J Vet Sci.  2017 Sep;18(3):387-397. 10.4142/jvs.2017.18.3.387.

Gintonin, an exogenous ginseng-derived LPA receptor ligand, promotes corneal wound healing

• Kim HJ
• Kim JY
• Lee BH
• Choi SH
• Rhim H
• Kim HC
• Ahn SY
• Jeong SW
• Jang M
• Cho IH
• Nah SY

• KMID: 2412457
• DOI: http://doi.org/10.4142/jvs.2017.18.3.387

Abstract

Ginseng gintonin is an exogenous ligand of lysophosphatidic acid (LPA) receptors. Accumulating evidence shows LPA helps in rapid recovery of corneal damage. The aim of this study was to evaluate the therapeutic efficacy of gintonin in a rabbit model of corneal damage. We investigated the signal transduction pathway of gintonin in human corneal epithelium (HCE) cells to elucidate the underlying molecular mechanism. We next evaluated the therapeutic effects of gintonin, using a rabbit model of corneal damage, by undertaking histochemical analysis. Treatment of gintonin to HCE cells induced transient increases of [Ca²âº](i) in concentration-dependent and reversible manners. Gintonin-mediated mobilization of [Ca²âº](i) was attenuated by LPA1/3 receptor antagonist Ki16425, phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca²âº chelator BAPTA-AM. Gintonin facilitated in vitro wound healing in a concentration-dependent manner. When applied as an eye-drop to rabbits with corneal damage, gintonin rapidly promoted recovery. Histochemical analysis showed gintonin decreased corneal apoptosis and increased corneal cell proliferation. We demonstrated that LPA receptor activation by gintonin is linked to in vitro and in vivo therapeutic effects against corneal damage. Gintonin can be applied as a clinical agent for the rapid healing of corneal damage.

Keyword

corneal injuries; ginseng; gintonin; human corneal epithelium cells; lysophosphatidic acid receptor

MeSH Terms

Animals
Blotting, Western/veterinary
Calcium/metabolism
Cells, Cultured
Cornea/drug effects/pathology
Corneal Injuries/*drug therapy/pathology
Dose-Response Relationship, Drug
Humans
Male
Plant Extracts/*therapeutic use
Rabbits
Receptors, Lysophosphatidic Acid/drug effects
Wound Healing/*drug effects
Plant Extracts
Receptors, Lysophosphatidic Acid
Calcium

Figure

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  Fig. 1 Effects of gintonin (GT) on intracellular calcium transients in human corneal epithelium (HCE) cells. (A) Representative trace obtained after application of the indicated concentration of gintonin. (B) Concentration-response relationship curve for gintonin-induced [Ca2+]i transients in HCE cells. Each point represents the mean ± SEM (n = 3–4). (C) Representative traces of gintonin-mediated [Ca2+]i transients in the absence or presence of LPA1/3 receptor antagonist Ki16425. (D) Histograms representing net increases of gintonin-mediated [Ca2+]i transients calculated from traces obtained in the absence or presence of various antagonists or blockers (*p < 0.05, n = 5–6).

• 

  Fig. 2 Effects of gintonin on extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in human corneal epithelium (HCE) cells. (A) Gintonin stimulated ERK1/2 phosphorylation in a concentration-dependent manner; however, a decrease in ERK1/2 phosphorylation was observed at a high concentration of gintonin (10 µg/mL). Cells were stimulated for 5 min with vehicle (control) or the indicated concentration of gintonin, and ERK1/2 phosphorylation was evaluated as described in the Materials and Methods section. The upper panels represent basal and different concentrations of gintonin-stimulated ERK1/2 phosphorylation obtained by immunoblotting from a representative experiment performed with HCE cells. (B) Data are presented as percentage increases of basal ERK1/2 phosphorylation, where the basal amount of ERK1/2 phosphorylation in untreated cells was assigned a value of 100% (*p < 0.01, compared with the gintonin-untreated control). (C) Gintonin-mediated ERK1/2 phosphorylation was time-dependent; ERK1/2 phosphorylation peaked 5 min after gintonin treatment and decreased after 60 min to the basal level. Cells were stimulated for the indicated time with gintonin (1 µg/mL). (D) Data are presented as fold increases of basal ERK1/2 phosphorylation, where the basal amount of ERK1/2 phosphorylation in untreated cells was assigned a value of 100% (*p < 0.01, compared with the gintonin-untreated control). Data shown represent the mean ± SEM values of duplicate analyses from each of three separate experiments.

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  Fig. 3 Effects of gintonin (GT) on human corneal epithelium cell proliferation. (A) Gintonin stimulated cell proliferation in a concentration-dependent manner. (B) Gintonin-mediated cell proliferation was time-dependent. Cells were treated with the indicated concentrations of gintonin and subjected to XTT assay as described in the Materials and Methods section. Data are presented as the mean ± SEM (n = 6; *p < 0.05, **p < 0.01, compared with untreated controls).

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  Fig. 4 Gintonin (GT) stimulates migration of human corneal epithelium cells. (A) Confluent cells were scratched and incubated with gintonin at the indicated concentrations in DMEM for 20 to 24 h. (B) The relative migration was quantified by comparing the migration of the treated cells to that of the untreated cells (control). The data are presented as the mean ± SEM (n = 3–6) (*p < 0.05, compared to untreated controls). Scale bars = 100 µm.

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  Fig. 5 Fluorescein staining of eyes at 48 h post-operation. (A) Ethanol-exposed control group. (B) Ethanol exposure + gintonin-treated group. (C) Ethanol exposure + Solcorin-treated group.

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  Fig. 6 Therapeutic effect of gintonin in ethanol-induced corneal damage. Paraffinized sections of corneas from normal control (A, F, and K), ethanol-exposed (B, G, and L), ethanol-exposed and gintonin (200 µg/eye)-treated (C, H, and M), ethanol-exposed and Solcorin-treated (D, I, and N), and gintonin alone-treated (E, J, and O) groups at 2 days after ethanol exposure were stained with H&E (A–E), immunostained with proliferating cell nuclear antigen (PCNA) antibody (F–J), or stained with a TUNEL kit (K–O). (A–E) Gintonin treatment partially recovered the damaged epithelium and decreased infiltration of inflammatory cells in the stroma (B and C). Solcorin showed effects similar to those of gintonin (D). Arrows in (A) indicate the epithelium in proliferating and migrating stages. (F–J) Decreased PCNA immunoreactivity was increased in the gintonin-treated group compared with that observed in the ethanol-exposed group and was similar to that of the Solcorin-treated group. (K–O) The increased number of apoptotic cells was decreased in the gintonin-treated group compared with that in the ethanol-exposed group and was similar to that of the Solcorin-treated group. (E, J, and O) Treatment with gintonin alone did not affect the histological structure of the corneas. Epithelium (e), anterior limiting membrane (a), and stroma (s). Scale bars = 100 µm.

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