• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.350-356
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• 2017

We previously reported that the CopA3 peptide (LLCIALRKK, ${\small{D}}-form$) originally isolated from the Korean dung beetle has antimicrobial and immunosuppressive effects. However, the high cost of producing the synthetic peptide, especially the ${\small{D}}-form$, has limited the development of CopA3 for therapeutic purposes. Here, we investigated whether the CopA3 deletion derivative, CopA5, which is composed of only five amino acids (LLCIA) and has the ${\small{L}}-form$ structure, could inhibit the lipopolysaccharide (LPS)-induced activation of macrophages. Peritoneal exudate macrophages (PEM) were isolated from mice and exposed to LPS in the presence or absence of CopA5, and biomarkers of macrophage activation were measured. Our results revealed that LPS-induced nitric oxide (NO) production, tumor necrosis factor $(TNF)-{\alpha}$ secretion, and phagocytic activity of PEM were significantly inhibited by CopA5 treatment. Similar to CopA3, the structurally modified CopA5 peptide had no cell toxicity (as assessed by measurement of cell viability loss and apoptosis) in PEM. Moreover, the LPS-induced upregulation of the activating phosphorylation of signal transducer and activator of transcription 1 (STAT1) was markedly inhibited by CopA5 treatment. These results suggest that, similar to CopA3, CopA5 inhibits macrophage activation by inhibiting STAT1 phosphorylation and blocking the release of NO and $TNF-{\alpha}$. CopA5 may therefore prove therapeutically useful in the realm of immune suppression.

• The Korean Journal of Physiology and Pharmacology
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• 제23권6호
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• pp.493-499
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• 2019

Macrophage-associated inflammation is crucial for the pathogenesis of diverse diseases including metabolic disorders. Rhodanthpyrone (Rho) is an active component of Gentiana rhodantha, which has been used in traditional Chinese medicine to treat inflammation. Although synthesis procedures of RhoA and RhoB were reported, the biological effects of the specific compounds have never been explored. In this study, the anti-inflammatory activity and mechanisms of action of RhoA and RhoB were studied in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment with RhoA and RhoB decreased inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW 264.7 cells and in thioglycollate-elicited mouse peritoneal macrophages. In addition, it downregulated transcript levels of several inflammatory genes in LPS-stimulated RAW 264.7 cells, including inflammatory cytokines/chemokines (Tnfa, Il6, and Ccl2) and inflammatory mediators (Nos2 and Ptgs2). Macrophage chemotaxis was also inhibited by treatment with the compounds. Mechanistic studies revealed that RhoA and RhoB suppressed the nuclear factor $(NF)-{\kappa}B$ pathway, but not the canonical mitogen activated protein kinase pathway, in LPS-stimulated condition. Moreover, the inhibitory effect of RhoA and RhoB on inflammatory gene expressions was attenuated by treatment with an $NF-{\kappa}B$ inhibitor. Our findings suggest that RhoA and RhoB play an anti-inflammatory role at least in part by suppressing the $NF-{\kappa}B$ pathway during macrophage-mediated inflammation.

• 대한한의학회지
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• 제40권2호
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• pp.51-62
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• 2019

Objectives: This study was designed to evaluate the macrophages polarization of traditional Korean medicine on cardiac pain about Geum-Gwe-Yo-Ryak's two prescriptions including Kwaruhaebaekbanha-tang (KHB) and Kwaruhaebaekpaekju-tang (KHP). Materials and methods: Flow cytometry analysis was used to measure the changes in the ratio of M1 type and M2 type macrophages. Protein expression of nuclear factor-like 2 (Nrf2), heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were measured by Western Blot, and ABCA1 and SR-B1 were detected by real time PCR (RT-PCR). Intracellular lipid accumulation was measured by Oil Red O staining (ORO staining). Results: KHB and KHP increase anti-oxidative activity related protein levels including Nrf2 and HO-1. Furthermore, KHB and KHP inhibit lipid accumulation on intracellular levels through induction of ATP binding receptor cassette subfamily A member 1 (ABCA1) and scavenging receptor class B member 1 (SR-B1), respectively. Finally, KHB and KHP also blocked pro-inflammatory mediators including tumor necrosis factor-alpha ($TNF{\alpha}$) and interleukin-6 (IL-6), iNOS and COX-2 expression. Conclusion: This study suggests that KHB and KHP potently regulate the M1/M2 macrophage polarization.

