(A) synergistic interaction between transcription factors nuclear factor-κB and signal transducers and activators of transcription 3 promotes gastric cancer cell migration and invasion

윤지연 서울대학교 대학원 2014 국내박사

Purpose: The transcription factor nuclear factor-κB (NF-κB) has been implicated in gastric cancer metastasis, but the underlying molecular mechanisms remain unclear. The present study investigated the role of the interaction between NF-κB and signal transducers and activators of transcription 3 (STAT3) in controlling metastatic potential of gastric cancer cells. Experimental design: Immunohistochemistry for NF-κB p65 (RelA), phospho-Tyr705-STAT3 (pSTAT3), or matrix metalloproteinase 9 (MMP9) was performed on tissue array slides containing 255 gastric carcinoma specimens. NF-κB inhibition in SNU-638 and MKN1 gastric cancer cell lines were performed by transduction with a retroviral vector containing NF-κB repressor mutant of IκBα, and STAT3 was silenced by RNA interference. Luciferase reporter assay, double immunofluorescence staining and immunoblotting were also performed. Cell migration and invasion were determined by wound-healing assay and invasion assay, respectively. Results: NF-κB and STAT3 were constitutively activated and were positively correlated (P = 0.038) in gastric cancer tissue specimens. In cell culture experiments, NF-κB inhibition reduced STAT3 expression and activation, whereas STAT3 silencing did not affect NF-κB activation. Moreover, both NF-κB inhibition and STAT3 silencing decreased gastric cancer cell migration and invasion in a synergistic manner. In addition, both NF-κB activation and STAT3 activation were positively correlated with MMP9 in gastric cancer tissues (P = 0.001 and P = 0.022, respectively), decreased E-cadherin expression and increased Snail and MMP9 expressions in cultured cells. Conclusion: NF-κB and STAT3 are positively associated and synergistically contribute to the metastatic potential of gastric cancer cells. Thus, dual use of NF-κB and STAT3 inhibitors may enhance the efficacy of the anti-metastatic treatment of gastric cancer.

The clinicopathological and prognostic significances of nuclear factor-kappa B (NF-κB) activation in papillary thyroid carcinoma : 갑상선 유두암에서 Nuclear factor-kappa B (NF-κB)의 임상-병리학적 중요성 및 예후에 미치는 영향

Pyo, Jung-Soo 을지대학교 대학원 2014 국내박사

Purpose: Nuclear factor-κB (NF-κB) is involved in proliferation, angiogenesis, and metastasis in various malignancies; however, little is known about the role of NF-κB and the associated pathway in papillary thyroid carcinoma (PTC). The purpose of this study is to elucidate the clinicopathological and prognostic significances of the NF-κB signaling pathway in PTC. Materials and Methods: One-hundred twenty two patients with conventional PTC were investigated NF-κB RelA nuclear expression by immunohistochemistry and the correlation between NF-κB RelA nuclear expression and clinicopathological parameters including proliferation index, BRAFV600E mutation, cullin-1 expression and the presence of psammoma body (PB). Results: Among 122 conventional PTCs, NF-κB RelA nuclear expression was found in 91 PTCs (74.6%). There were significant differences in extrathyroidal extension (P = 0.031), nodal metastasis (P = 0.021) and BRAFV600E mutation (P = 0.039), between NF-κB-positive and negative PTCs. NF-κB RelA nuclear expression was showed more frequent in overt PTCs (larger than 1 cm) than papillary thyroid microcarcinomas (PMCs) (P = 0.001). The expressions of cullin-1, which is involved in ubiquitination of phosphorylated IκBα, were found in 84 of 108 PTCs (77.8%). The nuclear expressions of NF-κB RelA were highly showed in cullin-1-positive PTCs than in cullin-1-negative PTCs (83.3% vs. 54.2%, respectively) (P = 0.003). The proliferation index was strongly associated with NF-κB nuclear expression (P = 0.045) and cullin-1 expression (P = 0.009), but not with BRAFV600E mutation (P = 0.141). The presence of PBs was found in 83 of 122 PTCs (68.0%) and significantly correlated with tumor size group and lymph node metastasis (P = 0.018 and P < 0.001, respectively), but not with any other clinicopathological parameters. Extratumoral PBs were only identified in 39 of 83 PTCs with PBs (47.0%) and significantly correlated with higher incidences of tumor multifocality, extrathyroidal extension, and nodal metastasis compared to PTCs with no extratumoral PBs (P < 0.001, P = 0.045 and P < 0.001, respectively). There was significant correlation between NF-κB nuclear expression and the presence of PBs (P = 0.023). Indeed, the presence of intratumoral PBs was significantly associated with NF-κB nuclear expression (P = 0.008). Conclusion: Taken together, these results suggest that NF-κB RelA activation may contribute to tumor growth and aggressiveness and PB formation of PTC after tumor transformation. The expression pattern of NF-κB may serve as a prognostic marker and a potential therapeutic target.

