• The Korean Journal of Physiology and Pharmacology
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• v.17 no.1
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• pp.65-71
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• 2013

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body $Mg^{2+}$ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-${\kappa}B$ ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of $Mg^{2+}$. Knock-down of TRPM7 by siTRPM7 reduced intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increases by 0 mM $[Mg^{2+}]_e$ in HEK293 cells and inhibited the generation of RANKL-induced $Ca^{2+}$ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced $[Ca^{2+}]_i$ oscillations that triggers the late stages of osteoclastogenesis.

• IMMUNE NETWORK
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• v.22 no.5
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• pp.40.1-40.24
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• 2022

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs, focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations, gene expression, and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma, including severe type 2 inflammation, airway fibrosis, and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF^-CD11c^-CD11b⁺ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages, especially M2a and M2c. Furthermore, MSCs downregulated the excessive accumulation of Ly6c^- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c^- monocytes promoted the infiltration of MoM and Th2 inflammation, that of MSC-exposed Ly6c^- monocytes did not. Ex vivo Ly6c^- MoMs upregulated M2-related genes, which were reduced by MSC treatment. Molecules secreted by Ly6c^- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts, which were also suppressed by MSC treatment. In conclusion, intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c^- monocytes could be a therapeutic target for asthma management.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.1
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• pp.25-30
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• 2010

In order to improve the functional benefits and storage properties of soybean tofu, fermented tofu was developed using Pleurotus eryngii mycelia. The immune activities of water and methanol extracts of the tofu were investigated. The optimal medium for the growth of Pleurotus eryngii mycelia was PD broth medium and the optimal fermentation period for the tofu was 7 days. The water and methanol extracts of the fermented tofu induced the proliferation of spleen cells at above $0.01 {\mu}g/mL$. The water extract increased IL-2, IFN-$\gamma$ production, while the methanol extract increased IFN-$\gamma$ synthesis. The water and methanol extracts of the fermented tofu induced the NO production in RAW264.7 macrophage cells at above $1 {\mu}g/mL$ and above $10 {\mu}g/mL$ concentration, respectively. The extracts also significantly increased the production of IL-6, TNF-$\alpha$, IL-1$\beta$ and GM-CSF in the cells. These results suggest that the tofu fermented with Pleurotus eryngii mycelia could be developed as a functional tofu.

• Journal of Veterinary Science
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• v.22 no.2
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• pp.16.1-16.13
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• 2021

Background: Preconditioning with inflammatory stimuli is used to improve the secretion of anti-inflammatory agents in stem cells from variant species such as mouse, human, and dog. However, there are only few studies on feline stem cells. Objectives: This study aimed to evaluate the immune regulatory capacity of feline adipose tissue-derived (fAT) mesenchymal stem cells (MSCs) pretreated with interferon-gamma (IFN-γ). Methods: To assess the interaction of lymphocytes and macrophages with IFN-γ-pretreated fAT-MSCs, mouse splenocytes and RAW 264.7 cells were cultured with the conditioned media from IFN-γ-pretreated MSCs. Results: Pretreatment with IFN-γ increased the gene expression levels of cyclooxygenase-2, indoleamine 2,3-dioxygenase, hepatocyte growth factor, and transforming growth factor-beta 1 in the MSCs. The conditioned media from IFN-γ-pretreated MSCs increased the expression levels of M2 macrophage markers and regulatory T-cell markers compared to those in the conditioned media from naive MSCs. Further, prostaglandin E₂ (PGE₂) inhibitor NS-398 attenuated the immunoregulatory potential of MSCs, suggesting that the increased PGE₂ levels induced by IFN-γ stimulation is a crucial factor in the immune regulatory capacity of MSCs pretreated with IFN-γ. Conclusions: IFN-γ pretreatment improves the immune regulatory profile of fAT-MSCs mainly via the secretion of PGE₂, which induces macrophage polarization and increases regulatory T-cell numbers.

• Parasites, Hosts and Diseases
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• v.59 no.6
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• pp.557-564
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• 2021

Macrophages play a key role in chronic inflammation, and are the most abundant immune cells in the tumor microenvironment. We investigated whether an interaction between inflamed prostate cancer cells stimulated with Trichomonas vaginalis and macrophages stimulates the proliferation of the cancer cells. Conditioned medium was prepared from T. vaginalis-infected (TCM) and uninfected (CM) mouse prostate cancer (PCa) cell line (TRAMP-C2 cells). Thereafter conditioned medium was prepared from macrophages (J774A.1 cell line) after incubation with CM (MCM) or TCM (MTCM). When TRAMP-C2 cells were stimulated with T. vaginalis, protein and mRNA levels of CXCL1 and CCL2 increased, and migration of macrophages toward TCM was more extensive than towards CM. Macrophages stimulated with TCM produced higher levels of CCL2, IL-6, TNF-α, their mRNAs than macrophages stimulated with CM. MTCM stimulated the proliferation and invasiveness of TRAMP-C2 cells as well as the expression of cytokine receptors (CCR2, GP130, CXCR2). Importantly, blocking of each cytokine receptors with anti-cytokine receptor antibody significantly reduced the proliferation and invasiveness of TRAMP-C2 cells. We conclude that inflammatory mediators released by TRAMP-C2 cells in response to infection by T. vaginalis stimulate the migration and activation of macrophages and the activated macrophages stimulate the proliferation and invasiveness of the TRAMP-C2 cells via cytokine-cytokine receptor binding. Our results therefore suggested that macrophages contribute to the exacerbation of PCa due to inflammation of prostate cancer cells reacted with T. vaginalis.

• Journal of Haehwa Medicine
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• v.9 no.2
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• pp.315-324
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• 2001

To investigate the capacity of anti-inflammatory cytokines and biological response modifiers (BRMs) to induce IL-$1{\beta}$, IL-6, TNF-${\alpha}$ gene overexpression from mouse macrophages, we isolated the resident peritoneal macrophages from BALB/c mouse (8 week old) and incubated for 6 h with lipopolysaccaride (LPS) and Moschus moschiferus (MOMS) extracts. Analysis of inflammatory cytokines gene expression was carried out by RT-PCR amplification. Amplified PCR products were electrophoresed on 1.2% agarose gel, and the analysis (Ht) was used to 1D-density program. 1. LPS and MOMS extract treatments resulted in a significant decrease in IL-$1{\beta}$, IL-6, TNF-${\alpha}$ mRNA expression level compared with the LPS treatment. 2. Among four sample of MOMS, Inhibitory effects of MOMS-A and MOMS-D for inflammatory cytokines gene expression were to be fine compared with the MOMS-Band MOMS-C. According to the above data, Because the anti- tumoral and anti-inflammatory response activities of macrophage are known to be dependent on the production of inflammatory cytokines (IL-$1{\beta}$, IL-6, TNF-${\alpha}$) by macrophages, we suggest that evaluations of BRM for the reduction of inflammatory cytokines production by macrophages are important for clinical application.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.43 no.2
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• pp.157-164
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• 2017

Stevia (Stevia rebaudiana) is a perennial plant of the genus Stevia, originated in South America. It stores many forms of glycosides, mainly stevioside and rebaudioside A, in which steviol is the basic structure. Steviol glycosides, widely used as sweeteners, are superior to sugar in sweetness. Recently, it has been reported that steviol glycosides are involved not only in the skin whitening and anti-inflammatory effect but also in enhancing skin barrier function through tight junction regulation. Thus, we examined anti-inflammatory effect of rebaudioside A and tried to identify its potential for improving atopic dermatitis as cosmetic ingredients. To investigate the anti-inflammatory effect, cell viability and mRNA expression level of inflammation-related cytokines were measured using mouse macrophage RAW264.7 cells. Cell counting kit 8 (CCK-8) assay was carried out to measure cell viability and the maximum concentration without cytotoxicity was set to $250{\mu}M$. A quantitative real-time RT-PCR method was used for the study of the inflammatory suppression of rebaudioside A. Rebaudioside A inhibited expression of inducible nitric oxide synthase (iNOS) up to 47% and COX-2 up to 41% compared to LPS treated condition. NO synthesis was decreased by rebaudioside A. Also, mRNA expression of interleukin (IL)-$1{\alpha}$, IL-$1{\beta}$ and IL-6 in LPS-stimulated RAW264.7 cells was decreased to 40%, 45% and 59%, respectively, as a concentration-dependent manner. In conclusion, rebaudioside A inhibited the inflammatory response by regulation of cytokine gene expression. From these results, we expect that steviol glycoside, such as rebaudioside A, can be used as a material for improving atopic dermatitis in the future.