• Animal Bioscience
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• 제34권3_spc호
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• pp.463-470
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• 2021

Objective: This experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP. Methods: Nitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit. Results: P700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 μM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700. Conclusion: In animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

• 대한한방부인과학회지
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• 제34권3호
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• pp.15-28
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• 2021

Objectives: This study was designed to examine the anti-cancer activity by innate immunomodulating and anti-inflammatory effects of liriopis tuber water extract (LPE). Methods: Cell cytotoxicity was tested with 4T1 mouse mammary carcinoma cells, spleen cells, macrophage, and RAW264.7 cells. To investigate innate immunomodulating effects of LPE on macrophage, we measured tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), and interleukin-10 (IL-10). To investigate innate immunomodulating effects of LPE on RAW264.7 cell, we measured TNF-α, interleukin-6 (IL-6). In addition, TNF-α and nitric oxide (NO) induced by lipopolysaccharide (LPS) were measured after treating with LPE to observe innate immunomodulating effect of LPE on RAW264.7 cell. Also, mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) were examined by western blot analysis. Results: In an in vitro cytotoxicity analysis, LPE affected tumor cell growth above specific concentration. As compared with the control group, the production of TNF-α, IL-12, and IL-10 were increased in macrophage. As compared with the control group, the production of TNF-α and IL-6 were increased in RAW 264.7 cell. The expression of TNF-α and NO induced by LPS after treating LPE was decreased. In addition, treatment of RAW 264.7 cell with LPE increased the phosphorylation levels of p-extracellular signal-regulated kinase (p-ERK), p-Jun N-terminal kinase (p-JNK), and p-p38. Conclusions: LPE might have impact on the anti-cancer effect by activation of innate immune system and inflammation control.

• BMB Reports
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• 제57권2호
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• pp.92-97
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• 2024

Elevated blood glucose is associated with an increased risk of atherosclerosis. Data from the current study showed that glucosamine (GlcN), a normal glucose metabolite of the hexosamine biosynthetic pathway (HBP), promoted lipid accumulation in RAW264.7 macrophage cells. Oleic acid- and lipopolysaccharide (LPS)-induced lipid accumulation was further enhanced by GlcN in RAW264.7 cells, although there was no a significant change in the rate of fatty acid uptake. GlcN increased acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), scavenger receptor class A, liver X receptor, and sterol regulatory element-binding protein-1c (SREBP-1c) mRNA expression, and; conversely, suppressed ATP-binding cassette transporter A1 (ABCA-1) and ABCG-1 expression. Additionally, GlcN promoted O-GlcNAcylation of nuclear SREBP-1 but did not affect its DNA binding activity. GlcN stimulated phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Rapamycin, a mTOR-specific inhibitor, suppressed GlcN-induced lipid accumulation in RAW264.7 cells. The GlcN-mediated increase in ACC and FAS mRNA was suppressed, while the decrease in ABCA-1 and ABCG-1 by GlcN was not significantly altered by rapamycin. Together, our results highlight the importance of the mTOR signaling pathway in GlcN-induced macrophage lipid accumulation and further support a potential link between mTOR and HBP signaling in lipogenesis.

• IMMUNE NETWORK
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• 제22권4호
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• pp.33.1-33.17
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• 2022

Suppressors of cytokine signaling (SOCS) have emerged as potential regulators of macrophage function. We have investigated mechanisms of SOCS3 action on type 2 macrophage (M2) differentiation induced by glucocorticoid using human monocytic cell lines and mouse bone marrow-derived macrophages. Treatment of THP1 monocytic cells with dexamethasone (Dex) induced ROS generation and M2 polarization promoting IL-10 and TGF-β production, while suppressing IL-1β, TNF-α and IL-6 production. SOCS3 over-expression reduced, whereas SOCS3 ablation enhanced IL-10 and TGF-β induction with concomitant regulation of ROS. As a mediator of M2 differentiation, glucocorticoid-induced leucine zipper (GILZ) was down-regulated by SOCS3 and up-regulated by shSOCS3. The induction of GILZ and IL-10 by Dex was dependent on ROS and p38 MAPK activity. Importantly, GILZ ablation led to the inhibition of ROS generation and anti-inflammatory cytokine induction by Dex. Moreover, GILZ knock-down negated the up-regulation of IL-10 production induced by shSOCS3 transduction. Our data suggest that SOCS3 targets ROS- and p38-dependent GILZ expression to suppress Dex-induced M2 polarization.

