• Journal of Applied Biological Chemistry
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• v.64 no.4
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• pp.447-451
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• 2021

In this study, we discovered for the first time that linarin, a flavonoid compound, enhances melanin biosynthesis in B16F10 cells, and subsequently elucidated the underlying mechanism of linarin-induced melanogenesis. Linarin showed no cytotoxicity at a concentration of 42 μM and significantly increased intracellular tyrosinase activity and melanin content in B16F10 cells. Mechanistic analysis showed that linarin increased the expression of tyrosinase, tyrosinase-related protein 1 (TRP-1), and microphthalmia-associated transcription factor (MITF) that are related to melanogenesis. Moreover, linarin decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT). Finally, we evaluated the effect of the structure-activity relationship of linarin and its aglycone on melanogenesis. The results indicated that linarin enhances the expression of melanogenic proteins by activating MITF expression via the modulation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B signaling pathways in B16F10 cells, thereby enhancing melanogenesis.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.171-177
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• 2022

The precise homeostatic regulation of local auxin accumulation in xylem precursors of cambium stem cell tissues is one of the most important mechanisms for plant vascular patterning and radial secondary growth. Walls are thin (WAT1), a novel intracellular auxin transporter, contributes directly to the auxin accumulation maxima in xylem precursors. According to recent research, the auxin signaling activated pathway-related gene network was significantly enriched during the secondary growth of Panax ginseng storage roots. These imply that during P. ginseng root secondary growth, specific signaling mechanisms for local auxin maxima in the vascular cambial cells are probably triggered. This study identified four WAT1-like genes, PgWAT1-1/-2 and PgWAT2-1/-2, in the P. ginseng genome. Their expression levels were greatly increased in nitratetreated storage roots stimulated for secondary root growth. PgWAT1-1 and PgWAT2-1 were similar to WAT1 from Arabidopsis and tomato plants in terms of their subcellular localization at a tonoplast and predicted transmembrane topology. We discovered that overexpression of PgWAT1-1 and PgWAT2-1 was sufficient to compensate for the secondary growth defects observed in slwat1-copi loss of function tomato mutants. This critical information from the PgWAT1-1 and PgWAT2-1 genes can potentially be used in future P. ginseng genetic engineering and breeding for increased crop yield.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1268-1280
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• 2023

Echinacoside (ECH) is a naturally occurring phenylethanoid glycoside, isolated from Echinacea angustifolia, and this study aimed to analyze its effect on thermogenesis and its interaction with dopaminergic receptors 1 and 5 (DRD1 and DRD5) in 3T3-L1 white adipocytes and mice models. We employed RT-PCR, immunoblot, immunofluorescence, a staining method, and an assay kit to determine its impact. ECH showed a substantial increase in browning signals in vitro and a decrease in adipogenic signals in vivo. Additionally, analysis of the iWAT showed that the key genes involved in beiging, mitochondrial biogenesis, and ATP-dependent thermogenesis were upregulated while adipogenesis and lipogenesis genes were downregulated. OXPHOS complexes, Ca²⁺ signaling proteins as well as intracellular Ca²⁺ levelswere also upregulated in 3T3-L1 adipocytes following ECH treatment. This was collectively explained by mechanistic studies which showed that ECH mediated the beiging process via the DRD1/5-cAMP-PKA and subsequent downstream molecules, whereas it co-mediated the α1-AR-signaling thermogenesis via the DRD1/5/SERCA2b/RyR2/CKmt pathway in 3T3-L1 adipocytes. Animal experiments revealed that there was a 12.28% reduction in body weight gain after the ECH treatment for six weeks. The effects of ECH treatment on adipose tissue can offer more insights into the treatment of obesity and metabolic syndrome.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.2
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• pp.43-49
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• 2008

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin ($1{\mu}M$ to $100{\mu}M$) for 5 min inhibited glutamate ($100{\mu}M$, 1 min) induced $[Ca^{2+}]_i$ increase, concentration-dependently. Pretreatment with apigenin ($30{\mu}M$) for 5 min significantly decreased the $[Ca^{2+}]_i$ responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, $10{\mu}M$, 1 min) and N-methyl-D-aspartate (NMDA, $100{\mu}M$, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the $[Ca^{2+}]_i$ response induced by 50 mM KCl solution, decreased the $[Ca^{2+}]_i$ responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxy-phenylglycine (DHPG, 100 $[Ca^{2+}]_i$, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced $[Ca^{2+}]_i$ responses. Furthermore, treatment with apigenin ($30{\mu}M$) significantly inhibited the amplitude and frequency of 0.1 mM $[Mg^{2+}]_o$-induced $[Ca^{2+}]_i$ spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