• The Korea Journal of Herbology
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• v.29 no.2
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• pp.61-67
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• 2014

Objectives : Aconiti Lateralis Preparata Radix (Aconitum Carmichaeli, AC) and Cinnamomi Cortex (Cinnamomi Cortex, CC) have been treated to elderly for kidney yang enhancement in Korean traditional medicine. In this study, the effects of water extract of AC and CC on RANKL (Receptor Activator for Nuclear Factor ${\kappa}B$ Ligand)-induced osteoclast differentiation were evaluated in culture system. Methods : MTT assay was used to evaluate the potential cytotoxicity of AC and CC extracts in bone macrophage marrows (BMMs) stimulated with M-CSF. TRAP (tartrate-resistant acid phosphatase) staining and TRAP activity were performed to know the inhibitory effect on osteoclast differentiation. The protein expression levels of nuclear factors such as activated T cell(NFAT)c1, c-Fos, MAPKs and ${\beta}$-actin in cell lysates treated with AC and CC extracts were analysed by western blotting. Results : AC, CC extracts and their co-administration inhibited significantly RANKL-induced osteoclast differentiation in BMMs in a dose dependent manner without toxicity. Each AC and CC extracts inhibited the phosphorylation of p38. Also, AC and CC extracts, respectively, inhibited the protein expression of c-Fos and NFATc1 more than Co-administration of AC and CC even if all treatments did. It was observed that RANKL-induced degradation of I-${\kappa}B$ is significantly suppressed by all treatments. Conclusions : Taken together, It was concluded that AC and CC have beneficial effect on osteoporosis by inhibition of osteoclast differentiation. Thus, Atractylodis AC and CC could be a treatment option for osteoporosis.

• Food Science and Biotechnology
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• v.15 no.2
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• pp.270-276
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• 2006

We evaluated the effects of extracts of Tsunokaori tangor peel on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin $E_2\;(PGE_2)$ in RAW 264.7 cells. The ethyl acetate fraction of Tsunokaori tangor peel (EA-TTP) markedly inhibited the production of NO and $PGE_2$ in LPS-stimulated RAW 264.7 cells. Consistent with these findings, the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins were down-regulated in a dose-dependent manner. Additionally, EA-TTP decreased the expression iNOS mRNA but not COX-2 mRNA. To determine the upstream signaling mechanism for the down-regulation of LPS-induced iNOS expression, we investigated the effect of EA-TTP on the degradation and re-synthesis of $I{\kappa}B{\alpha}$. EA-TTP dose-dependently delayed $I{\kappa}B{\alpha}$ degradation and increased $I{\kappa}B{\alpha}$ re-appearance following degradation, suggesting this as the mechanism by which EA-TTP suppressed iNOS gene expression. The EA-TTP also dose-dependently reduced the expression of the cellular stress-response protein heme oxygenase-1, and inhibited the LPS-induced sustained activation of extracellar signal-regulated kinase (ERK).

• Biomolecules & Therapeutics
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• v.22 no.4
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• pp.295-300
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• 2014

The actin cytoskeleton plays an important role in macrophage-mediated inflammatory responses by modulating the activation of Src and subsequently inducing nuclear factor (NF)-${\kappa}B$ translocation. In spite of its critical functions, few papers have examined how the actin cytoskeleton can be regulated by the activation of toll-like receptor (TLR). Therefore, in this study, we further characterized the biological value of the actin cytoskeleton in the functional activation of macrophages using an actin cytoskeleton disruptor, cytochalasin B (Cyto B), and explored the actin cytoskeleton's involvement in morphological changes, cellular attachment, and signaling events. Cyto B strongly suppressed the TLR4-mediated mRNA expression of inflammatory genes such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-${\alpha}$, and inducible nitric oxide (iNOS), without altering cell viability. This compound also strongly suppressed the morphological changes induced by lipopolysaccharide (LPS), a TLR4 ligand. Cyto B also remarkably suppressed NO production under non-adherent conditions but not in an adherent environment. Cyto B did not block the co-localization between surface glycoprotein myeloid differentiation protein-2 (MD2), a LPS signaling glycoprotein, and the actin cytoskeleton under LPS conditions. Interestingly, Cyto B and PP2, a Src inhibitor, enhanced the phagocytic uptake of fluorescein isothiocyanate (FITC)-dextran. Finally, it was found that Cyto B blocked the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at 1 min and the phosphorylation of heat shock protein 27 (HSP27) at 5 min. Therefore, our data suggest that the actin cytoskeleton may be one of the key components involved in the control of TLR4-mediated inflammatory responses in macrophages.