Inhibitory effects of Centella asiatica on the productions of NO, PGE2, TNF-α, IL-1β, and IL-6 via the suppression of NF-κB pathway in LPS-induced macrophages

조백건 경희대학교 대학원 2009 국내박사

Therapeutic effect of transducible Smad3 and p65 transcription modulation domain in inflammatory diseases

박성동 Graduate School, Yonsei University 2015 국내박사

Smad3는 TGF-β가 T 세포의 증식과 분화 뿐만 아니라 염증성 사이토카인의 억제와 같은 다양한 방법을 통해 T 세포의 기능을 조절하는데 있어서 중추적 역할을 하는 전사인자이다. TGF-β는 Smad3 의존적 경로를 통한 Treg 세포의 분화와 Smad3 비의존적 경로를 통한 TH17 세포의 분화를 위해 필요하다. 그러나 TGF-β의 상충된 기능 때문에 TGF-β를 이용한 염증성 질환의 치료 접근은 여전히 문제를 안고 있었다. 그래서 우리는 세포투과성 Smad3(MH1) (tSmad3(MH1))만을 이용하여 TH1, TH2, TH17 그리고 Treg 세포의 기능을 조절할 수 있는 새로운 치료 방법을 개발하였다. tSmad3(MH1)은 CD4+ T 세포의 핵 내에 효과적으로 전달 가능한 재조합 단백질이다. tSmad3(MH1)는 각각 T-bet, GATA3 또는 RORγt에 의해 유도된 TH1, TH2 또는 TH17 관련 사이토카인을 상당히 억제하였고, 반면에 Foxp3에 의해 유도된 Treg 관련 사이토카인을 많이 증가시킴으로써 미접촉 T 세포의 TH1, TH2, TH17 또는 Treg 으로의 분화를 조절한다. tSmad3(MH1)는 전신 홍반 루푸스와 류마티스 관절염의 임상적 심각성과 발생 정도를 약화 시켰고, 이는 치료적인 측면뿐만 아니라 예방적 차원에서도 효과를 보였다. 그러므로 tSmad3(MH1)는 TH1, TH2 또는 TH17 세포와 관련된 염증성 질환의 치료를 위한 새로운 치료 단백질 약물이 될 수 있다. 이러한 접근은 전사 인자가 발병에 중요 역할을 하는 다양한 염증 질병들을 치료하는 데에 적용될 수 있다. 내재면역과 적응면역 반응의 주요 조절자로 잘 알려져 있는 NF-κB의 활성을 억제하는 것이 패혈증과 같은 급성 염증 반응을 치료하는데 있어서 가장 중요한 치료전략이다. NF-κB의 기능을 저해하기 위해, 우리는 p65의 전사 조절 도메인을 Hph-1-PTD에 결합시켜 세포투과성 p65-TMD (tp65-TMD)를 제작하여 세포핵까지 효과적으로 전달하였으며 그것이 내인성 p65 매개 전사복합체의 기능을 막아 NF-κB의 기능 억제자로서의 역할을 제대로 할 수 있다는 것을 보였다. tp65-TMD는 lipopolysaccharide (LPS)에 의해 자극되어진 BV2 미세아교세포에서 분비되는 TNF-α, IL-1β, 그리고 IL-6와 같은 전염증성 사이토카인을 억제하였다. tp65-TMD는 T 세포 활성화와 관련된 ZAP-70, p38, JNK, 그리고 ERK와 같은 신호 매개자의 tyrosine 인산화와 T 세포의 CD69 발현에는 영향을 주지 못하였지만, NF-κB와 IL-2의 전사 활성을 효과적으로 억제함으로서 IL-2, IFN-γ, IL-4, IL-17A 그리고 IL-10과 같은 T 세포 특이적 사이토카인의 분비를 저해하였다. 이를 통해, LPS에 의해 유도되는 급성 패혈증 동물 모델에서 tp65-TMD를 전신투여한 결과 전염증성 사이토카인의 분비를 억제하여 쥐의 생존률이 현저하게 증가되었음을 확인하였다. 그러므로 tp65-TMD는 전사인자가 병의 발병에 중요 역할을 하는 다양한 염증성 질환의 치료에 새로운 치료법으로 사용될 수 있을 것이고, 더 나아가 유전적 변형이 필요 없이 p65의 새로운 기능을 발견하는 데 사용할 수 있을 것이다. Smad3 can mediate transforming growth factor (TGF-β) regulation for T cells functions through multiple mechanisms, such as suppression of T cells proliferation and differentiation, as well as inflammatory cytokines. TGF-β is required for the differentiation of both regulatory T (Treg) cells via Smad3-dependent pathway and T helper 17 (TH17) cells via Smad3-independent pathways. However, an approach to treat inflammatory diseases using TGF-β has still caused problems due to the conflicted function of TGF-β. Here, we