Korean J Physiol Pharmacol.  2017 Mar;21(2):189-195. 10.4196/kjpp.2017.21.2.189.

Comparison of cytotoxicities and wound healing effects of diquafosol tetrasodium and hyaluronic acid on human corneal epithelial cells

• Lee JH
• Lee JS
• Kim S
• Lee JE

• KMID: 2371037
• DOI: http://doi.org/10.4196/kjpp.2017.21.2.189

Abstract

This study aimed to compare the cellular toxicities of three clinically used dry eye treatments; 3% diquafosol tetrasodium and hyaluronic acid at 0.3 and 0.18%. A methyl thiazolyltetrazoiun (MTT)-based calorimetric assay was used to assess cellular proliferation and a lactate dehydrogenase (LDH) leakage assay to assess cytotoxicity, using Human corneal epithelial cells (HCECs) exposed to 3% diquafosol tetrasodium, 0.3% hyaluronic acid (HA), or 0.18% HA or 1, 6 or 24 h. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy, and wound widths were measured 24 h after confluent HCECs were scratched. Diquafosol had a significant, time-dependent, inhibitory effect on HCEC proliferation and cytotoxicity. HCECs treated with diquafosol detached more from the bottoms of dishes and damaged cells showed degenerative changes, such as, reduced numbers of microvilli, vacuole formation, and chromatin of the nuclear remnant condensed along the nuclear periphery. All significantly stimulated reepithelialization of HCECs scratched, which were less observed in diquafosol. Therefore, epithelial toxicity should be considered after long-term usage of diquafosol and in overdose cases, especially in dry eye patients with pre-existing punctated epithelial erosion.

Keyword

Diquafosol; Dry eye syndromes; Hyaluronic acid; Toxicity

MeSH Terms

Cell Proliferation
Chromatin
Dry Eye Syndromes
Epithelial Cells*
Humans*
Hyaluronic Acid*
L-Lactate Dehydrogenase
Microscopy
Microscopy, Electron
Microvilli
Vacuoles
Wound Healing*
Wounds and Injuries*
Chromatin
Hyaluronic Acid
L-Lactate Dehydrogenase

Figure

• 

  Fig. 1 Metabolic activities of human corneal epithelial cells as determined by methyl thiazolyl tetrazolium (MTT) assay after 1, 6, or 24 hours of treatment with 3% diquafosol, or 0.3% or 0.18% hyaluronic acid (HA).Cell viability significantly decreased after treatment for 1 hour with 30% diluted diquafosol and after 6 hours of treatment with 10% or 20% diluted diquafosol. The two HAs did not significantly change cell viability. Survival rates are provided as mean±SEMs and *means significantly different (p<0.05).

• 

  Fig. 2 Lactate dehydrogenase (LDH) activities of human corneal epithelial cells after treatment with 3% diquafosol or 0.3% or 0.18% hyaluronic acid (HA) for 1, 6, or 24 hours.Diquafosol increased LDH activities all times versus HAs, and significantly increased LDH activity after 24 hours. Survival rates are presented as means±SEMs and * means significantly different (p<0.05).

• 

  Fig. 3 Inverted phase-contrast micrographs of human corneal epithelial cells exposed to 0.3% diquafosol or 0.3% or 0.18% hyaluronic acid (HA) (original magnification, ×200).Many epithelial cells were visible in control culture media (A). HCECs were more detached from dishes after treatment with 0.3% diquafosol (B) than after treatment with 0.3% (C) or 0.18% (D) HA.

• 

  Fig. 4 Transmission electron micrographs of human corneal epithelial cells exposed to 0.3% diquafosol, or 0.3% or 0.1% hyaluronic acid (HA) (bar length=2 mm: original magnification, ×3000~4000).Normal corneal epithelial cells (A) showing microvilli, homogenous cytoplasm, and intact cell and nuclear membranes. Cells treated with diquafosol (B) showing loss of microvilli, condensed nuclear remnant along nuclear peripheries (white arrowheads), and cytoplasmic vacuole formation presumed to be due to swollen cytoplasmic organelles (black arrowheads). When exposed to 0.3% (C) or 0.18% (D) HA, corneal epithelial cells appeared normal with the exception of well-developed cytoplasmic vacuoles (white arrows).

• 

  Fig. 5 The closure of HCEC wounds in response to 3% diquafosol, or 0.3% or 0.18% hyaluronic acid (HA).The effects of diquafosol and HAs on wound closure are expressed as percentage reductions in average wound width 24 hours after confluent HCECs were scratched in the absence (A) or presence of diquafosol (B) or HAs (C&D) (2 mg/mL). Results are the means±SDs (n=3) of percentage reductions in wound widths, which were determined by measuring average widths at 10 positions. HAs and diquafosol were found to significantly stimulate wound closure versus control, and HAs had greater effects than diquafosol.

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