• Journal of Physiology & Pathology in Korean Medicine
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• v.36 no.1
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• pp.1-6
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• 2022

The tuber of Pinellia ternata (Thunb.) Brei (TPT) used in traditional Oriental medicine for the treatment of cough, sputum, vomiting, and insomnia, possesses antioxidant, antibacterial, and anti-inflammatory effects. Although recent studies have reported the anticancer effects of TPT in several cancer cells, it is still unclear whether TPT regulates tumor-associated macrophage (TAM) characterized by the immunosuppressive M2 macrophage phenotype. Our results showed that the ethanol extract of TPT (ETPT) suppressed the migration of RAW264.7 mouse macrophage cells and THP-1 human monocytes differentiated into macrophages towards the conditioned media (CM) collected from lung cancer cells, suggesting that ETPT would attenuate the recruitment of macrophages into tumors. In addition, ETPT suppressed the interleukin (IL)-4 or IL-6-induced M2 macrophage polarization in RAW264.7 cells. ETPT treatment not only downregulated the mRNA expression of M2 macrophage markers including arginase-1, mannose receptor C type 1 (MRC-1), and IL-10, but also inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and STAT6, general regulators of M2 macrophage polarization. Finally, the transwell assay results showed that the CM from M2-polarized RAW264.7 cells increased the migration of mouse lewis lung carcinoma (LLC) cells, while those from RAW264.7 cells co-treated with ETPT and IL-6 significantly reduced the migration of LLC cells. Taken together, our observations clearly demonstrate that ETPT suppressed the cancer cell migration by regulating macrophage recruitment and M2 macrophage polarization.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.408-417
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• 2022

Background: Korean Red Ginseng extract (KRGE) has been used as a health supplement and herbal medicine. Astrocytes are one of the key cells in the central nervous system (CNS) and have bioenergetic potential as they stimulate mitochondrial biogenesis. They play a critical role in connecting the brain vasculature and nerves in the CNS. Methods: Brain samples from KRGE-administered mice were tested using immunohistochemistry. Treatment of human brain astrocytes with KRGE was subjected to assays such as proliferation, cytotoxicity, Mitotracker, ATP production, and O₂ consumption rate as well as western blotting to demonstrate the expression of proteins related to mitochondria functions. The expression of hypoxia-inducible factor-1α (HIF-1α) was diminished utilizing siRNA transfection. Results: Brain samples from KRGE-administered mice harbored an increased number of GFAP-expressing astrocytes. KRGE triggered the proliferation of astrocytes in vitro. Enhanced mitochondrial biogenesis induced by KRGE was detected using Mitotracker staining, ATP production, and O₂ consumption rate assays. The expression of proteins related to mitochondrial electron transport was increased in KRGE-treated astrocytes. These effects were blocked by HIF-1α knockdown. The factors secreted from KRGE-treated astrocytes were determined, revealing the expression of various cytokines and growth factors, especially those related to angiogenesis and neurogenesis. KRGE-treated astrocyte conditioned media enhanced the differentiation of adult neural stem cells into mature neurons, increasing the migration of endothelial cells, and these effects were reduced in the background of HIF-1α knockdown. Conclusion: Our findings suggest that KRGE exhibits prophylactic potential by stimulating astrocyte mitochondrial biogenesis through HIF-1α, resulting in improved neurovascular function.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.3
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• pp.231-240
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• 2023

Fabry disease is a lysosomal storage disorder characterized by the lysosomal accumulations of glycosphingolipids in a variety of cytotypes, which include endothelial cells. The disease is inherited and originates from an error in glycosphingolipid catabolism caused by insufficient α-galactosidase A activity, which causes uncontrolled progressive storage of intracellular globotriaosylceramide (Gb3) in the vasculature and extracellular accumulation of lyso-Gb3 (a deacetylated soluble form of Gb3). Necrosis can lead to inflammation, which exacerbates necrosis and creates a positive feedback loop that triggers necroinflammation. However, the role played by necroptosis, a form of programmed necrotic cell death, in the cell-to-cell inflammatory reaction between epithelial and endothelial cells is unclear. Thus, the present study was undertaken to determine whether lyso-Gb3 induces necroptosis and whether necroptosis inhibition protects endothelial dysfunction against lyso-Gb3 inflamed retinal pigment epithelial cells. We found lyso-Gb3 induced necroptosis of a retinal pigment epithelial cell line (ARPE-19) in an autophagy-dependent manner and that conditioned media (CM) from ARPE-19 cells treated with lyso-Gb3 induced the necroptosis, inflammation, and senescence of human umbilical vein endothelial cells. In addition, a pharmacological study showed CM from lyso-Gb3 treated ARPE-19 cells induced endothelial necroptosis, inflammation, and senescence were significantly inhibited by an autophagy inhibitor (3-MA) and by two necroptosis inhibitors (necrostatin and GSK-872), respectively. These results demonstrate lyso-Gb3 induces necroptosis via autophagy and suggest that lyso-Gb3 inflamed retinal pigment epithelial cells trigger endothelial dysfunction via the autophagy-dependent necroptosis pathway. This study suggests the involvement of a novel autophagy-dependent necroptosis pathway in the regulation of endothelial dysfunction in Fabry disease.

• Journal of Physiology & Pathology in Korean Medicine
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• v.38 no.1
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• pp.32-37
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• 2024

The aim of this study was to investigate the role of Daesiho-tang (Da Chai Hu-Tang) water extract (DSTE) in regulating chronic stress-induced cancer progression, focusing on its activity in modulating tumor-associated macrophages (TAMs). Different stimuli can polarize TAMs into immune-stimulating M1 macrophages or immunosuppressive M2 macrophages. During cancer progression, M2 phenotype increases and supports tumor growth, angiogenesis and metastasis. Notably, chronic stress-induced catecholamines promote M2 macrophage polarization. In this study, we investigated whether DSTE regulates norepinephrine (NE)-induced M2 macrophage polarization in RAW 264.7 mouse macrophage cells. Even though NE itself did not increase the expression of M2 markers, the conditioned media of NE-treated 4T1 mouse breast cancer cells (NE CM) significantly up-regulated M2 markers in RAW 264.7 cells, suggesting that NE-regulated cancer cell secretome stimulated M2 polarization. However, such increase was abrogated by DSTE. NE CM also induced phosphorylation of signal transducer and activator of transcription 6 (STAT6) in RAW 264.7 cells, which was clearly reversed by pretreatment with DSTE, demonstrating that DSTE inhibited M2 polarization by inactivating STAT6. Finally, M2-polarized RAW264.7 cells by NE CM markedly increased the migration of 4T1 cells. However, such increase was completely reversed by co-treating RAW264.7 cells with NE CM and DSTE, indicating that DSTE attenuated cancer cell migration by blocking M2 polarization. Taken together, our results suggest a probable use of DSTE for cancer patients under chronic stress by regulating M2 macrophage polarization.

• IMMUNE NETWORK
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• v.16 no.3
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• pp.189-194
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• 2016

Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity.

• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.270-279
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• 2024

Macrophages are versatile immune cells that play crucial roles in tissue repair, immune defense, and the regulation of immune responses. In the context of skeletal muscle, they are vital for maintaining muscle homeostasis but macrophage-induced chronic inflammation can lead to muscle dysfunction, resulting in skeletal muscle atrophy characterized by reduced muscle mass and impaired insulin regulation and glucose uptake. Although the involvement of macrophage-secreted factors in inflammation-induced muscle atrophy is well-established, the precise intracellular signaling pathways and secretion factors affecting skeletal muscle homeostasis require further investigation. This study aimed to explore the regulation of macrophage-secreted factors and their impact on muscle atrophy and glucose metabolism. By employing RNA sequencing (RNA-seq) and proteome array, we uncovered that factors secreted by lipopolysaccharide (LPS)-stimulated macrophages upregulated markers of muscle atrophy and pro-inflammatory cytokines, while concurrently reducing glucose uptake in muscle cells. The RNA-seq analysis identified alterations in gene expression patterns associated with immune system pathways and nutrient metabolism. The utilization of gene ontology (GO) analysis and proteome array with macrophage-conditioned media revealed the involvement of macrophage-secreted cytokines and chemokines associated with muscle atrophy. These findings offer valuable insights into the regulatory mechanisms of macrophage-secreted factors and their contributions to muscle-related diseases.

