• Journal of Ginseng Research
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• v.45 no.2
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• pp.264-272
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• 2021

Background: Gintonin is a ginseng-derived exogenous G-protein-coupled lysophosphatidic acid (LPA) receptor ligand, which exhibits in vitro and in vivo functions against Alzheimer disease (AD) through lysophosphatidic acid 1/3 receptors. A recent study demonstrated that systemic treatment with gintonin enhances paracellular permeability of the blood-brain barrier (BBB) through the LPA1/3 receptor. However, little is known about whether gintonin can enhance brain delivery of donepezil (DPZ) (Aricept), which is a representative cognition-improving drug used in AD clinics. In the present study, we examined whether systemic administration of gintonin can stimulate brain delivery of DPZ. Methods: We administered gintonin and DPZ alone or coadministered gintonin with DPZ intravenously or orally to rats. Then we collected the cerebral spinal fluid (CSF) and serum and determined the DPZ concentration through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results: Intravenous, but not oral, coadministration of gintonin with DPZ increased the CSF concentration of DPZ in a concentration- and time-dependent manner. Gintonin-mediated enhancement of brain delivery of DPZ was blocked by Ki16425, a LPA1/3 receptor antagonist. Coadministration of vascular endothelial growth factor (VEGF) + gintonin with DPZ similarly increased CSF DPZ concentration. However, gintonin-mediated enhancement of brain delivery of DPZ was blocked by axitinip, a VEGF receptor antagonist. Mannitol, a BBB disrupting agent that increases the BBB permeability, enhanced gintonin-mediated enhancement of brain delivery of DPZ. Conclusions: We found that intravenous, but not oral, coadministration of gintonin facilitates brain delivery of DPZ from plasma via LPA1/3 and VEGF receptors. Gintonin is a potential candidate as a ginseng-derived novel agent for the brain delivery of DPZ for treatment of patients with AD.

• Biomolecules & Therapeutics
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• v.14 no.1
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• pp.25-29
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• 2006

We tested effects of bioactive lysophospholipids including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosylphosphorylcholine (SPC), and sphingosine I-phosphate (S1P) on membrane potential in C6 glioma cells to understand action mechanism of the lysophospholipids. Membrane potential was estimated by measuring fluorescence change of DiBAC-loaded glioma cells. LPA largely increased membrane potential and the increase was gradually diminished. LPC also increased the membrane potential, however, the increase sustained. SPC induced smaller increase of membrane potential than LPC. SIP was not able to change the membrane potential. We tested effects of suramin and pertussis toxin on lysophospholipid-induced membrane potential increase. However, there wasn't any effect. The membrane potential increase was partially diminished in $Na^+$-free media, suggesting $Na^+$ influx as a component of membrane potential changes. Thus, involvement of $Na^+$ influx in the increase of membrane potential by lysophospholipids and independence of suramin-sensitive GPCRs and pertussis toxin-sensitive G proteins are found in this study.

• Biomolecules & Therapeutics
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• v.25 no.4
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• pp.354-361
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• 2017

Transcriptional co-activator with a PDZ-binding motif (TAZ) is an important factor in lysophosphatidic acid (LPA)-induced promotion of migration and proliferation of human mesenchymal stem cells (MSCs). The expression of TAZ significantly increased at 6 h after LPA treatment, and TAZ knockdown inhibited the LPA-induced migration and proliferation of MSCs. In addition, embryonic fibroblasts from TAZ knockout mice exhibited the reduction in LPA-induced migration and proliferation. The LPA1 receptor inhibitor Ki16425 blocked LPA responses in MSCs. Although TAZ knockdown or knockout did not reduce LPA-induced phosphorylation of ERK and AKT, the MEK inhibitor U0126 or the ROCK inhibitor Y27632 blocked LPA-induced TAZ expression along with the reduction in the proliferation and migration of MSCs. Our data suggest that TAZ is an important mediator of LPA signaling in MSCs in the downstream of MEK and ROCK signaling.

• Journal of Ginseng Research
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• v.45 no.3
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• pp.390-400
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• 2021