• Korean Journal of Food Science and Technology
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• v.53 no.6
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• pp.722-730
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• 2021

In this study, we aimed to elucidate the intracellular signaling pathways for macrophage activation by the polysaccharide GBW-II purified from ginseng berry. GBW-II consists of 14 different sugars, including rarely observed sugars such as 2-O-methyl-xylose, apiose, aceric acid, 2-keto-3-deoxy-D-manno-2-octulosonic acid, and 2-keto-3-deoxy-D-lyxo-2-heptulosaric acid, which are typical RG-II component sugars. GBW-II enhanced the production of IL-6 and TNF-α in RAW 264.7 cells. In experiments evaluating specific inhibitor activity, it was found that the production of IL-6 was suppressed by inhibitors of SB, PD, and BAY, and the production of TNF-α was suppressed by PD and BAY. The experiments with neutralizing antibodies showed that TLR4 was involved in the stimulation of IL-6 production by GBW-II in RAW 264.7 cells, whereas TNF-α production was regulated through SR and TLR2. These results suggest that GBW-II activates the MAPK and NF-κB pathways via several macrophage receptors, including SR, TLR2, and TLR4, and subsequently induces the secretion of IL-6 and TNF-α.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.3
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• pp.357-364
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• 1999

We investigated the role of $Ca^{2+}$ and protein kinases/phosphatases in the stimulatory effect of insulin on glucose transport. In isolated rat adipocytes, the simple omission of $CaCl_2$ from the incubation medium significantly reduced, but did not abolish, insulin-stimulated 2-deoxy glucose (2-DG) uptake. Pre-loading adipocytes with intracellular $Ca^{2+}$ chelator, 5,5'-dimethyl bis (o-aminophenoxy)ethane-N,N,N'N' tetraacetic acetoxymethyl ester (5,5'-dimethyl BAPTA/AM) completely blocked the stimulation. Insulin raised intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ about 1.7 times the basal level of $72{\pm}5$ nM, and 5,5'-dimethyl BAPTA/AM kept it constant at the basal level. This correlation between insulin-induced increases in 2-DG uptake and $[Ca^{2+}]_i$ indicates that the elevation of $[Ca^{2+}]_i$ may be prerequisite for the stimulation of glucose transport. Studies with inhibitors (ML-9, KN-62, cyclosporin A) of $Ca^{2+}-calmodulin$ dependent protein kinases/phosphatases also indicate an involvement of intracellular $Ca^{2+}.$ Additional studies with okadaic acid and calyculin A, protein phosphatase-1 (PP-1) and 2A (PP-2A) inhibitors, indicate an involvement of PP-1 in insulin action on 2-DG uptake. These results indicate an involvement of $Ca^{2+}-dependent$ signaling pathway in insulin action on glucose transport.

• Journal of Ginseng Research
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• v.47 no.6
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• pp.706-713
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• 2023

Background and objective: The ability to inhibit aggregation has been demonstrated with synthetically derived ginsenoside compounds G-Rp (1, 3, and 4) and ginsenosides naturally found in Panax ginseng 20(S)-Rg3, Rg6, F4, and Ro. Among these compounds, Rk3 (G-Rk3) from Panax ginseng needs to be further explored in order to reveal the mechanisms of action during inhibition. Methodology: Our study focused to investigate the action of G-Rk3 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin α_IIbβ₃ using flow cytometry, intracellular calcium mobilization, dense granule secretion, and thromboxane B₂ secretion. In addition, we checked the regulation of phosphorylation on PI3K/MAPK pathway, and thrombin-induced clot retraction was also observed in platelets rich plasma. Key Results: G-Rk3 significantly increased amounts of cyclic adenosine monophosphate (cAMP) and led to significant phosphorylation of cAMP-dependent kinase substrates vasodilator-stimulated phosphoprotein (VASP) and inositol 1,4,5-trisphosphate receptor (IP₃R). In the presence of G-Rk3, dense tubular system Ca²⁺ was inhibited, and platelet activity was lowered by inactivating the integrin αIIb/β₃ and reducing the binding of fibrinogen. Furthermore, the effect of G-Rk3 extended to the inhibition of MAPK and PI3K/Akt phosphorylation resulting in the reduced secretion of intracellular granules and reduced production of TXA₂. Lastly, G-Rk3 inhibited platelet aggregation and thrombus formation via fibrin clot. Conclusions and implications: These results suggest that when dealing with cardiovascular diseases brought upon by faulty aggregation among platelets or through the formation of a thrombus, the G-Rk3 compound can play a role as an effective prophylactic or therapeutic agent.

