• Journal of Ginseng Research
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• v.43 no.2
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• pp.218-225
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• 2019

Background: Streptococcus pneumoniae, more than 90 serotypes of which exist, is recognized as an etiologic agent of pneumonia, meningitis, and sepsis associated with significant morbidity and mortality worldwide. Immunization with a pneumococcal pep27 mutant (${{\Delta}}pep27$) has been shown to confer comprehensive, long-term protection against even nontypeable strains. However, ${{\Delta}}pep27$ is effective as a vaccine only after at least three rounds of immunization. Therefore, treatments capable of enhancing the efficiency of ${{\Delta}}pep27$ immunization should be identified without delay. Panax ginseng Mayer has already been shown to have pharmacological and antioxidant effects. Here, the ability of Korean Red Ginseng (KRG) to enhance the efficacy of ${{\Delta}}pep27$ immunization was investigated. Methods: Mice were treated with KRG and immunized with ${{\Delta}}pep27$ before infection with the pathogenic S. pneumoniae strain D39. Total reactive oxygen species production was measured using lung homogenates, and inducible nitric oxide (NO) synthase and antiapoptotic protein expression was determined by immunoblotting. The phagocytic activity of peritoneal macrophages was also tested after KRG treatment. Results: Compared with the other treatments, KRG significantly increased survival rate after lethal challenge and resulted in faster bacterial clearance via increased phagocytosis. Moreover, KRG enhanced ${{\Delta}}pep27$ vaccine efficacy by inhibiting reactive oxygen species production, reducing extracellular signal-regulated kinase apoptosis signaling and inflammation. Conclusion: Taken together, our results suggest that KRG reduces the time required for immunization with the ${{\Delta}}pep27$ vaccine by enhancing its efficacy.

• Nutrition Research and Practice
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• v.15 no.5
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• pp.568-578
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• 2021

BACKGROUND/OBJECTIVES: Psidium guajava L. (guava) leaves have been shown to exhibit hypoglycemic and antidiabetic effects in rodents. This study investigated the effects of guava leaf extract on adipogenesis, glucose uptake, and lipolysis of adipocytes to examine whether the antidiabetic properties are mediated through direct effects on adipocytes. MATERIALS/METHODS: 3T3-L1 cells were treated with 25, 50, 100 ㎍/mL of methanol extract from guava leaf extract (GLE) or 0.1% dimethyl sulfoxide as a control. Lipid accumulation was evaluated with Oil Red O Staining and AdipoRed assay. Immunoblotting was performed to measure the expression of adipogenic transcription factors, fatty acid synthase (FAS), and AMP-activated protein kinase (AMPK). Glucose uptake under basal or insulin-stimulated condition was measured using a glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose. Lipolysis from fully differentiated adipocytes was measured by free fatty acids release into the culture medium in the presence or absence of epinephrine. RESULTS: Oil Red O staining and AdipoRed assay have shown that GLE treatment reduced lipid accumulation during adipocyte differentiation. Mitotic clonal expansion, an early essential event for adipocyte differentiation, was inhibited by GLE treatment. GLE inhibited the expression of transcription factors involved in adipocyte differentiation, such as peroxisome proliferator-activated receptor 𝛄 (PPAR𝛄), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein-1c (SREBP-1c). FAS expression was also decreased while the phosphorylation of AMPK was increased by GLE treatment. In addition, GLE increased insulin-induced glucose uptake into adipocytes. In lipid-filled mature adipocytes, GLE enhanced epinephrine-induced lipolysis but reduced basal lipolysis dose-dependently. CONCLUSIONS: The results show that GLE inhibits adipogenesis and improves adipocyte function by reducing basal lipolysis and increasing insulin-stimulated glucose uptake in adipocytes, which can be partly associated with antidiabetic effects of guava leaves.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.692-698
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• 2019