• Tuberculosis and Respiratory Diseases
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• v.48 no.2
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• pp.223-235
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• 2000

Background: Liquid ventilation is associated with decreased inflammatory response in an injured lung. This study was performed to investigate if whether perfluorocarbon(PFC) can decrease chemokine expression in airway epithelial cells. Methods: A549 cells were used for airway epithelial cells and perfluorodecalin for PFC. To expose cells to PFC, lower chamber of Transwell$^{(R)}$plate was used. This study was performed in two parts. In the first part, we examined whether PFC could decrease chemokine expression in airway epithelial cells through inhibition of other inflammatory cells. Peripheral blood mononuclear cells(PBMC's) were isolated and stimulated with lipopolysaccharide(LPS, 10 ${\mu}g/mL$) for 24 hours with or without exposure to PFC. Then A549 cells were stimulated with conditioned media(CM) containing the culture supernatants of PBMC. After 24 hours, the expressions of interleukin-8(IL-8) and RANTES were measured. In the second part of the study, we studied whether PFC could directly suppress chemokine expression in airway epithelial cells. A549 cells were stimulated for 24 hours with interleukin-l$\beta$ and/or tumor necrosis factor-$\alpha$ with or without exposure to PFC, and then the chemokine expression was measured. Northern analysis was used to measure the mRNA expression, and ELISA was used for immunoreactive protein measurements in culture supernatant. Results: 1. IL-8 and RANTES mRNA expression and immunoreactive protein production were increased significantly by CM from LPS-stimulated PBMC in A459 cells compared to with CM from unstimulated PBCM (p<0.05), but exposure of PFC had no significant effect on either mRNA expression or immunoreactive protein expression. 2. IL-8 and RANTES mRNA expression and immunoreactive protein production were increased significantly by IL-1$\beta$ and TNF-$\alpha$ in A549 cells(p<0.05), but exposure of PFC had no significant effect on neither either mRNA expression nor immunoreactive protein production. Conclusion : Decreased chemokine expression of airway epithelial cells may not be involved in decreased inflammatory response observed in liquid ventilation. Further studies on possible mechanisms of decreased inflammatory response are warranted.

• International Journal of Oral Biology
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• v.33 no.4
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• pp.179-186
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• 2008

Several lines of evidence suggest that osteocytes play a critical role in bone remodeling. Both healthy and apoptotic osteocytes can send signals to other bone surface cells such as osteoblasts, osteoclasts, osteoclast precursors, and bone lining cells through canalicular networks. Osteocytes responding to mechanical strain may also send signals to other cells. To determine the role for osteocytes an mechanical strain in bone remodeling, we examined the effects of fluid flow shear stress on osteoclast precursor cell and osteoblast proliferation and recruitment induced by osteocytes. In addition, the effects of fluid flow shear stress on osteocyte M-CSF, RANKL, and OPG mRNA expression were also examined. MLO-Y4 cells were used as an in vitro model for osteocytes, RAW 264.7 cells and MOCP-5 cells as osteoclast precursors, and 2T3 cells as osteoblasts. MLO-Y4 cells conditioned medium (Y4-CM) was collected after 24h culture. For fluid flow experiments, MLO-Y4 cells were exposed to 2h of pulsatile fluid flow (PFF) at 2, 4, 8, $16{\pm}0.6\;dynes/cm^2$ using the Flexcell $Streamer^{TM}$ system. For proliferation assays, MOCP-5, RAW 264.7, and 2T3 cells were cultured with control media or 10-100% Y4 CM. Cells were cultured for 3d, and then cells were counted. RAW 264.7 and 2T3 cell migration was assayed using transwells with control media or 10-100% Y4-CM. M-CSF, RANKL and OPG in MLO-Y4 mRNA expression was determined by semiquantitative RT-PCR. Y4-CM increased osteoclast precursor proliferation and migration, but decreased 2T3 cell proliferation and migration. CM from MLO-Y4 cells exposed to PFF caused decreased RAW 267.4 cell proliferation and migration and 2T3 migration compared to control Y4-CM. However, Y4-CM from cells exposed to PFF had no effect on 2T3 osteoblastic cell proliferation. PFF decreased RNAKL mRNA and increased OPG mRNA in MLO-Y4 cells compared to control(without PFF). PFF had no effect on M-CSF mRNA expression in MLO-Y4 cells. These results suggest that osteocytes can regulate bone remodeling by communication with osteoclast precursors and osteoblasts and that osteocytes can communicate mechanical signals to other cells.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.89-89
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• 1995

