treatments, but was inhibited by pLTA pretreatment. Neutralizing antibodies against TLR2, TLR4, and CD14 showed that these receptors were important in LPS-mediated or aLTA-mediated PAFR expression. PAFR expression is mainly regulated by the nuclear factor kappa B signaling pathway. Blocking PAF binding to PAFR using a PAFR inhibitor indicated that LPS-mediated or aLTA-mediated PAF expression affected TNF-${\alpha}$ production. In the mouse small intestine, pLTA inhibited PAFR, TLR2, and TLR4 expression that was induced by heat-labile toxin. Our data suggested that pLTA has an anti-inflammatory effect by inhibiting the expression of PAFR that was induced by pathogenic ligands.