developed a novel therapeutic strategy to modulate the functions of TH1, TH2, TH17 or Treg cells using only the transducible Smad3(MH1) (tSmad3(MH1)), which can be delivered effectively into the nucleus of CD4+ T cells. tSmad3(MH1) significantly inhibited TH1, TH2 or TH17-associated cytokines induced by T-bet, GATA3 or RORγt, although it substantially increased Treg-related cytokine induced by Foxp3, thereby regulated the differentiation of naive T cells into TH1, TH2, TH17 or Treg cells. The clinical severity and incidence of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were attenuated by tSmad3(MH1) in preventive as well as therapeutic manner. Therefore, tSmad3(MH1) can be novel therapeutic protein drug for the treatment of inflammatory diseases associated with TH1, TH2 or TH17 cells. This strategy can be applied to treat various diseases where a transcription factor has a key role in pathogenesis. Suppression of nuclear factor-κB (NF-κB) activation, which is best known as a major regulator of innate and adaptive immune responses, is a potent strategy for the treatment of endotoxic sepsis. To inhibit NF-κB functions, we designed the intranuclear transcription modulation domain (TMD) of RelA (p65), called tp65-TMD, which can be delivered effectively into the nucleus without influencing the cell viability and work as interactomic inhibitors via disruption of the endogenous p65-mediated transcription complex. tp65-TMD effectively inhibited the secretion of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in stimulated BV2 microglia cells by lipopolysaccharide (LPS). tp65-TMD cannot inhibit tyrosine phosphorylation of signaling mediators such as ZAP-70, p38, JNK, and ERK involved in T cell activation, but is capable of suppressing the transcriptional activity of NF-κB upon T-cell receptor (TCR) stimulation. The transduced tp65-TMD in T cell did not affect the expression of CD69, however, it significantly inhibited the secretion of T cell-specific cytokines such as IL-2, IFN-γ, IL-4, IL-17A, and IL-10. Systemic administration of tp65-TMD showed a significant therapeutic effect on LPS-induced sepsis model by inhibiting pro-inflammatory cytokines secretion. Therefore, tp65-TMD can be a novel therapeutics for the treatment of various inflammatory diseases, including endotoxic sepsis, where a transcription factor has a key role in pathogenesis and further allows us to discover new function of p65 without the need for genetic alteration.

Deoxynivalenol induces inflammation by activating NF-κB and inflammasome pathway

Molagoda, Ilandarage Menu Neelaka 제주대학교 일반대학원 2018 국내석사

The tricothecene toxin deoxynivalenol (DON) is one of the most prevalent and hazardous fungal secondary metabolites which contaminates serial based foods and causing toxicity to human. Many previous studies reported that the amounts of DON only existed in foods; however, whether DON influences on deleterious effects such as the pro-inflammatory responses in the cellular levels has not been understood. Therefore, we evaluated whether DON gives any effects on the pro-inflammatory responses in vitro in murine macrophage RAW264.7 cell. In the current study, we observed that low concentrations of DON (below 1000 nM) exhibited no substantial influence on cell viability and cellular morphological modification. Nevertheless, over at 400 nM DON slightly increased sub-G1 populations (4.65 ± 0.26% at 400 nM, 7.37 ± 0.22% at 800 nM and 7.61 ± 0.61% at 1000 nM DON, respectively) compared to the untreated control (1.66 ± 0.14%); however, the populations were lower than those of LPS-treated group (10.61 ± 0.08%). Then, we found that DON upregulated inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) at the transcription and translation levels, which consequently increased nitric oxide (NO) and prostaglandin E2 production. In addition, DON dose-dependently enhanced the expression of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-12 and tumor necrosis factor-α (TNF-α). DON also remarkably upregulated the nuclear translocation of NF-κB in a concentration-dependent way. Therefore, we suggest that DON could causes pro-inflammatory conditions in macrophages and thereby may lead to inflammatory disorders in human. Deoxynivalenol (DON) is one of the most common fungal toxins which contaminate food grains and cereal-derived products. However, whether DON induces inflammasome-mediated inflammation is still unknown. In the current study, we examined whether DON stimulates IL-1β secretion in BV2 microglia cells by activating NLRP3/ACS-mediated inflammasome. Treatment with high doses of DON (over 800 nM) decreased cell proliferation; however, no significant apoptotic sub-G1 was observed, which indicates that DON induces BV2 cells at a stagnant stage. We also found that DON significantly upregulated IL-1β expression in a dose-dependent manner from 0.5 h to 6 h as much as comparing to treatment with LPS and ATP, which was regulated by nuclear factor-κB (NF-κB) activation. In addition, IL-1β was remarkably secreted in the presence of DON at 24 h and a caspase-I inhibitor significantly prohibited DON-mediated IL-1β secretion, which suggest that caspase-1, which is an effector molecule of inflammasome, is an important upregulator of IL-1β. Thus, components of inflammasome such as ASC and NLRP3 substantially increased by DON treatment and transition knockdown of ASC and NLRP3 significantly downregulated DON-mediated IL-1β expression and secretion, which means that DON stimulates NLRP3/ASC-mediated inflammasome, which consequently promotes IL-1β expression and secretion in BV2 microglia cells. Taken together, these data suggest that DON induces IL-1β expression in BV2 microglial cells by activating the NF-κB signaling pathway and subsequently upregulated NLRP3/ASC-mediated inflammasome-mediated IL-1β secretion. Therefore, DON could induce inflammatory diseases or disorders by activating inflammasome-mediated IL- 1β.

조골세포에서 Peroxisome proliferators-activated receptor-g 작용제인 15-Deoxy-D12,14-prostaglandin J2의 Akt와 NF-κB 기전을 통한 인터루킨-6의 분비 억제 : The 15-Deoxy-D12,14 - prostaglandin J2, peroxisome proliferators-activated receptor-g agonist, inhibits interleukin-6 sec