Background: We recently showed that gintonin, an active ginseng ingredient, exhibits antibrain neurodegenerative disease effects including multiple target mechanisms such as antioxidative stress and antiinflammation via the lysophosphatidic acid (LPA) receptors. Amyotrophic lateral sclerosis (ALS) is a spinal disease characterized by neurodegenerative changes in motor neurons with subsequent skeletal muscle paralysis and death. However, pathophysiological mechanisms of ALS are still elusive, and therapeutic drugs have not yet been developed. We investigate the putative alleviating effects of gintonin in ALS. Methods: The G93A-SOD1 transgenic mouse ALS model was used. Gintonin (50 or 100 mg/kg/day, p.o.) administration started from week seven. We performed histological analyses, immunoblot assays, and behavioral tests. Results: Gintonin extended mouse survival and relieved motor dysfunctions. Histological analyses of spinal cords revealed that gintonin increased the survival of motor neurons, expression of brain-derived neurotrophic factors, choline acetyltransferase, NeuN, and Nissl bodies compared with the vehicle control. Gintonin attenuated elevated spinal NAD(P) quinone oxidoreductase 1 expression and decreased oxidative stress-related ferritin, ionized calcium-binding adapter molecule 1-immunoreactive microglia, S100β-immunoreactive astrocyte, and Olig2-immunoreactive oligodendrocytes compared with the control vehicle. Interestingly, we found that the spinal LPA1 receptor level was decreased, whereas gintonin treatment restored decreased LPA1 receptor expression levels in the G93A-SOD1 transgenic mouse, thereby attenuating neurological symptoms and histological deficits. Conclusion: Gintonin-mediated symptomatic improvements of ALS might be associated with the attenuations of neuronal loss and oxidative stress via the spinal LPA1 receptor regulations. The present results suggest that the spinal LPA1 receptor is engaged in ALS, and gintonin may be useful for relieving ALS symptoms.

• Animal cells and systems
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• v.1 no.4
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• pp.641-646
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• 1997

Lysophosphatidic acid (LPA; 1-acyl-glycerol-3-phosphate) has been known as an intercellular phospholipid messenger with a wide range of biological activities. In this study, the effect of LPA on both the proliferation and differentiation of rat E63 myoblasts has been investigated. In the serum-free Insulin-Transferrin-Selenium (ITS) media, the proliferation of E63 cells was largely restricted. Addition of LPA into the ITS media strongly promoted the cell proliferation and resulted in two to four fold increase of cell number. Furthermore, it appeared to increase the percent fusion in a dose-dependent manner up to 15 ug/ml. The synthesis of myosin heavy chain (MHC) was increased by LPA as well. These results indicate that LPA is able to promote both cell proliferation and differentiation in rat E63 myoblasts. Suramin, known to have uncoupling activity on growth factor-receptor interaction, was tested for antagonistic activity in myoblast proliferation and differentiation. Myoblasts grown in the ITS medium containing LPA were able to proliferate well even in the presence high concentration of suramin whereas myoblast differentiation was completely blocked by 30 ug/ml of suramin. The inhibitory effect of suramin on the myoblast differentiation was completely reversible by removing the suramin. This result indicates that the intracellular signaling pathway of LPA leading to cell proliferation might be distinct from that leading to cell differentiation on E63 myoblasts. Also, the antagonistic effect of suramin suggests that the differentiation activity elicited by LPA might be mediated by a specific G protein-coupled receptor.

• Biomolecules & Therapeutics
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• v.15 no.3
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• pp.150-155
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• 2007

In the present study, we have tested the effect of dioleoyl phosphatidic acid (PA) on intracellular $Ca_{2+}$ concentration ($[Ca^{2+}]_{i}$) in two human colon cancer cell lines (HCT116 and HT29). PA and lysophosphatidic acid (LPA), a bioactive lysolipid, increased $[Ca^{2+}]_{i}$ in both HCT116 and HT29 cell lines. Increases of $[Ca^{2+}]_{i}$ by PA and LPA were more robust in HCT116 cells than in HT29 cells. A specific inhibitor of phospholipase C (U73122), however, was not inhibitory to the cell responses. Pertussis toxin, a specific inhibitor of $G_{i/o}$ type G proteins, however, had an inhibitory effect on the responses except for an LPA-induced one in HT29 cells. Ruthenium red, an inhibitor of the ryanodine receptor, was not inhibitory on the responses, however, 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor, completely inhibited both lipid-induced $Ca^{2+}$ increases in both cell types. Furthermore, by using Ki16425 and VPC32183, two structurally dissimilar specific antagonists for $LPA_{1}/LPA_{3}$ receptors, an involvement of endogenous LPA receptors in the $Ca^{2+}$ responses was observed. Ki16425 completely inhibited the responses but the susceptibility to VPC32183 was different to PA and LPA in the two cell types. Expression levels of five LPA receptors in the HCT116 and HT29 cells were also assessed. Our data support the notion that PA could increase $[Ca^{2+}]_{i}$ in human colon cancer cells, probably via endogenous LPA receptors, G proteins and $IP_{3}$ receptors, thereby suggesting a role of PA as an intercellular lipid mediator.

