• 한국자원식물학회지
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• 제34권5호
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• pp.411-419
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• 2021

본 연구에서 금화규 뿌리 추출물(AMR)이 마우스 대식세포인 RAW264.7 세포의 활성화 유도를 통한 면역증진 활성과 마우스 지방전구세포인 3T3-L1 세포의 지질축적억제를 통한 항비만 활성을 평가하였다. 금화규 뿌리 추출물(AMR)은 전반적으로 RAW264.7 세포에서 TLR2/TLR4의 자극을 통해 p38과 JNK를 활성화시켜 NO, iNOS, IL-1𝛽, IL-6, TNF-𝛼와 같은 면역증진 인자의 발현을 증가시키는 것으로 판단된다. 그러나 IL-6의 경우, p38과 JNK 활성화에 의존하지 않는 것으로 확인되어 TLR2/4에 의한 다른 신호전달이 관여하는 것으로 사료되어 추가적인 연구가 필요하다. 또한, 금화규 뿌리 추출물(AMR)은 PPAR𝛾의 과대발현을 억제하여 지방전구세포의 성숙한 지방세포로의 분화를 억제하고, 성숙한 지방세포에서 CEBP𝛼, PPAR𝛾, perilipin-1, FABP4, adiponectin의 발현을 억제하여 지방세포 내 지질 형성 및 축적을 억제하는 것으로 판단된다. 본 연구를 통해 구명된 결과들은 금화규 뿌리 추출물(AMR)이 향후 면역증진 및 항비만을 위한 보조제 또는 건강 기능성 식품과 의약품으로의 개발 및 활용이 가능할 것으로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제19권4호
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• pp.365-372
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• 2015

Aripiprazole (ARI) is a commonly prescribed medication used to treat schizophrenia and bipolar disorder. To date, there have been no studies regarding the molecular pathological and immunotoxicological profiling of aripiprazole. Thus, in the present study, we prepared two different formulas of aripiprazole [Free base crystal of aripiprazole (ARPGCB) and cocrystal of aripiprazole (GCB3004)], and explored their effects on the patterns of survival and apoptosis-regulatory proteins under acute toxicity and cytotoxicity test conditions. Furthermore, we also evaluated the modulatory activity of the different formulations on the immunological responses in macrophages primed by various stimulators such as lipopolysaccharide (LPS), pam3CSK, and poly(I:C) via toll-like receptor 4 (TLR4), TLR2, and TLR3 pathways, respectively. In liver, both ARPGCB and GCB3004 produced similar toxicity profiles. In particular, these two formulas exhibited similar phospho-protein profiling of p65/nuclear factor $(NF)-{\kappa}B$, c-Jun/activator protein (AP)-1, ERK, JNK, p38, caspase 3, and bcl-2 in brain. In contrast, the patterns of these phospho-proteins were variable in other tissues. Moreover, these two formulas did not exhibit any cytotoxicity in C6 glioma cells. Finally, the two formulations at available in vivo concentrations did not block nitric oxide (NO) production from activated macrophage-like RAW264.7 cells stimulated with LPS, pam3CSK, or poly(I:C), nor did they alter the morphological changes of the activated macrophages. Taken together, our present work, as a comparative study of two different formulas of aripiprazole, suggests that these two formulas can be used to achieve similar functional activation of brain proteins related to cell survival and apoptosis and immunotoxicological activities of macrophages.

• BMB Reports
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• 제56권6호
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• pp.359-364
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• 2023

KAI1/CD82, a membrane tetraspanin protein, can prevent various cancers and retinal disorders through its anti-angiogenic and anti-metastatic capacity. However, little is known about its anti-inflammatory effect and molecular mechanism. Therefore, the present study aimed to inLPSvestigate effect of a recombinant protein of the large extracellular domain of human KAI1 (Gly 111-Leu 228, rhKAI1) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM) and to identify its underlying mechanism. Our data showed that rhKAI1 suppressed expression levels of classically macrophages (M1) phenotype-related surface markers F4/80+CD86+ in LPS-stimulated BMDM and RAW264.7 cells. In addition, LPS markedly increased mRNA expression and release levels of pro-inflammatory cytokines and mediators such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2, whereas these increases were substantially down-regulated by rhKAI1. Furthermore, LPS strongly increased expression of NF-κB p65 in the nuclei and phosphorylation of ERK, JNK, and p38 MAPK. However, nuclear translocation of NF-κB p65 and phosphorylation of JNK were greatly reversed in the presence of rhKAI1. Especially, rhKAI1 markedly suppressed expression of toll-like receptor (TLR4) and prevented binding of LPS with TLR4 through molecular docking predict analysis. Importantly, Glu 214 of rhKAI1 residue strongly interacted with Lys 360 of TLR4 residue, with a binding distance of 2.9 Å. Taken together, these findings suggest that rhKAI1 has an anti-inflammatory effect on LPS-polarized macrophages by interacting with TLR4 and down-regulating the JNK/NF-κB signaling pathway.

• 생명과학회지
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• 제21권5호
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• pp.664-670
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• 2011

Heat shock protein (HSP)은 외부적인 자극에 반응하여 세포를 보호하는 역할을 한다. 또한 HSP90은 혈관질환에서 처럼 세포가 사멸되거나 손상을 입는 경우 세포 밖으로 유리된다. 그러나 지금까지 세포 밖 HSP90이 혈관평활근세포에 어떠한 영향을 주는지에 대한 연구는 미약하다. 따라서 본 연구는 혈관평활근세포에서 HSP90이 CXCL10 발현에 대한 영향과 그 기전을 규명하였다. HSP90에 노출된 혈관평활근세포에서 CXCL10 transcript가 증가하고, CXCL10 단백질의 분비가 증가되었다. HSP90에 의한 CXCL10 분비는 Toll-like receptor (TLR)-2/-4 억제제인 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine (OxPAPC)과 NADPH oxidase 억제제인 diphenyleneiodium 그리고 Akt 경로를 억제하는 LY294002와 Akti IV에 의하여 크게 감소되었다. 또한 TLR-4의 dimerization을 저해하는 curcumin 역시 HSP90에 의한 CXCL10의 분비를 억제하였다. 전사인자인 nuclear factor kappa B(NF-${\kappa}$B)의 생물학적 억제제인 inhibitory kappa B (I${\kappa}$B)와 NF-${\kappa}$B 억제작용이 있는 rasveratrol은 HSP90에 의한 CXCL10 분비를 억제하였다. 이러한 결과는 혈관평활근세포에서 HSP90에 의한 CXCL10의 발현에 TLR-4와 Akt 및 NF-${\kappa}$B가 관여함을 의미한다.

• 생명과학회지
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• 제24권6호
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• pp.664-670
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• 2014

비브리오균(Vibrio vulnificus)은 심각하고 치명적인 감염을 일으킬 수 있는 호염성의 식중독균이지만, 숙주세포 내에서 염증반응을 일으키는 분자적 기작은 아직 잘 알려지지 않았다. 본 연구에서는 오염된 식품 섭취를 통해 유입되는 비브리오균의 소장 특이적 염증 반응 위치와 기작을 알아보기 위해, 7주령의 수컷 마우스에 비브리오균($1{\times}10^9CFU$)을 16시간 동안 경구 투여하였다. 그 결과 비브리오균은 주로 회장(ileum) 부위에서 비브리오균(WT) 수가 가장 많이 증가하였고, 공장(Jejunum), 근위부대장(proximal colon), 원위부 대장(distal colon)에도 유의적으로 군집현성이 이루어짐을 알 수 있었지만, 십이지장(duodenum)과 비장(spleen), 그리고 간(liver) 조직들에서는 관찰되지 않았다. 특히 비브리오균의 표적기관인 회장상피조직에서는 비브리오균(WT)이 침입 시 융모 안으로 다량의 염증세포들이 유입되었고, 융포의 폭이 넓어지고 길이가 짧아지는 전형적인 조직학적 염증 반응을 보여주었다. 비브리오균이 유도한 조직 특이적 염증반응기작을 알아보기 위해, 비브리오에 감염된 회장상피조직으로부터 단백질과 mRNA를 분리하였다. 비브리오균은 숙주세포의 중추적 신호전달 단백질인 protein kinase C (PKC)의 인산화 및 $PKC{\alpha}$의 세포막이동을 촉진시켰고, mitogen-activated protein kinase (MAPK) 중 extracellular signal-regulated kinases (ERK)와 c-Jun N-terminal kinases (JNK)의 인산화를 유도하였지만, p38 MAPK 인산화에는 영향을 미치지 않았다. 특히 비브리오균은 inhibitory factor-kappa B (I-${\kappa}B$)의 활성을 촉진시킴으로써 nuclear factor-kappa B (NF-${\kappa}B$)의 인산화를 유도하였다. 마지막으로 비브리오균(WT)에 감염된 회장의 경우, 정상마우스에 비해 염증성 cytokine인 interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-${\alpha}$의 mRNA 수준이 유의적으로 증가되었다. 염증매개 수용체인 toll like receptor (TLR)-4, TLR-5, TLR-9의 mRNA의 발현 또한 비브리오균 처리에 의해 증가되어 있음이 관찰되었다. 종합적으로 오염된 식품 섭취를 통해 유입되는 비브리오균은 회장상피세포를 표적으로 염증반응을 일으키며, 그 기작은 PKC, ERK1/2, 그리고 JNK의 인산화를 통한 NF-${\kappa}B$ 활성의 촉진이며, 이로 인한 다양한 염증 매개 단백질 발현의 증가를 통해 이루어진다고 할 수 있다.

• 생명과학회지
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• 제29권9호
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• pp.977-985
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• 2019

쑥부쟁이는 국화과에 속하는 다년생 식물로서 다양한 질병의 예방 및 치료에 오랫동안 사용되어 왔다. 최근 연구에서 쑥부쟁이 잎 추출물이 항산화 및 항염증 효과가 있는 것으로 알려져 있지만, 정확한 효능 평가에 관한 연구는 여전히 미비한 실정이다. 본 연구에서는 RAW 264.7 대식세포에서 쑥부쟁이 잎 에탄올 추출물(EEAY)의 항산화 효능이 항염증 효능과 연관이 있는지의 여부를 조사하였다. 본 연구의 결과에 의하면, EEAY는 $H_2O_2$ 처리에 의한 RAW 264.7 세포의 세포 독성을 유의적으로 억제시켰으며, 이는 Nrf2 및 HO-1의 발현 증가와 관련이 있음을 보여 주었다. 또한 EEAY는 $H_2O_2$에 의한 apoptosis를 유의적으로 억제하였으며, 이는 caspase-3의 활성 억제에 따른 PARP의 분해 차단과 연관성이 있었다. 그리고 EEAY는 대표적인 항 염증성 사이토카인인 IL-10의 발현 및 생산을 증가시켰으며, 이는 전사 및 번역 수준에서의 TLR-4 및 Myd88 발현 증가와 관련이 있었다. 아울러 EEAY는 LPS에 의한 염증성 매개인자인 NO의 생성 증가를 현저히 억제하였으며, EEAY에 의한 NO 생성의 억제 효과는 HO-1 유도제인 hemin에 의해 더욱 증가되었다. 따라서 본 연구의 결과는 EEAY에 의한 산화적 및 염증성 스트레스에 대한 RAW 264.7 대식세포의 보호 효과에 최소한 Nrf2/HO-1 신호 경로의 활성화가 관여할 가능성을 보여주었다.