박익수 인제대학교 2010 국내석사

ABSTRACT The 15-Deoxy-D12,14-prostaglandin J2, peroxisome proliferators-activated receptor g agonist, inhibits interleukin-6 secretion in MC3T3E-1 osteoblasts via the Akt and NF-κB pathways Ik-Su Park (Advisor:Prof. Sang-Jun Park, M.D) Department of Medicine Graduate School, Inje University Objective Periodontitis is a chronic inflammatory disease that results in gingival inflammation and periodontal tissue destruction and is accompanied by alveolar bone resorption and eventual tooth loss. Gram-negative bacterium possesses and produces lipopolysaccharide (LPS) molecules that play a role in the production of interleukin-6 (IL-6) during inflammatory process, as well as in bone resorption. 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2), peroxisome proliferators-activated receptor gamma (PPARg) activator, has been shown to have anti-inflammatory effects. Therefore, we examined the effect of 15d-PGJ2 on production of IL-6 by stimulating mouse osteoblast-like cells with LPS. Methods This study investigated mRNA and protein expression of IL-6 using reverse transcription-polymerase chain reaction (RT-PCR) and sandiwich ELISA inMC3T3E-1 cells, respectively. I also investigated the transcription factor of IL-6 in MC3T3E-1 cellsusing electric mobility shift assay (EMSA). I identified the LPS-stimulated IL-6 expression is involved signal pathway using RT-PCR and Western blot analysis. Results Our results indicate that 15d-PGJ2 inhibits LPS-induced IL-6 production in a concentration-dependent manner in MC3T3E-1 cells, without appreciable cytotoxicity. To further examine the mechanism responsible for the inhibition of IL-6 production by 15d-PGJ2, we examined the effect of 15d-PGJ2 on nuclear factorkB (NFkB) activation and the phosphorylation of protein kinase B (Akt). 15d-PGJ2 treatment clearly reduced the DNA binding activity of NF-kB in LPS-stimulated MC3T3E-1 cells, an effect that was mediated by inhibiting the degradation of inhibitor kB (IkB) and nuclear translocation of NF-kB p65 subunit. In addition, 15d-PGJ2 attenuated the LPS-mediated Akt pathway. These effects of 15d-PGJ2 were not abrogated by the PPARg antagonist, GW9662, indicating that they are PPARg-independent actions. Conclusion Collectively, these results suggest that 15d-PGJ2 possess a potent suppressive effect on inflammatory responses of MC3T3E-1 via the Akt and NF-κB pathways independent of PPARg and suggest that thiscompound may offer some insight into the development of a new therapeutic approach to the prevention and treatment of periodontal diseases. Key Words : Periodontitis 15-deoxy-D12,14-prostaglandin J2 Interleukin-6 Nuclear factor-κB Protein kinase B 국문초록 조골세포에서 Peroxisome proliferators-activated receptor-g 작용제인 15-Deoxy-D12,14-prostaglandin J2의 Akt와 NF-κB 기전을 통한 인터루킨-6의 분비 억제 박익수 (지도교수: 박상준) 인제대학교 대학원 의학과 목 적 치주염은 만성 염증성 질환으로서 잇몸염증과 치아 주위 조직 파괴가 일어나며, 치조골 흡수로 결국에는 치아를 잃게 된다. 그람네거티브 박테리아가 분비하는 내독소는 염증반응뿐 만 아니라 치조골 흡수에 중요한 역할을 하는 인터루킨-6의 분비를 유도한다. 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2), peroxisome proliferators-activated receptor gamma-g (PPARg) activator의 항-염증효과는이미 밝혀져 있다. 따라서 본 저자는 마우스 15d-PGJ2 를 이용하여 내독소로 자극한 조골세포주에서 치주염과 관련된 인자인 인터루킨-6의 분비 조절의 효과와 기전을 알아보고자 한다. 방 법 15d-PGJ2의 치주염 억제 효과를 알아보기 위하여 마우스 조골세포주에 내독소로 자극하여 인터루킨-6를 sandwich ELISA와 RT-PCR로 발현 정도를 측정하였고, 인터루킨-6 발현에 관련된 조절 기전을 조사하였다. 결 과 마우스 조골세포주에서 인터루킨-6 발현에 대한 15d-PGJ2의 효과는 독성이 없이 농도 의존적이었다. 이러한 억제효과는 인터루킨-6의 발현은 새포내에서 Akt 기전과 NF-kB 핵전사인자를 통하여 조절 되어진다. 결 론 마우스 조골세포주에서 15d-PGJ2는 Akt 기전과 NF-kB 핵전사인자조절을 통하여 치주질환의 원인중의 하나인 인터루킨-6의 분비를 조절한다. 이러한 결과로서, 치주질환 예방과 치료에 15d-PGJ2의 적용 가능성을 제시한다. 주제어 : 치주염 15-deoxy-D12,14-prostaglandin J2 Interleukin-6 Nuclear factor-κB Protein kinase B

Neuroprotective effects of sulfuretin on oxidative stress and inflammatory response in neurodegenerative diseases