Surface epithelial cells isolated from hamster tracheas and grown on a thick collagen gel become a highly enriched population of mucus-secreting cells. Epithelial cells from tracheas of hamsters were collected using enzymatic procedures and cultured under various conditions. The medium used consisted of a 1:1 mixture of medium 199 and Dulbecco's modified Eagle's (DME) medium which was conditioned before use. Insulin, transferrin, hydrocortisone, epidermal growth factor, and extract from bovine hypothalamus were used as supplement. Due to relatively low basal rates of min secretion from in vitro cultures, cultures are generally radiolabeled using $^3$H-glucosamine as a metabolic precursor. The radiolabeled mucinsreleased are quantitated by precipitation with TCA/PTA. Using this cell culture system, we investigated mucin release of goblet cells by altering the media bathing the apical surface of hamster tracheal surface epithelial(HTSE) cells. Acidic media added sulfuric acid caused sigcificant increases in mucin relesse (155${\pm}$20% at pH 4 and 146${\pm}$16% at, pH 5). Ammonium hydroxide also increased mucin release at pH 9.0(156${\pm}$17%) and pH 10(295${\pm}$9%) respectively. This additional mucin release seems to be associated with cell membrane damage as indicated by release of cellular LDH. SP stimulates secretion of mucin in cultured HTSE cells(154${\pm}$16% at 1${\times}$10$\^$-6/M and 165${\pm}$25% at 1${\times}$10$\^$-5/M. PAF at 5${\times}$10$\^$-6/M and 5${\times}$10$\^$-5/M enhanced by HTSE cells in vitro 168${\pm}$34% and 259${\pm}$30% of mucin secretion, respectively. The increase in mucin release by PAF and SP was not secondary to cell damage or necrosis. SP and PAF may be in mediating mucous secretion induced by inflammation irritantion and infection.

• Tuberculosis and Respiratory Diseases
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• v.50 no.5
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• pp.549-556
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• 2001

Background : IGF-I is an important mitogen in many types of malignancies. Tumors also express many IGF binding proteins, which modulate IGF action. The propose of this study was to evaluate the effect of IGF-I and IGFBP on cell proliferation in mouse lung cancer cells (3LL). Methods : The cellular proliferation of 3LL with the treatment of growth factors was evaluated using MTT assay. Western ligand blot was performed in order to determine whether 3LL cells secrete IGFBPs and we evaluated the effect of IGFBP on cellular proliferation. Results : The treatment of 3LL cells with IGF-I increased cellular proliferation in a serum free media. Western ligand blot of conditioned medium of 3LL with $^{125}I$-IGF-I demonstrated one single major band with an estimated molecular mass of 24 kDa. This band was identified as IGFBP-4 with immunoblot analysis using antisera. The addition of anti-IGFBP-4 antibody to abrogate the effect of IGFBP-4 resulted in increased cellular proliferation suggesting that IGFBP-4 inhibits cell growth. Conclusion : IGF-I increases cellular proliferation, however the secreted IGFBP-4 has an inhibitory function on cell growth in 3LL. These findings suggest that IGF-I and IGFBP are involved in the cell proliferation.