• Journal of Ginseng Research
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• v.43 no.2
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• pp.305-311
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• 2019

Background: Gintonin is a ginseng-derived exogenous ligand of the G protein-coupled lysophosphatidic acid (LPA) receptor. We previously reported that gintonin stimulates gliotransmitter release in primary cortical astrocytes. Astrocytes play key roles in the functions of neurovascular systems. Although vascular endothelial growth factor (VEGF) is known to influence the normal growth and maintenance of cranial blood vessels and the nervous system, there is little information about the effect of gintonin on VEGF regulation in primary astrocytes, under normal and hypoxic conditions. Methods: Using primary cortical astrocytes of mice, the effects of gintonin on the release, expression, and distribution of VEGF were examined. We further investigated whether the gintonin-mediated VEGF release protects astrocytes from hypoxia. Results: Gintonin administration stimulated the release and expression of VEGF from astrocytes in a concentration- and time-dependent manner. The gintonin-mediated increase in the release of VEGF was inhibited by the LPA1/3 receptor antagonist, Ki16425; phospholipase C inhibitor, U73122; inositol 1,4,5- triphosphate receptor antagonist, 2-APB; and intracellular $Ca^{2+}$ chelator, BAPTA. Hypoxia further stimulated astrocytic VEGF release. Gintonin treatment stimulated additional VEGF release and restored cell viability that had decreased due to hypoxia, via the VEGF receptor pathway. Altogether, the regulation of VEGF release and expression and astrocytic protection mediated by gintonin under hypoxia are achieved via the LPA receptor-VEGF signaling pathways. Conclusion: The present study shows that the gintonin-mediated regulation of VEGF in cortical astrocytes might be neuroprotective against hypoxic insults and could explain the molecular basis of the beneficial effects of ginseng on the central nervous system.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.325-333
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• 2016

Background: Ginseng extracts are known to have angiogenic effects. However, to date, only limited information is available on the molecular mechanism underlying the angiogenic effects and the main components of ginseng that exert these effects. Human umbilical-vein endothelial cells (HUVECs) are used as an in vitro model for screening therapeutic agents that promote angiogenesis and wound healing. We recently isolated gintonin, a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand, from ginseng. LPA plays a key role in angiogenesis and wound healing. Methods: In the present study, we investigated the in vitro effects of gintonin on proliferation, migration, and tube formation of HUVECs, which express endogenous LPA1/3 receptors. Results: Gintonin stimulated proliferation and migration of HUVECs. The LPA1/3 receptor antagonist, Ki16425, short interfering RNA against LPA1 or LPA3 receptor, and the Rho kinase inhibitor, Y-27632, significantly decreased the gintonin-induced proliferation, migration, and tube formation of HUVECs, which indicates the involvement of LPA receptors and Rho kinase activation. Further, gintonin increased the release of vascular endothelial growth factors from HUVECs. The cyclooxygenase-2 inhibitor NS-398, nuclear factor kappa B inhibitor BAY11-7085, and c-Jun N-terminal kinase inhibitor SP600125 blocked the gintonin-induced migration, which shows the involvement of cyclooxygenase-2, nuclear factor kappa B, and c-Jun N-terminal kinase signaling. Conclusion: The gintonin-mediated proliferation, migration, and vascular-endothelial-growth-factor release in HUVECs via LPA-receptor activation may be one of in vitro mechanisms underlying ginsenginduced angiogenic and wound-healing effects.

• Biomolecules & Therapeutics
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• v.22 no.2
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• pp.129-135
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• 2014

Previously, we reported that lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, can increase intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) via type 1 lysophosphatidic acid (LPA) receptor ($LPA_1$) and CD97, an adhesion G-protein-coupled receptor (GPCR), in MDA-MB-231 breast cancer cells. Furthermore, LPE signaling was suggested as like $LPA_1/CD97-G_{i/o}$ proteins-phospholipase $C-IP_3-Ca^{2+}$ increase in these cells. In the present study, we further investigated actions of LPE not only in the $[Ca^{2+}]_i$ increasing effect but also in cell proliferation and migration in MDA-MB-231 breast cancer cells. We utilized chemically different LPEs and a specific inhibitor of $LPA_1$, AM-095 in comparison with responses in SK-OV3 ovarian cancer cells. It was found that LPE-induced $Ca^{2+}$ response in MDA-MB-231 cells was evoked in a different manner to that in SK-OV3 cells in terms of structural requirements. AM-095 inhibited LPE-induced $Ca^{2+}$ response and cell proliferation in MDA-MB-231 cells, but not in SK-OV3 cells, supporting $LPA_1$ involvement only in MDA-MB-231 cells. LPA had significant effects on cell proliferation and migration in MDA-MB-231 cells, whereas LPE had less or no significant effect. However, LPE modulations of MAPKs (ERK1/2, JNK and p38 MAPK) was not different to those by LPA in the cells. These data support the involvement of LPA1 in LPE-induced $Ca^{2+}$ response and cell proliferation in breast MDA-MB-231 cells but unknown GPCRs (not $LPA_1$) in LPE-induced responses in SK-OV3 cells. Furthermore, although LPE and LPA utilized $LPA_1$, LPA utilized more signaling cascades than LPE, resulting in stronger responses by LPA in proliferation and migration than LPE in MDA-MB-231 cells.