• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.812-827
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• 2024

Phloroglucinol (PG) is one of the abundant isomeric benzenetriols in brown algae. Due to its polyphenolic structure, PG exhibits various biological activities. However, the impact of PG on anagen signaling and oxidative stress in human dermal papilla cells (HDPCs) is unknown. In this study, we investigated the therapeutic potential of PG for improving hair loss. A non-cytotoxic concentration of PG increased anagen-inductive genes and transcriptional activities of β-Catenin. Since several anagen-inductive genes are regulated by β-Catenin, further experiments were performed to elucidate the molecular mechanism by which PG upregulates anagen signaling. Various biochemical analyses revealed that PG upregulated β-Catenin signaling without affecting the expression of Wnt. In particular, PG elevated the phosphorylation of protein kinase B (AKT), leading to an increase in the inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9. Treatment with the selective phosphoinositide 3-kinase/AKT inhibitor, LY294002, restored the increased AKT/GSK3β/β-Catenin signaling and anagen-inductive proteins induced by PG. Moreover, conditioned medium from PG-treated HDPCs promoted the proliferation and migration of human epidermal keratinocytes via the AKT signaling pathway. Subsequently, we assessed the antioxidant activities of PG. PG ameliorated the elevated oxidative stress markers and improved the decreased anagen signaling in hydrogen peroxide (H₂O₂)-induced HDPCs. The senescence-associated β-galactosidase staining assay also demonstrated that the antioxidant abilities of PG effectively mitigated H₂O₂-induced senescence. Overall, these results indicate that PG potentially enhances anagen signaling and improves oxidative stress-induced cellular damage in HDPCs. Therefore, PG can be employed as a novel therapeutic component to ameliorate hair loss symptoms.

• Journal of Ginseng Research
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• v.47 no.6
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• pp.714-725
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• 2023

Background: Our previous investigation indicated that the preparation of Panax ginseng Meyer (P. ginseng) inhibited melanogenesis. It comprised salicylic acid (SA), protocatechuic acid (PA), p-coumaric acid (p-CA), vanillic acid (VA), and caffeic acid (CA). In this investigation, the regulatory effects of P. ginseng phenolic acid monomers on melanin production were assessed. Methods: In vitro and in vivo impact of phenolic acid monomers were assessed. Results: SA, PA, p-CA and VA inhibited tyrosinase (TYR) to reduce melanin production, whereas CA had the opposite effects. SA, PA, p-CA and VA significantly downregulated the melanocortin 1 receptor (MC1R), cycle AMP (cAMP), protein kinase A (PKA), cycle AMP-response element-binding protein (CREB), microphthalmia-associated transcription factor (MITF) pathway, reducing mRNA and protein levels of TYR, tyrosinase-related protein 1 (TYRP1), and TYRP2. Moreover, CA treatment enhanced the cAMP, PKA, and CREB pathways to promote MITF mRNA level and phosphorylation. It also alleviated MITF protein level in α-MSH-stimulated B16F10 cells, comparable to untreated B16F10, increasing the expression of phosphorylation glycogen synthase kinase 3β (p-GSK3β), β-catenin, p-ERK/ERK, and p-p38/p38. Furthermore, the GSK3β inhibitor promoted p-GSK3β and p-MITF expression, as observed in CA-treated cells. Moreover, p38 and ERK inhibitors inhibited CA-stimulated p-p38/p38, p-ERK/ERK, and p-MITF increase, which had negative binding energies with MC1R, as depicted by molecular docking. Conclusion: P. ginseng roots' phenolic acid monomers can safely inhibit melanin production by bidirectionally regulating melanin synthase transcription. Furthermore, they reduced MITF expression via MC1R/cAMP/PKA signaling pathway and enhanced MITF post-translational modification via Wnt/mitogen-activated protein kinase signaling pathway.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.127-138
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• 2023

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (Tau_RD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC₅₀ of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 Tau_RD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 Tau_RD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 Tau_RD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

• Journal of Periodontal and Implant Science
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• v.47 no.5
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• pp.273-291
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• 2017

Purpose: Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods: Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results: The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase $kinase-3{\beta}$ ($GSK-3{\beta}$) and total ${\beta}-catenin$ protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) pathways were activated. Conclusions: SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.1
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• pp.43-48
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• 2012

Glutamate excitotoxicity is emerging as a contributor to degeneration of spinal cord motoneurons in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin protects motoneurons against chronic glutamate excitotoxicity through the activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-$3{\beta}$ pathways. Previous studies suggest that activated microglia actively participate in the pathogenesis of ALS motoneuron degeneration. However, it is still unknown whether ghrelin exerts its protective effect on motoneurons via inhibition of microglial activation. In this study, we investigate organotypic spinal cord cultures (OSCCs) exposed to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration, to determine if ghrelin prevents microglial activation. Exposure of OSCCs to THA for 3 weeks produced typical motoneuron death, and treatment of ghrelin significantly attenuated THA-induced motoneuron loss, as previously reported. Ghrelin prevented THA-induced microglial activation in the spinal cord and the expression of pro-inflammatory cytokines tumor necrosis factor-${\alpha}$ and interleukin-$1{\beta}$. Our data indicate that ghrelin may act as a survival factor for motoneurons by functioning as a microglia-deactivating factor and suggest that ghrelin may have therapeutic potential for the treatment of ALS and other neurodegenerative disorders where inflammatory responses play a critical role.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.155-161
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• 2014

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta ($GSK3{\beta}$) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the $GSK3{\beta}$ kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

• Journal of Veterinary Science
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• v.23 no.1
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• pp.4.1-4.15
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• 2022

Background: Flavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the antiadipogenic effects of flavonoids are unclear. Objectives: The present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells. Methods: The intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis. Results: The flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators. Conclusion: The overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

• Biomedical Science Letters
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• v.9 no.2
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• pp.59-65
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• 2003

When cells are exposed to proteotoxic stresses such as heat shock, amino acid analogs, and heavy metals, they increase the synthesis of the heat shock proteins (HSPs) by activating the heat shock transcription factor 1 (HSF1), whose activity is controlled via multiple steps including homotrimerization, nuclear translocation, DNA binding, and hyperphosphorylation. Under unstressed conditions, the HSF1 activity is repressed through its constitutive phosphorylation by glycogen synthase kinase 3$\beta$ (GSK3$\beta$), extracellular regulated kinase 1/2 (ERK1/2), and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). However, the protein kinase (s) responsible for HSF1 hyperphosphorylation and activation is not yet identified. In the present study, we observed that profile of p38 mitogen-activated protein kinase (p38MAPK) activation in response to heat shock was very similar to those of HSF1 hyperphosphorylation and nuclear translocation. Therefore, we investigated whether p38MAPK is involved in the heat shock-induced HSF1 activation and HSP expression. Here we show that the p38MAPK inhibitors, SB202190 and SB203580, but not other inhibitors including the MEK1/2 inhibitor PD98059 and the PI3-K inhibitor LY294002 and wortmannin, suppress HSF1 hyperphosphorylation in response to heat shock and L-azetidine 2-carboxylic acid (Azc), but not to heavy metals. Furthermore, heat shock-induced HSF1-DNA binding and HSP72 expression was specifically prevented by the p38MAPK inhibitors, but not by the MEK1/2 inhibitor and the PI3-K inhibitors. These results suggest that SB202190- and SB203580-sensitive p38MAPK may positively regulate HSP gene regulation in response to heat shock and amino acid analogs.

• Biomolecules & Therapeutics
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• v.23 no.1
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• pp.26-30
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• 2015

Wnt/${\beta}$-catenin signaling pathway was mutated in about 90% of the sporadic and hereditary colorectal cancers. The abnormally activated ${\beta}$-catenin increases the cancer cell proliferation, differentiation and metastasis through increasing the expression of its oncogenic target genes. In this study, we identified an inhibitor of ${\beta}$-catenin dependent Wnt pathway from rhizomes of Atractylodes macrocephala Koidzumi (Compositae). The active compound was purified by activity-guided purification and the structure was identified as 2,8-dimethyl-6-hydroxy-2-(4-methyl-3-pentenyl)-2H-chromene (atractylochromene, AC). AC suppressed b-catenin/Tcell factor transcriptional activity of HEK-293 reporter cells when they were stimulated by Wnt3a or inhibitor of glycogen synthase kinase-$3{\beta}$. AC down-regulated the nuclear level of ${\beta}$-catenin through the suppression of galectin-3 mediated nuclear translocation of ${\beta}$-catenin in SW-480 colon cancer cells. Furthermore, AC inhibits proliferation of colon cancer cell. Taken together, AC from A. macrocephala might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.1
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• pp.53-61
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• 2018

Ethyl linoleate is an unsaturated fatty acid used in many cosmetics for its various attributes, such as antibacterial and anti-inflammatory properties and clinically proven to be an effective anti-acne agent. In this study, we investigated the effect of ethyl linoleate on the melanogenesis and the mechanism underlying its action on melanogenesis in B16F10 murine melanoma cells. Our results revealed that ethyl linoleate significantly inhibited melanin content and intracellular tyrosinase activity in ${\alpha}$-MSH-induced B16F10 cells, but it did not directly inhibit activity of mushroom tyrosinase. Ethyl linoleate inhibited the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein 1 (TRP1) in governing melanin pigment synthesis. We observed that ethyl linoleate inhibited phosphorylation of Akt and glycogen synthase kinase $3{\beta}$ ($GSK3{\beta}$) and reduced the level of ${\beta}-catenin$, suggesting that ethyl linoleate inhibits melanogenesis through $Akt/GSK3{\beta}/{\beta}-catenin$ signal pathway. Therefore, we propose that ethyl linoleate may be useful as a safe whitening agent in cosmetic and a potential therapeutic agent for reducing skin hyperpigmentation in clinics.