• Molecules and Cells
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• 제25권2호
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• pp.253-257
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• 2008

Acrolein is a highly electrophilic ${\alpha},{\beta}$-unsaturated aldehyde present in a number of environmental sources, especially cigarette smoke. It reacts strongly with the thiol groups of cysteine residues by Michael addition and has been reported to inhibit nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) activation by lipopolysaccharide (LPS). The mechanism by which it inhibits $NF-{\kappa}B$ is not clear. Toll-like receptors (TLRs) play a key role in sensing microbial components and inducing innate immune responses, and LPS-induced dimerization of TLR4 is required for activation of downstream signaling pathways. Thus, dimerization of TLR4 may be one of the first events involved in activating TLR4-mediated signaling pathways. Stimulation of TLR4 by LPS activates both myeloid differential factor 88 (MyD88)- and TIR domain-containing adapter inducing $IFN{\beta}$ (TRIF)-dependent signaling pathways leading to activation of $NF-{\kappa}B$ and IFN-regulatory factor 3 (IRF3). Acrolein inhibited $NF-{\kappa}B$ and IRF3 activation by LPS, but it did not inhibit $NF-{\kappa}B$ or IRF3 activation by MyD88, inhibitor ${\kappa}B$ kinase $(IKK){\beta}$, TRIF, or TNF-receptor-associated factor family member-associated $NF-{\kappa}B$ activator (TANK)-binding kinase 1 (TBK1). Acrolein inhibited LPS-induced dimerization of TLR4, which resulted in the down-regulation of $NF-{\kappa}B$ and IRF3 activation. These results suggest that activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain chemicals with a structural motif that enables Michael addition.

• 대한화장품학회지
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• 제32권3호
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• pp.135-140
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• 2006

Nucler factor-kappa B (NF-kappaB) 프로모터는 염증성 질환을 유도하는 호염증성 시토카인의 발현에 중요한 역할을 수행하는 전사인자 중의 하나이다. 본 실험에서는 200 여종의 식물추출물들로부터 항염효능이 있는 추출물을 선발하기 위해 NF-kappaB 리포터 실험을 수행하였다. NF-kappaB 리포터 실험결과, 12종의 식물추출물, 즉 개나리, 고추잎, 박하, 뱀딸기, 뽕나무, 삼백초, 솔잎, 양애줄기, 약쑥, 어성초, 왕벚꽃가지, 조릿대 등이 lipopolysaccharide (LPS)에 의해 유도된 NF-kappaB 프로모터 활성을 농도의존적으로 억제하는 것을 확인하였다. 이들 12종의 식물추출물이 호염증성 시토카인 발현에도 동일한 효과를 나타내는지 알아보기 위해 tumor necrosis factor-alpha (TNF alpha)와 인터루킨-8에 대한 ELISA실험을 실시하였다. ELISA실험 결과, NF-kappaB 리포터 실험결과와 동일하게, TNF-alpha와 인터루킨-8 생산이 12종 식물추출물 모두에서 감소됨을 관찰하였다. 이러한 실험결과는, 12종의 식물 추출물에서 보여지는 호염증성 시토카인 억제효과가 NF-kappaB 프로모터 활성억제를 통해 이루어지고 있음을 시사한다. 또한, 이들 12종 식물은 diphenyl-p-picrylhydrazyl (DPPH) assay를 통해 살펴본 결과 높은 항산화 활성도 있음을 확인하였다. 이상의 결과로부터, 12종의 식물 추출물은 염증성 피부질환 전용 화장품 제형에서 항염 및 자극완화 소재로 응용될 수 있음을 확인하였다.

• IMMUNE NETWORK
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• 제2권3호
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• pp.137-141
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• 2002

Among the members of the inhibitor of apoptosis (IAP) protein family, only Livin and survivin have been reported to have variant forms. We have found a variant form of c-IAP2 through the interaction with the X protein of HBV using the yeast two-hybrid system. In contrast to the wild-type c-IAP2, the variant form has two stretches of sequence in the RING domain that are repeated in the C-terminus that would disrupt the RING domain. We demonstrate that the variant form has an inhibitory effect on TNF-mediated $NF-{\kappa}B$ activation unlike the wild-type c-IAP2, which increases TNFmediated $NF-{\kappa}B$ activation. These results suggest that this variant form has different activities from the wild-type and the RING domain may be involved in the regulation of TNF-induced $NF-{\kappa}B$ activation.

• The Korean Journal of Physiology and Pharmacology
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• 제6권6호
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• pp.319-325
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• 2002

The induction of interleukin-6 (IL-6) using combined proinflammatory agents $(LPS/IFN-{\gamma}\;or\;TNF-{\alpha}/IFN-{\gamma})$ was studied in relation to p38 mitogen-activated protein kinase (MAPK) and $NF-{\kappa}B$ transcriptional factor in primary neonatal cardiomyocytes. When added to cultures of cardiomyocytes, the combined agents $(LPS/IFN-[\gamma}\;or\;TNF-{\alpha}/IFN-{\gamma})$ had stimulatory effect on the production of IL-6 and the elevation was significantly reduced by SB203580, a specific p38 MAPK inhibitor. SB203580 inhibited protein production and gene expression of IL-6 in a concentration-dependent manner. In this study, $IFN-{\gamma}$ enhancement of $TNF-{\alpha}-induced\;NF-{\kappa}B$ binding affinity as well as p38 MAP kinase activation was observed. However, a specific inhibitor of p38 MAPK, SB203580, had no effect on $TNF-{\alpha}/IFN-{\gamma}\;or\;LPS/IFN-{\gamma}-induced\;NF-{\kappa}B$ activation. This study strongly suggests that these pathways about $TNF-{\alpha}/IFN-{\gamma}$ or $LPS/IFN-{\gamma}-activated$ IL-6 release can be primarily dissociated in primary neonatal cardiomyocytes.

• Journal of Acupuncture Research
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• 제21권6호
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• pp.19-36
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• 2004

Objective : The purpose of this study was to investigate the effect of Bee Venom and Melittin Solution on the lipopolysaccharide(LPS) and sodium nitroprusside(SNP)-induced expression of prostaglandin $E_2(PGE_2)$, cyclooxygenase-2(COX-2), nuclear factor kappa B($NF-{\kappa}B$) and nuclear factor kappa B($NF-{\kappa}B$) dependent luciferase activity in RAW 264.7 cells, a murine macrophage cell line. Methods : The expression of PGE2 was determined by determination of $PEG_2$, COX-2 was by western blotting with corresponding antibodies, $NF-{\kappa}B$ was by gel mobility shift assay method and $NF-{\kappa}B$ dependent luciferase activity was investigated by luciferase assay in RAW 264.7 cells. Results : 1. LPS and SNP-induced expression of $PEG_2$ was significant after 24hour. 2. The 0.5, 1 and $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly LPS-induced expression of $PEG_2$ and, the $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly SNP-induced expression of $PEG_2$ compared with control, respectively. The 0.5 and $1{\mu}g/mL$ of bee venom could not significantly inhibit SNP-induced expression of $PEG_2$ compared with control. 3. The $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly LPS and SNP-induced expression of COX-2 compared with control, respectively. The 0.5 and $1{\mu}g/mL$ of bee venom inclined to decrease LPS and SNP-induced expression of COX-2 compared with control. 4. The 0.5, 1 and $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly LPS and SNP-induced expression of $NF-{\kappa}B$ compared with control, respectively. 5. The 0.5, 1 and $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly LPS-induced expression of $NF-{\kappa}B$ dependent luciferase activity and the 1 and $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly SNP-induced expression of $NF-{\kappa}B$ dependent luciferase activity compared with control, respectively. The $NF-{\kappa}B$ inhibitor also inhibited significantly LPS and SNP-induced expression of $NF-{\kappa}B$ dependent luciferase activity compared with control. 6. The 0.5, 1 and $5{\mu}g/mL$ of bee venom and the 5 and $10{\mu}g/mL$ of melittin solution inhibited significantly LPS + IFN-${\gamma}$, TNF-${\alpha}$ and LPS + TNF-${\alpha}$-induced expression of $NF-{\kappa}B$ dependent luciferase activity compared with control, respectively. The $NF-{\kappa}B$ inhibitor also inhibited significantly LPS and SNP-induced expression of $NF-{\kappa}B$ dependent luciferase activity compared with control. Conclusions : These results suggest the inhibitory action of bee venom and melittin solution on the inflammatory mediators such as $PEG_2$, COX-2 and $NF-{\kappa}B$.

• Archives of Pharmacal Research
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• 제23권1호
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• pp.54-58
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• 2000

To elucidate the molecular mechanisms for the suppression of LPS-induced nitric oxide (NO) production by a dehydrocostus lactone (DL) from Saussurea lappa, we examined the preventive effect of this compound on $NF-{\kappa}B$ activation in LPS-treated RAW 264.7 macrophages and U937 human monocytic cells. The results suggest that the suppression of NO production is mediated by the inhibitory action on the i-NOS gene expression through the inactivation of $NF-{\kappa}B$ and this sesquiterpene lactone can act as a pharmacological inhibitor of the $NF-{\kappa}B$ activation.

• 대한의생명과학회지
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• 제20권4호
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• pp.244-249
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• 2014

Arazyme is a metalloprotease secreted by Aranicola proteolyticus that was previously shown to suppress cytokine expression of keratinocytes and endothelial cells and inhibit histopathological features in an atopic dermatitis-like animal model. However, the regulatory effects of arazyme in other allergic diseases have yet to be elucidated. In this study, we investigated whether arazyme is effective against neutrophil apoptosis in allergic diseases such as allergic rhinitis and asthma. Arazyme inhibited neutrophil apoptosis of normal subjects in a dose-dependent manner. However, the antiapoptotic effect of arazyme was reversed by LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of MEK, and BAY-11-7085, an inhibitor of NF-${\kappa}B$. Arazyme induced activation of NF-${\kappa}B$ via PI3K/Akt/ERK pathway. The anti-apoptotic effect of arazyme is associated with inhibition of cleavage of caspase 3 and caspase 9. Arazyme inhibited constitutive apoptosis of neutrophil in a dose-dependent manner in allergic subjects, and its mechanism was shown to be associated with PI3K/Akt/ERK/NF-${\kappa}B$. The results presented here improve our understanding of neutrophil apoptosis regulation and will facilitate development of drugs for treatment of allergic diseases.

• Tuberculosis and Respiratory Diseases
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• 제48권2호
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• pp.166-179
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• 2000