• International Journal of Stem Cells
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• v.15 no.4
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• pp.384-394
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• 2022

Background and Objectives: Dental pulp stem cells (DPSCs) play an important role in the repair of tooth injuries. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a Na⁺-coupled HCO₃^- transporter encoded by the solute carrier 4A4 (SLC4A4) gene and plays a crucial role in maintaining the pH of DPSCs. Our previous research confirmed that NBCe1 is highly expressed in odontoblasts during the development of the tooth germ. Therefore, in this study, we aimed to investigate the effect of NBCe1 on odontogenic differentiation of DPSCs and further clarify the underlying mechanisms. Methods and Results: DPSCs were isolated and identified, and the selective NBCe1 inhibitor S0859 was used to treat DPSCs. We used a cell counting Kit-8 assay to detect cell proliferative ability, and intracellular pH was assessed using confocal microscopy. Odontogenic differentiation of DPSCs was analyzed using real-time PCR and Alizarin Red S staining, and the NF-κB pathway was assessed using western blotting. Our results indicated that 10 µM S0859 was the optimal concentration for DPSC induction. Intracellular pH was decreased upon treatment with S0859. The mRNA expressions of DSPP, DMP1, RUNX2, OCN, and OPN were upregulated in the NBCe1 inhibited group compared to the controls. Moreover, NBCe1 inhibition significantly activated the NF-κB pathway, and a NF-κB inhibitor reduced the effect of NBCe1 on DPSC differentiation. Conclusions: NBCe1 inhibition significantly promotes odontogenic differentiation of DPSCs, and this process may be regulated by activating the NF-κB signaling pathway.

• Journal of Life Science
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• v.28 no.9
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• pp.1016-1021
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• 2018

Clostridium difficile toxin A causes pseudomembranous colitis. The pathogenesis of toxin A-induced colonic inflammation includes toxin A-dependent epithelial cell apoptosis, resulting in the loss of barrier function provided by epithelial cells against luminal pathogens. Toxin A-dependent epithelial cell apoptosis has been linked to toxin A-induced production of reaction oxygen species and subsequent p38MAPK activation; $p21^{CIP1/WAF1}$ upregulation-dependent cell cycle arrest; cytoskeletal disaggregation; and/or the induction of Fas ligand on epithelial cells. However, the molecular mechanisms underlying toxin A-induced apoptosis remain poorly understood. This study tested whether toxin A could block the Wnt signaling pathway, which is involved in gut epithelial cell proliferation, differentiation and antiapoptotic progression. Toxin A treatment of nontransformed human colonocytes (NCM460) rapidly reduced ${\beta}$-catenin protein, an essential component of the Wnt signaling pathway. Exposure of mouse ileum to toxin A also significantly reduced ${\beta}$-catenin protein levels. MG132 inhibition of proteasome-dependent protein degradation resulted in the recovery of toxin A-mediated reduction of ${\beta}$-catenin, indicating that toxin A may activate intracellular processes, such as $GSK3{\beta}$, to promote degradation of ${\beta}$-catenin. Immunoblot analysis showed that toxin A increased active phosphorylation of $GSK3{\beta}$. Because the Wnt signaling pathway is essential for gut epithelial cell proliferation and anti-apoptotic processes, our results suggest that toxin A-mediated inhibition of the Wnt signaling pathway may be required for maximal toxin A-induced apoptosis of gut epithelial cells.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.5
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• pp.731-738
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• 2016

Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that $100{\mu}g/mL$ LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ's expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway.