Atypical formations of gintonin lysophosphatidic acids as new materials and their beneficial effects on degenerative diseases.

Fresh ginseng is prone to spoilage due to its high moisture content. For long-term storage, most fresh ginsengs are dried to white ginseng (WG) or steamed for hours at high temperature/pressure and dried to form Korean Red ginseng (KRG). They are further processed for ginseng products when subjected to hot water extraction/concentration under pressure. These WG or KRG preparation processes affect ginsenoside compositions and also other ginseng components, probably during treatments like steaming and drying, to form diverse bioactive phospholipids. It is known that ginseng contains high amounts of gintonin lysophosphatidic acids (LPAs). LPAs are simple lipid-derived growth factors in animals and humans and act as exogenous ligands of six GTP-binding-protein coupled LPA receptor subtypes. LPAs play diverse roles ranging from brain development to hair growth in animals and humans. LPA-mediated signaling pathways involve various GTP-binding proteins to regulate downstream pathways like [Ca2+]i transient induction. Recent studies have shown that gintonin exhibits anti-Alzheimer's disease and anti-arthritis effects in vitro and in vivo mediated by gintonin LPAs, the active ingredients of gintonin, a ginseng-derived neurotrophin. However, little is known about how gintonin LPAs are formed in high amounts in ginseng compared to other herbs. This review introduces atypical or non-enzymatic pathways under the conversion of ginseng phospholipids into gintonin LPAs during steaming and extraction/concentration processes, which exert beneficial effects against degenerative diseases, including Alzheimer's disease and arthritis in animals and humans via LPA receptors.

Visualization of the binding between gintonin, a Panax ginseng-derived LPA receptor ligand, and the LPA receptor subtypes and transactivation of the EGF receptor.

Background: Gintonin is a ginseng-derived exogenous G-protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin exerts its neuronal and non-neuronal in vitro and in vivo effects through LPA receptor subtypes. However, it is unknown whether gintonin can bind to the plasma membrane of cells and can transactivate the epidermal growth factor (EGF) receptor. In the present study, we examined whether gintonin-biotin conjugates directly bound to LPA receptors and transactivated the EGF receptor. Methods: We designed gintonin-biotin conjugates through gintonin biotinylation and examined whether gintonin-biotin conjugate binding sites co-localized with the LPA receptor subtype binding sites. We further examined whether gintonin-biotin transactivated the EGF receptor via LPA receptor regulation via phosphor-EGF and cell migration assays. Results: Gintonin-biotin conjugates elicit [Ca2+]i transient similar to that observed with unbiotinylated gintonin in cultured PC3 cells, suggesting that biotinylation does not affect physiological activity of gintonin. We proved that gintonin-biotin conjugate binding sites co-localized with the LPA1/6 receptor binding sites. Gintonin-biotin binding to the LPA1 receptor transactivates the epidermal growth factor (EGF) receptor through phosphorylation, while the LPA1/3 receptor antagonist, Ki16425, blocked phosphorylation of the EGF receptor. Additionally, an EGF receptor inhibitor AG1478 blocked gintonin-biotin conjugate-mediated cell migration. Conclusions: We observed the binding between ginseng-derived gintonin and the plasma membrane target proteins corresponding to the LPA1/6 receptor subtypes. Moreover, gintonin transactivated EGF receptors via LPA receptor regulation. Our results suggest that gintonin directly binds to the LPA receptor subtypes and transactivates the EGF receptor. It may explain the molecular basis of ginseng physiology/pharmacology in biological systems.

Protective Effects of Gintonin on Reactive Oxygen Species-Induced HT22 Cell Damages: Involvement of LPA1 Receptor-BDNF-AKT Signaling Pathway.

Gintonin is a kind of ginseng-derived glycolipoprotein that acts as an exogenous LPA receptor ligand. Gintonin has in vitro and in vivo neuroprotective effects; however, little is known about the cellular mechanisms underlying the neuroprotection. In the present study, we aimed to clarify how gintonin attenuates iodoacetic acid (IAA)-induced oxidative stress. The mouse hippocampal cell line HT22 was used. Gintonin treatment significantly attenuated IAA-induced reactive oxygen species (ROS) overproduction, ATP depletion, and cell death. However, treatment with Ki16425, an LPA1/3 receptor antagonist, suppressed the neuroprotective effects of gintonin. Gintonin elicited [Ca2⁺]i transients in HT22 cells. Gintonin-mediated [Ca2⁺]i transients through the LPA1 receptor-PLC-IP3 signaling pathway were coupled to increase both the expression and release of BDNF. The released BDNF activated the TrkB receptor. Induction of TrkB phosphorylation was further linked to Akt activation. Phosphorylated Akt reduced IAA-induced oxidative stress and increased cell survival. Our results indicate that gintonin attenuated IAA-induced oxidative stress in neuronal cells by activating the LPA1 receptor-BDNF-TrkB-Akt signaling pathway. One of the gintonin-mediated neuroprotective effects may be achieved via anti-oxidative stress in nervous systems.

Gintonin facilitates brain delivery of donepezil, a therapeutic drug for Alzheimer disease, through lysophosphatidic acid 1/3 and vascular endothelial growth factor receptors.

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.

Ginseng gintonin attenuates the disruptions of brain microvascular permeability and microvascular endothelium junctional proteins in an APPswe/PSEN-1 double-transgenic mouse model of Αlzheimer's disease.

It has been previously indicated that gintonin, which is a novel exogenous ginseng-derived lysophosphatidic acid (LPA) receptor ligand, restores memory dysfunctions in an APPswe/PSEN-1 double-transgenic mouse model of Alzheimer's disease (AD Tg mice) by attenuating ß-amyloid plaque deposition, recovering cholinergic dysfunctions and upregulating hippocampal neurogenesis in the cortex and hippocampus. Although ß-amyloid plaque depositions in AD is accompanied with disruptions of brain microvessels, including the brain-blood barrier (BBB), it is unknown whether gintonin exerts protective effects on brain microvascular dysfunctions in AD Tg mice. In the present study, the effects of gintonin-enriched fraction (GEF) on the changes in ß-amyloid plaque depositions, brain permeability of Evans blue, and microvascular junctional proteins were investigated in AD Tg mice. Long-term oral administration of GEF reduced ß-amyloid plaque depositions in the cortex and hippocampus of AD Tg mice. GEF treatment also reduced the permeability of Evans blue through BBB and decreased immunoreactivity of platelet endothelial cell adhesion molecule-1 (a marker of BBB disruption) in the cortex and hippocampus of AD Tg mice in a dose-dependent manner. However, GEF elevated the protein expression of occludin, claudin-5 and zonula occludens-1, which are tight-junction proteins. The present results demonstrated that long-term oral GEF treatment not only attenuates ß-amyloid plaque depositions in the brain but also exhibits protective effects against microvascular disruptions in AD Tg mice. Finally, GEF exhibits anti-AD effects through attenuation of ß-amyloid plaque depositions and protection against brain microvascular damage in an AD animal model.

Effects of a gintonin-enriched fraction on hair growth: an in vitro and in vivo study.

BACKGROUND: Ginseng has been widely used as a health-promoting tonic. Gintonin present in ginseng acts as a lysophosphatidic acid (LPA) receptor ligand that activates six LPA receptor subtypes. The LPA6 subtype plays a key role in normal hair growth, and mutations in the LPA6 receptor impair normal human hair growth. Currently, human hair loss and alopecia are concerning issues that affect peoples' social and day-to-day lives. OBJECTIVE: We investigated the in vitro and in vivo effects of a gintonin-enriched fraction (GEF) on mouse hair growth. METHODS: Human hair follicle dermal papilla cells (HFDPCs) and six-week-old male C57BL/6 mice were used. The mice were divided into the four groups: control, 1% minoxidil, 0.75% GEF, and 1.5% GEF. The dorsal hair was removed to synchronize the telogen phase. Each group was treated topically, once a day, for 15 days. We analyzed hair growth activity and histological changes. RESULTS: GEF induced transient [Ca2+]i, which stimulated HFDPC proliferation and caused 5-bromo-2'-deoxyuridine (BrdU) incorporation in a concentration-dependent manner. GEF-mediated HFDPC proliferation was blocked by the LPA receptor antagonist and Ca2+ chelator. HFDPC treatment with GEF stimulated vascular endothelial growth factor release. Topical application of GEF and minoxidil promoted hair growth in a dose-dependent manner. Histological analysis showed that GEF and minoxidil increased the number of hair follicles and hair weight. CONCLUSION: Topical application of GEF promotes mouse hair growth through HFDPC proliferation. GEF could be one of the main components of ginseng that promote hair growth and could be used to treat human alopecia.

Gintonin attenuates depressive-like behaviors associated with alcohol withdrawal in mice.

BACKGROUND: Panax ginseng Meyer extracts have been used to improve mood and alleviate symptoms of depression. However, little is known about the extracts' active ingredients and the molecular mechanisms underlying their reported anti-depressive effects. METHODS: Gintonin is an exogenous lysophosphatidic acid (LPA) receptor ligand isolated from P. ginseng. BON cells, an enterochromaffin cell line, and C57BL/6 mice were used to investigate whether gintonin stimulates serotonin release. Furthermore, the effects of gintonin on depressive-like behaviors following alcohol withdrawal were evaluated using the forced swim and tail suspension tests. RESULTS: Treatment of BON cells with gintonin induced a transient increase in the intracellular calcium concentration and serotonin release in a concentration- and time-dependent manner via the LPA receptor signaling pathway. Oral administration of the gintonin-enriched fraction (GEF) induced an increase in the plasma serotonin concentration in the mice. Oral administration of the GEF in mice with alcohol withdrawal decreased the immobility time in two depression-like behavioral tests and restored the alcohol withdrawal-induced serotonin decrease in plasma levels. LIMITATIONS: We cannot exclude the possibility that the gintonin-mediated regulation of adrenal catecholamine release in the peripheral system, and acetylcholine and glutamate release in the central nervous system, could also contribute to the alleviation of depressive-like behaviors. CONCLUSION: The GEF-mediated attenuation of depressive-like behavior induced by alcohol withdrawal may be mediated by serotonin release from intestinal enterochromaffin cells. Therefore, the GEF might be responsible for the ginseng extract-induced alleviation of depression-related symptoms.

Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the ß-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the ß-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.

rbCLCA1 is a putative metalloprotease family member: localization and catalytic domain identification.

Here, we identify the rat brain (rb) CLCA1 metalloprotease motif and its role in rbCLCA1 processing. GFP tagging or c-myc tagging adjacent to the rbCLCA1 signal sequence was used to detect rbCLCA1 expression and localization patterns if they matched those of other CLCA family members. Immunoblot analysis revealed that massive deletion of the metalloprotease motif affects the protein cleavage process by restricting two cleavage products to only one product. rbCLCA1 as well as the mutant proteins H155A, E156Q, H159A, D166A, E167A, E170A, and D171A overexpressed in HEK293T cells showed plasma membrane localization; and intracellular localizations of H159A and E167A were observed in permeabilized and non-permeabilized conditions. C-terminally GFP-tagged rbCLCA1 showed either ER localization or overall signal within the cells rather than on the cell surface. Cell surface biotinylation analysis was used to show that rbCLCA1, H155A, E156Q, D166A, E170A, and D171A reach the cell surface while little H159A and E167A reach the cell surface. Taken together, our findings indicate that the amino acids H159 and E167 in the rbCLCA1 metalloprotease motif are important in rbCLCA1 processing for localization to the cell surface. Our data demonstrate that rbCLCA1 localization is dependent on the H159 and E167, suggesting either the metalloprotease motif including H159 and E167 may be the key site for rbCLCA1 cellular processing or that a novel rbCLCA1 regulation mechanism exists with a metalloprotease activity.

Isolation and Expression Profile of the Ca-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis.

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.

Cloning and heterologous expression of new xANO2 from Xenopus laevis.

We have successfully isolated a novel anoctamin (xANO2), Ca(2+)-activated chloride channel (ANO1, TMEM16A), from Xenopus laevis. The cDNA sequence was determined to belong to the anoctamin family by comparison with the xTMEM16A sequence in a previous report. Full length cDNA synthesis was performed by repeating 5'- and 3'-rapid amplification of cDNA end (RACE). We successfully completed the entire cDNA sequence and transiently named this sequence xANO2. The xANO2 cDNA is 3884 base pair (bp) long and codes 980 amino acid (aa) proteins. According to an aa homology search using the Basic Local Alignment Search Tool (BLAST), xANO2 showed an overall identity of 92% to xTMEM16A (xANO1) independently sub-cloned in our laboratory. A primary sequence of xANO2 revealed typical characteristics of transmembrane proteins. In tissue distribution analysis, the gene products of anoctamins were ubiquitously detected by real-time PCR (RT-PCR). The expression profiles of each anoctamin were different among brain, oocytes, and digestive organs with relatively weak expression. To clarify the anoctamin activity, physiological studies were performed using the whole cell patch-clamp technique with HEK293T cells, enhanced green fluorescent protein (EGFP), and expression vectors carrying anoctamins. Characteristics typical of voltage-dependent chloride currents were detected in cells expressing both xANO2 and xTMEM16A but not with EGFP alone. Sensitive reactions to the anion channel blocker niflumic acid (NFA) were also revealed. Considering these results, xANO2 was regarded as a new TMEM16A belonging to the Xenopus anoctamin family.