• 한국식품영양학회지
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• 제30권6호
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• pp.1348-1358
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• 2017

To enhance the physiological activity of the Rhynchosia volubilis (RV), R. volubilis (RVHE-A) and R. volubilis-added herbal powder (RVHE-B) were fermented with a solid state culture of Hericium erinaceum mycelia (HE). The total isoflavone contents of the non-fermented RV-A ($489.9{\mu}g/g$) and RV-B ($571.1{\mu}g/g$) were remarkably increased in fermented RVHE-A ($1,836.4{\mu}g/g$) and RVHE-B ($1,276.7{\mu}g/g$). In particular, aglycone isoflavones such as daidzein and genistein were significantly higher in the RVHE-A than any other sample. When hot-water (HW) and EtOH extracts (E) were fractionated from the RV and RVHE, both extracts from the RVHE-A were higher than those from the RV-A in total polyphenol and flavonoid contents. However, the RVHE-B-HW showed a lower polyphenol and flavonoid content level than did RV-B-HW. RVHE-A-HW and -E also had more potent ABTS radical scavenging activity than any extract from the non-fermented RV and other ferments (RVHE-B). In the meanwhile, RVHE-A-HW potently stimulated the production of macrophage activation-related cytokines such as $TNF-{\alpha}$, IL-6 and IL-12 ($841.7{\pm}71.3pg/mL$, $3.9{\pm}0.1ng/mL$, $179.3{\pm}30.2pg/mL$) from peritoneal macrophage more than RV-A-HW ($92.5{\pm}1.5pg/mL$, $0.1{\pm}0.0ng/mL$, $37.4{\pm}5.4pg/mL$) as well as RVHE-B-HW ($557.0{\pm}21.3pg/mL$, $1.8{\pm}0.0ng/mL$, $90.0{\pm}10.0pg/mL$). However, all the EtOH extracts did not show significant activity. In addition, the RVHE-A-HW showed a significantly higher intestinal immune system modulating activity through Peyer's patch and GM-CSF production than did any other extract from RV and RVHE-B. In conclusion, these results suggest that the fermented R. volubilis with H. erinaceum mycelia possesses a possible use as an industrial application as functional food or material.

• 한국식품영양과학회지
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• 제26권2호
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• pp.254-260
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• 1997

본 연구에서는 in vitro와 in vivo에서 sarcoma-180 cells과 마우스를 이용하여 김치의 세포독성 효과와 면역계, 특히 대식세포의 탐식능의 활성 증강에 미치는 효과를 중심으로 김치의 항암기작을 연구하였다. 김치가 발효되기 전인 0주(생김치)와 최적의 숙성도를 나타내는 3주 김치$(5^{\circ}C)$를 메탄올, 헥산추출물과 MSF(methanol soluble fraction)로 분리 조제하여 실험에 이용하였다. 김치추출물은 탐식능 증진효과를 나타냈으며, 0주 생김치 보다 3주 발효 김치에서 더욱더 활성이 높았다. 이러한 실험결과를 요약하면 다음과 같다. 1. Sarcoma-180 종양세포에 대한 시료의 직접적인 작용에서는 김치추출물에 의하여 총 세포수가 크게 줄어드는 것을 볼 수 있었다. 3주 발효 김치추출물은 같은 농도에서 0주(생김치) 시료 보다 세포독성작용이 더 높았다. 2. Phagocytic activity는 in vitro와 in vivo에서 대조군에 비해 김치추출물의 모든 시료에서 탐식율이 높게 나타났다. In vitro에서는 대조군이 41.3%에 비해, 3주 김치의 메탄올추출물은 57.3%, 헥산추출물에서는 49.2%를 나타냈고, in vivo에서는 대조군이 24.3%에 비해, 3주 김치의 메탄올추출물은 57.0%, MSF에서는 55.0%였다. 3. In vitro에서는 메탄올추출물에서, in vivo에서는 3주 발효 김치의 메탄올추출물과 MSF에서 대식세포 당 평균 2개 이상의 C. albicans를 탐식하고 있음을 알 수 있었으며 0주(생김치) 보다 3주 김치 추출물에서 더욱더 탐식능 증진효과를 나타냈다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권6호
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.