Background: Breast cancer is a severe disease and the second leading cause of cancer death in women worldwide. To surmount this, various diagnosis and treatment options for breast cancer have been developed. One of the most effective strategies for cancer treatment is to induce apoptosis using naturally occurring compounds. Compound K (CK) is a ginseng saponin metabolite generated by human intestinal bacteria. CK has been studied for its cardioprotective, antiinflammatory, and liver-protective effects; however, the role of CK in breast cancer is not fully understood. Methods: To investigate the anticancer effects of CK in SKBR3 and MDA-MB-231 cells, cell viability assays and flow cytometry analysis were used. In addition, the direct targets of CK anticancer activity were identified using immunoblotting analysis and overexpression experiments. Invasion, migration, and clonogenic assays were carried out to determine the effects of CK on cancer metastasis. Results: CK-induced cell apoptosis in SKBR3 cells as determined through 3-(4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assays, propidium iodide (PI) and annexin V staining, and morphological changes. CK increased the cleaved forms of caspase-7, caspase-8, and caspase-9, whereas the expression of Bcl-2 was reduced by CK. In assays probing the cell survival pathway, CK activated only AKT1 and not AKT2. Moreover, CK inhibited breast cancer cell invasion, migration, and colony formation. Through regulation of AKT1 activity, CK exerts anticancer effects by inducing apoptosis. Conclusion: Our results suggest that CK could be used as a therapeutic compound for breast cancer.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.195-204
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• 2021

Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4) regulates the ubiquitination and degradation of c-Jun, mediating the lipopolysaccharide-induced cellular response. However, the upstream signaling pathway that regulates this process is unknown. In this study, we describe how endoplasmic reticulum (ER) stress reversely regulates sequestosome-1 (p62)and c-Jun protein levels. Furthermore, our study reveals that expression of p62 attenuates c-Jun protein levels through the ubiquitinproteasome system. Conversely, siRNA knockdown of p62 elevates c-Jun protein levels. Immunoprecipitation and immunoblotting experiments demonstrate that p62 interacts with c-Jun and CRBN to form a ternary protein complex. Moreover, we find that CRBN knockdown completely abolishes the inhibitory effect of p62 on c-Jun. Using brefeldin A as an inducer of ER stress, we demonstrate that the p62/c-Jun axis participates in the regulation of ER stress-induced apoptosis, and that CRBN is required for this regulation. In summary, we have identified an upstream signaling pathway, which regulates p62-mediated c-Jun degradation. Our findings elucidate the underlying molecular mechanism by which p62/c-Jun axis regulates the ER stress-induced apoptosis, and provide a new molecular connection between ER stress and apoptosis.

• International Journal of Oral Biology
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• v.46 no.1
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• pp.15-22
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• 2021

Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and Bcl-xL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

• Herbal Formula Science
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• v.30 no.1
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• pp.1-9
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• 2022

Objectives : This study investigated the protective effect of Ojeok-san (OJS) on cellular damage induced by oxidative stress and whether it induces changes in CYP450 expression. Methods : To investigate the protective effect, we used cells stimulated by oxidative stress caused by the combination treatment of AA+iron. Changes in CYP450 expression were detected by immunoblotting analysis using Huh7 cells. Results : We observed that OJS altered the expression of CYP1A2, CYP3A4, CYP2C19, CYP2D6, and CYP2E1. OJS increased cell viability against AA+iron-induced oxidative stress and inhibited mitochondrial dysfunction. OJS increased phosphorylation of LKB1, phosphorylation of AMPK, and phosphorylation of ACC, which are related to the LKB1-AMPK pathway. In addition, phosphorylation of LATS1 and phosphorylation of YAP, which are related to the Hippo-YAP pathway, were increased. Conclusions : Our results show that OJS has 1) the ability to protect hepatocytes against oxidative stress, and 2) the potential to induce changes in CYP450.

• Molecules and Cells
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• v.45 no.4
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• pp.257-272
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• 2022

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

• Journal of Korean Medicine for Obesity Research
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• v.22 no.1
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• pp.38-46
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• 2022