Kwon, Seunghwan Sungkyunkwan University 2013 국내박사

Sulfuretin, a flavonoid from the stem bark of Albizzia julibrissin and heartwood of Rhus verniciflua, has been shown to possess anti-oxidative, anti-inflammatory, and anti-cancer properties. Although it is reported that sulfuretin had several bioactivities, yet its action mechanisms in neurodegenerative diseases are unknown. The present study aimed to investigate the mechanisms of sulfuretin on protection of neuronal cells from oxidative stress induced by neurotoxin 6-hydroxydopamine (6-OHDA). Here, I also investigated the anti-inflammatory effects of sulfuretin on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated microglial BV-2 cells. Sulfuretin significantly inhibited neuronal cell death, neurotoxicity, apoptosis, and reactive oxygen species (ROS) production. Sulfuretin also strikingly attenuated 6-OHDA-induced mitochondrial dysfunction. Eventually, sulfuretin inhibited 6-OHDA-induced nuclear factor-κB (NF-κB) translocation to the nucleus induced by 6-OHDA. Further studies revealed that sulfuretin induced the activation of Nrf2, enhanced the expression of HO-1. The phosphorylation of mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K)/Akt can be induced by sulfuretin in SH-SY5Y cells, and the ability of sulfuretin to enhanced NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) activation partly attenuated by the SB203580 (p38 inhibitor) and LY294002 (PI3K/Akt inhibitor). Zinc protoporphyrin (ZnPP, HO-1 inhibitor) also completely reduced the cytoprotective ability of sulfuretin, indicating the vital role of HO-1. On the other hand, sulfuretin significantly inhibited LPS-induced production of pro-inflammatory response factors at both the protein and mRNA levels. Moreover, sulfuretin attenuated the accumulation of intracellular ROS in LPS-stimulated BV-2 cells. Our data also indicated that sulfuretin significantly suppressed activation of NF-κB translocation from the cytosol to nucleus. In additon, sulfuretin induced the nucleus translocation of Nrf2 activation and induction of HO-1 expression. Further studies revealed that phosphorylation of p38 MAPK and PI3K/Akt can be induced by sulfuretin in BV-2 cells, and the ability of sulfuretin to enhanced Nrf2/HO-1 activation partly attenuated by the SB203580 and LY294002 inhibitors. Zinc protoporphyrin also completely blocked the ability of NO release of sulfuretin, indicating the vital role of HO-1. The present results provide that sulfuretin protects SH-SY5Y cells against 6-OHDA-induced oxidative stress possibly through suppression of apoptosis, ROS reduction, mitochondrial protection, NF-κB modulations and Nrf2/HO-1 activation via MAPKs, PI3K/Akt, and glycogen synthese kinase-3 beta (GSK-3β) pathways. Moreover, this data suggested that inhibition of the NF-κB activation, c-Jun N-terminal kinase (JNK), p38 MAPKs, and PI3K/Akt phosphorylation signaling pathways may suppress LPS-stimulated pro-inflammatory responses and/or cytokines. Sulfuretin also exerts anti-neuroinflammatory activity through the nuclear translocation of Nrf2 signaling and induction of downstream HO-1 activation. In conclusion, sulfuretin may hold a potential role for neuroprotection and, therefore, may be used a therapeutic agent for neurodegerative diseases. 오늘날 예방의학과 치료의학의 발달로 노년층이 차지하는 비율이 점진적으로 증가함에 따라 퇴행성뇌질환 환자가 급격하게 증가하고 있다. 이러한 퇴행성뇌질환은 중추신경계내에 산화적 스트레스 및 염증반응에 따른 신경세포 및 미세아교세포의 손상으로 인해 신경세포사멸을 초래하며, 종래에는 인지능 장애, 언어장애, 및 운동능력 손실과 같은 행동학적 장애를 초래한다. 설퍼레틴은 자귀나무의 수피 또는 옻나무의 심목에서 발견되는 주요한 플라보노이드계 물질로서, 항산화, 항염증 및 항암효과가 탁월한 것으로 보고가 되었다. 그러나 현재까지 퇴행성뇌질환에서 설퍼레틴의 효능 및 작용 기전에 대한 보고는 전무하다. 따라서, 본 연구에서는 신경세포주에서 산화적 스트레스 및 염증반응에 의해 유도된 신경세포사멸 그리고 세포독성으로부터 설퍼레틴의 신경보호 효과 및 작용기전을 실험하였다. 본 연구 결과에서 설퍼레틴은 산화적 스트레스 및 염증반응에 의해 유도된 신경세포사멸, 신경독성, 염증반응 인자, 그리고 활성산소종의 생성을 현저하게 억제시켰다. 또한 설퍼레틴은 산화적 스트레스 그리고 염증반응에 의해 매개된 미토콘드리아 손상 및 싸이토카인의 생성을 유의적으로 억제시켰으며, 종래에는 산화적 스트레스 그리고 염증반응에 의해 유도된 NF-κB의 핵내로의 활성화를 현저하게 감소시켰다. 구체적으로 이러한 설퍼레틴의 산화적 스트레스 및 염증반응에 의해 유도된 신경세포사멸 그리고 세포독성 억제 효과는 mitogen-activated protein kinase (MAPK) 그리고 phosphoinositide-3-kinase(PI3K)/Akt 단백질들의 인산화의 활성화를 통한 NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)의 활성화에 의해서 유도된 설퍼레틴의 뇌신경보호효과임을 증명하였다. 본 연구 결과는 퇴행성뇌질환에서 설퍼레틴의 항산화 및 항염증을 통한 뇌신경보호효과는 MAPK 및 PI3K/Akt 기전을 경유하여 신경세포사멸의 억제, 활성산소종의 억제, 미토콘드리아의 보호, 싸이토카인 분비의 억제, NF-κB의 조절작용, 그리고 Nrf2/HO-1의 활성화에 의한 것임을 제시하였다. 따라서, 설퍼레틴은 퇴행성뇌질환 치료제로서 사용될 수 있을 것으로 예상되며, 이러한 주요기전 중의 하나는 설퍼레틴의 우수한 뇌신경보호 작용에 기인하는 것으로 사료된다.