• BMB Reports
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• 제49권10호
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• pp.554-559
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• 2016

Mycobacterium abscessus, a member of the group of non-tuberculous mycobacteria, has been identified as an emerging pulmonary pathogen in humans. However, little is known about the protective immune response of antigen-presenting cells, such as dendritic cells (DCs), which guard against M. abscessus infection. The M. abscessus gene MAB1843 encodes ᴅ-alanyl-ᴅ-alanine dipeptidase, which catalyzes the hydrolysis of ᴅ-alanyl-ᴅ-alanine dipeptide. We investigated whether MAB1843 is able to interact with DCs to enhance the effectiveness of the host's immune response. MAB1843 was found to induce DC maturation via toll-like receptor 4 and its downstream signaling pathways, such as the mitogen-activated protein kinase and nuclear factor kappa B pathways. In addition, MAB1843-treated DCs stimulated the proliferation of T cells and promoted Th1 polarization. Our results indicate that MAB1843 could potentially regulate the immune response to M. abscessus, making it important in the development of an effective vaccine against this mycobacterium.

• Journal of Ginseng Research
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• 제38권3호
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• pp.167-172
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• 2014

Background: Roles of immune reaction and toll-like receptor-4 (TLR-4) have widely been established in the pathogenesis of alcoholic liver disease (ALD). Methods: We evaluated the biologic efficacy of Korean Red Ginseng (KRG), urushiol, and probiotics (Lactobacillus rhamnosus R0011 and Lactobacillus acidophilus R0052) in mouse models of ALD. Sixty C57BL/6 mice were equally divided into six feeding groups for 10 weeks: normal diet, alcohol, control, alcohol + KRG, alcohol + urushiol, and alcohol + probiotics. Alcohol was administered via a LiebereDeCarli liquid diet containing 10% alcohol. TLR-4 expression, proinflammatory cytokines, and histology, as well as the results of liver function tests were evaluated and compared. Results: No between-group differences were observed with regard to liver function. TLR-4 levels were significantly lower in the KRG, urushiol, and probiotics groups than in the alcohol group ($0.37{\pm}0.06ng/mL$, $0.39{\pm}0.12ng/mL$, and $0.33{\pm}0.07ng/mL$, respectively, vs. $0.88{\pm}0.31ng/mL$; p < 0.05). Interleukin-$1{\beta}$ levels in liver tissues were decreased among the probiotics and KRG groups. The tumor necrosis factor-${\alpha}$ level of liver tissue was decreased in the KRG group. Conclusion: The pathological findings showed that alcohol-induced steatosis was significantly reduced by KRG and urushiol. As these agents improve immunologic capacity, they may be considered in potential anti-ALD treatments.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.686-694
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• 2008

The inhibitory effects of 5,6,3',5'-tetramethoxy 7,4'-hydroxyflavone (labeled as p7F) were elucidated on the productions of proinflammatory cytokines as well as inflammatory mediators in human synovial fibroblasts and macrophage cells. p7F inhibited IL-1${\beta}$ or TNF-${\alpha}$ induced expressions of inflammatory mediators (ICAM-1, COX-2, and iNOS). p7F also inhibited LPS-induced productions of nitric oxide and prostaglandin $E_2$ in RAW 264.7 cells. In order to investigate whether p7F would inhibit IL-1 signaling, p7F was added to the D10S Th2 cell line (which is responsive to only IL-1${\beta}$ and thus proliferates), revealing that p7F inhibited IL-1${\beta}$-induced proliferation of D10S Th2 cells in a dose-response manner. A flow cytometric analysis revealed that p7F reduced the intracellular level of free radical oxygen species in RAW 264.7 cells treated with hydrogen peroxide. p7F inhibited IkB degradation and NF-${\kappa}$B activation in macrophage cells treated with LPS, supporting that p7F could inhibit signaling mediated via toll-like receptor. Taken together, p7F has inhibitory effects on LPS-induced productions of inflammatory mediators on human synovial fibroblasts and macrophage cells and thus has the potential to be an anti-inflammatory agent for inhibiting inflammatory responses.

• IMMUNE NETWORK
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• 제11권1호
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• pp.79-94
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• 2011

Background: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-${\kappa}B$. Methods: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. Results: DPT promoted the activation of $CD8^+$ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-${\gamma}$ production and induction of cytotoxic T lymphocyte activity. Conclusion: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.