연구배경: 현재 사용되고 있는 많은 항암제는 암세포의 apoptosis를 유도함으로서 암세포를 사멸시키는 것이 주된 작용 기전이다. 항암제에 의한 치료 실패의 주된 원인은 암세포의 apoptosis에 대한 내성 획득에 의한 것으로 이해되고 있다. 최근의 보고에 의하면 많은 암세포주에서 apoptosis에 대한 내성 획득에 NF-${\kappa}B$의 활성화가 중요한 역할을 한다고 알려져 있지만 일부 세포에서는 상반된 결과를 보이고 있어 apoptosis에 대한 내성 획득과 NF-${\kappa}B$의 활성화와의 관련성은 세포에 따라 차이를 보이고 있다. 본 연구에서는 비소세포폐암 세포주가 TNF-$\alpha$ 유발 apoptosis에 저항을 나타내는 기전에서 NF-${\kappa}B$의 역할을 규명하고자 하였다. 방 법: 비소세포폐암세포주인 NCI-H157 세포에 TNF-$\alpha$ cycloheximide(CHX), TNF-$\alpha$와 CHX를 각각 투여하고 24시간 후 MTT assay로 세포생존율을 평가하였고 apoptosis의 발생 유무는 PARP에 대한 Westrn 분석으로 평가하였다. Ad5LacZ와 $Ad5I{\kappa}B{\alpha}SR$를 20시간 동안 감염시킨 각각의 세포를 1, 5, 10, 20, 50 ng/ml의 TNF-$\alpha$로 24, 48시간 자극 후 MTT assay를 시행하였고, 같은 농도의 TNF-$\alpha$로 24, 48시간 자극 후 PARP에 대한 Western 분석을 시행하였다. $I{\kappa}B{\alpha}$의 분해를 억제하는 proteasome inhibitor 인 MG132를 전처치하고 TNF-$\alpha$로 24, 48 시간 자극 후 MTT assay와 PARP에 대한 Westrn 분석을 시행하였다. $Ad5I{\kappa}B{\alpha}SR$를 감염시킨 세포를 TNF-$\alpha$로 자극한 후 NF-${\kappa}B$의 활성화를 EMSA로 평가하였다. 결 과: 1. TNF-$\alpha$ 단독 투여로는 세포 생존율의 감소와 apoptosis가 관찰되지 않았고 TNF-$\alpha$와 CHX를 같이 투여하였을 때는 세포 생존율의 감소와 함께 apoptosis가 유도되었다. 2. $Ad5I{\kappa}B{\alpha}SR$의 감염으로 $I{\kappa}B{\alpha}$가 과발현된 세포와 MG132를 전처치한 세포에서 TNF-$\alpha$ 자극에 의한 NF-${\kappa}B$의 활성화가 억제되었다. 3. $Ad5I{\kappa}B{\alpha}$ SR를 감염시킨 세포와 MG132를 전처치하여 NF-${\kappa}B$의 활성을 억제한 세포에서 TNF-$\alpha$ 자극 후 세포 생존율이 감소하고 apoptosis가 유도되었다. 결 론: 비소세포폐암 세포주의 TNF-$\alpha$ 유발 apoptosis에 대한 내성은 NF-${\kappa}B$의 활성화에 의해 생성되는 새로운 단백의 발현에 의한 것으로 생각되며, NF-${\kappa}B$ 활성화의 억제로 이를 극복할 수 있을 것으로 사료된다.

• Molecules and Cells
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• 제26권1호
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• pp.48-52
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• 2008

We here demonstrate an anti-inflammatory action of raloxifene, a selective estrogen receptor modulator, in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells. Treatment with raloxifene at micromolar concentrations suppressed the production of nitric oxide (NO) by down-regulating expression of the inducible nitric oxide synthase (iNOS) gene in LPS-activated cells. The decreased expression of iNOS and subsequent reduction of NO were due to inhibition of nuclear translocation of transcription factor NF-${\kappa}B$. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. In addition, pretreatment with raloxifene reduced LPS-induced Akt phosphorylation as well as NF-${\kappa}B$ DNA binding activity and NF-${\kappa}B$-dependent reporter gene activity. Thus our findings indicate that raloxifene exerts its anti-inflammatory action in LPS-stimulated macrophages by blocking the PI 3-kinase-Akt-NF-${\kappa}B$ signaling cascade, and eventually reduces expression of pro-inflammatory genes such as iNOS.

• International Journal of Oral Biology
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• 제39권1호
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• pp.15-22
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• 2014

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-${\kappa}B$, NF-${\kappa}B$-related genes, inflammatory cytokines, TNF-${\alpha}$ and IL-$1{\beta}$ in RAW 264.7 cells. NF-${\kappa}B$ inhibitor pretreatment significantly reduced the levels of TNF-${\alpha}$ and IL-$1{\beta}$ mRNA and protein. In addition, the Aa LPS-induced TNF-${\alpha}$ and IL-$1{\beta}$ expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-${\alpha}$ and IL-$1{\beta}$ expression through NF-${\kappa}B$ and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.