Anti-inflammatory effect of a fraction from leaves of Pyrus pyrifolia Nakai on LPS-induced THP-1 cells

이길혜 Graduate School, Korea University 2020 국내석사

Pyrus pyrifolia Nakai (P. pyrifolia) has been used traditionally for diseases such as phlegm, cough, hangover, and fever. The biological activities of leaves of Pyrus pyrifolia were not studied. The purpose of this study was to investigate the anti-inflammatory effect of a fraction from leaves of Pyrus pyrifolia (PP) in lipopolysaccharide (LPS)-induced THP-1 cells. Initially, I examined the Cyto X cell viability assay, which result is that PP was not cytotoxic at the range from 1.25μg/ml to 40μg/ml. Also, the enzyme-linked immunosorbent assay (ELISA) results demonstrate that PP decreased tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), interleukin-8 (IL-8), and interleukin-6 (IL-6) in protein level. In addition, the result of reverse transcription-polymerase chain reaction (RT-PCR) was that PP reduce TNF-α, IL-8, and MCP-1 in the RNA level. I also confirmed that PP restrains the phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, after stimulation with LPS, it was confirmed that the degradation of IκB-α was suppressed by PP, and the phosphorylation of IκB-α and p65 was suppressed by PP in a concentration-dependent manner. Also, PP increased heme oxygenase 1 (HO-1), which controls the production of inflammatory molecules, by activating nuclear factor erythroid 2-related factor 2 (Nrf2). These results show that PP could be used as an anti-inflammatory drug.

Anti-inflammatory effect of myristic acid through inhibiting NF-κB and MAPK signaling pathways in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

현지현 성균관대학교 일반대학원 2016 국내석사

Myristic acid (MA), which was named after the fruit of Myristica fragrans, has been widely used for cosmetic and topical medicinal preparations since it enhances skin absorption as ester isopropyl myristate. However, its anti-inflammatory effect and mechanism remain unknown. The aim of the present study was to investigate the anti-inflammatory effect and mechanism of MA using lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage cells. The MA suppressed LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor- α (TNF-α), and interleukin-1β (IL-1β) by enzyme inhibition, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression. In addition, MA inhibited nuclear factor (NF)-κB translocation by suppressing degradation of IκB-α and phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs). The results from the present study indicate that MA inhibits the production of various inflammatory mediators through NF-κB and MAPK signaling pathways. This finding explains the mechanism of anti-inflammatory effect of MA in LPS-stimulated RAW 264.7 macrophage cells and suggests MA is a potential therapeutic agent for inflammatory diseases.

Castanea seguinii Dode 메탄올 추출물의 LPS로 자극한 RAW264.7 대식세포에서 항염증 효과

임유림 충북대학교 2018 국내석사

Castanea extracts are known to have antioxidant properties and are used as a traditional medicine in China and Asia. However, the biological activity of Castanea seguinii Dode has remained to be fully elucidated. The present study investigated the anti-inflammatory effects of a Castanea seguinii Dode methanolic extract (CSME) on lipopolysaccharide-induced RAW264.7 macrophage cells. CSME inhibited the production of nitric oxide (NO) and the expression of inducible NO synthase. It also suppressed the production of the pro-inflammatory cytokines inteleukin-6 and tumor necrosis factor-α, as well as chemokine monocyte chemoattractant protein 1. In addition, CSME inhibited nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, while also downregulating transcription factor activator protein-1. Furthermore, CSME increased heme oxygenase 1 through the upregulation of NF (erythroid-derived 2)-like-2 (Nrf-2), which directly or indirectly affects inflammation. It also increased the phosphorylation of 5'-adenosine monophosphate-activated protein kinase (AMPK). In conclusion, CSME was demonstrated to exert its anti-inflammatory activities through the inhibition of the NF-κB and the MAPK signaling pathways, as well as the activation of Nrf-2 and AMPK. These results indicated that CSME may be a promising for development as a commercial anti-inflammatory medicine.