Objectives: Juknyeok (JN) is natural liquor extracted from bamboo stems (Phyllostachys bambusoides) and has been used as a traditional Korean medicine for improving vascular function, blood glucose, and treating stroke. Until now, the JN's lipid-lowering effect and underlying mechanism in adipocytes are poorly understood. The aim of this study was to scrutinize the effect of a standardized commercial JN on lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Methods: Lipid and triglyceride (TG) accumulation in differentiating 3T3-L1 preadipocytes were measured by Oil Red O staining and AdipoRed assay, respectively. Cell count analysis was used to ascertain 3T3-L1 cytotoxicity. Immunoblotting and Reverse transcription polymerase chain reaction analysis were used to assess protein and messenger RNA (mRNA) expression levels in 3T3-L1 cells, respectively. Results: Treatment with JN at 25 𝜇l/ml after pH calibration with 6.35 significantly reduced lipid and TG accumulation in differentiating 3T3-L1 preadipocytes without significant cytotoxicity. On mechanistic levels, JN markedly suppressed protein expression levels of CCAAT/enhancer-binding protein (C/EBP)-𝛽 and fatty acid synthase (FAS) during the differentiation of 3T3-L1 preadipocytes. However, JN did not affect the protein expression levels of C/EBP-𝛼, peroxisome proliferator-activated receptor-𝛽/𝛾, and phosphorylation levels of signal transducer and activator of transcription-3/5 in differentiating 3T3-L1 preadipocytes. JN also reduced leptin mRNA expression levels in differentiating 3T3-L1 preadipocytes. Conclusions: JN at 25 𝜇l/ml lowers lipid accumulation and TG content in differentiating 3T3-L1 cells, mediated through the reduced expression levels of C/EBP-𝛽 and FAS.

• Journal of Ginseng Research
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• v.46 no.5
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• pp.690-699
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• 2022

Background: Few studies reported the therapeutic effect of Korean Red Ginseng (KRG) in lung inflammatory diseases. However, the anti-inflammatory role and underlying molecular in cadmium-induced lung injury have been poorly understood, directly linked to chronic lung diseases (CLDs): chronic obstructive pulmonary disease (COPD), cancer etc. Therefore, in this study we aim to investigate the therapeutic activities of water extract of KRG (KRG-WE) in mouse cadmium-induced lung injury model. Method: The anti-inflammatory roles and underlying mechanisms of KRG-WE were evaluated in vitro under cadmium-stimulated lung epithelial cells (A549) and HEK293T cell line and in vivo in cadmium-induced lung injury mouse model using semi-quantitative polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), luciferase assay, immunoblotting, and FACS. Results: KRG-WE strongly ameliorated the symptoms of CdSO4-induced lung injury in mice according to total cell number in bronchoalveolar lavage fluid (BALF) and severity scores as well as cytokine levels. KRG-WE significantly suppressed the upregulation of inflammatory signaling comprising mitogen-activated protein kinases (MAPK) and their upstream enzymes. In in vitro study, KRG-WE suppressed expression of interleukin (IL)-6, matrix metalloproteinase (MMP)-2, and IL-8 while promoting recovery in CdSO₄-treated A549 cells. Similarly, KRG-WE reduced phosphorylation of MAPK and c-Jun/c-Fos in cadmium-exposed A549 cells. Conclusion: KRG-WE was found to attenuate symptoms of cadmium-induced lung injury and reduce the expression of inflammatory genes by suppression of MAPK/AP-1-mediated pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.6
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• pp.447-456
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• 2022

The present study was carried out to investigate the effect of Arctigenin on cell growth and the mechanism of cell death elicited by Arctigenin were examined in FaDu human pharyngeal carcinoma cells. To determine the apoptotic activity of Arctigenin in FaDu human pharyngeal carcinoma cells, cell viability assay, DAPI staining, caspase activation analysis, and immunoblotting were performed. Arctigenin inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Arctigenin-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was up-regulated by Arctigenin treatment. Moreover, caspase-8, a part of the extrinsic apoptotic pathway, was activated by Arctigenin treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria-dependent intrinsic apoptosis pathway, significantly decreased following Arctigenin treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9, and tumor suppressor -53 increased by Arctigenin treatments. In addition, Arctigenin activated caspase-3 and poly (ADP-ribose) polymerase (PARP) induced cell death. Arctigenin also inhibited the proliferation of FaDu cells by the suppression of p38, NF-κB, and Akt signaling pathways. These results suggest that Arctigenin may inhibit cell proliferation and induce apoptotic cell death in FaDu human pharyngeal carcinoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway.