Korean J Ophthalmol.  2020 Feb;34(1):1-10. 10.3341/kjo.2019.0046.

Effect of Diquafosol on Hyperosmotic Stress-induced Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Corneal Epithelial Cells

Affiliations
  • 1Myungmoon Bio, Hwaseong, Korea.
  • 2Department of Physiology, College of Korean Medicine Dongguk University, Gyeongju, Korea.
  • 3Binaree, Daegu, Korea.
  • 4Central Ophthalmic Clinic, Daegu, Korea. eyepark9@naver.com
  • 5Division of Biomedicinal & Cosmetics, College of Sciences & Technology, Mokwon University, Daejeon, Korea.
  • 6Developmental Biology Laboratory, Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, Korea. jcjung@knu.ac.kr

Abstract

PURPOSE
Diquafosol is a pharmaceutical drug used for dry eye treatment with a novel mechanism of action. It is a purinergic P2Y2 receptor agonist that promotes the secretion of tears and healing of corneal epithelial wounds. However, its inhibitory effect on hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs) remains unclear.
METHODS
A hyperosmotic stress model was established by transferring HCECs from isosmotic (312 mOsm/kg to hyperosmotic medium (500 mOsm/kg). HCECs were incubated with 500 mOsm/kg hyperosmotic medium for 30 minutes, and then treated with diquafosol (0.6-6 mg/mL) for 4 or 24 hours. Cells were then harvested and analyzed by western blot, immunocytochemistry, and real-time polymerase chain reaction to evaluate the expression of interleukin-6, tumor necrosis factor-alpha, and the phosphorylation status of nuclear factor-kappa B.
RESULTS
Diquafosol significantly decreased the mRNA and protein expression of hyperosmotic stress-induced tumor necrosis factor-alpha and interleukin-6. These results were supported by immunofluorescence staining and quantitative real-time polymerase chain reaction analysis. Furthermore, diquafosol inhibits nuclear factor-kappa B activation by suppressing the phosphorylation and degradation of the inhibitor of кB.
CONCLUSIONS
This study shows that diquafosol inhibits nuclear factor-kappa B signaling and inflammatory factors induced by hyperosmotic stress in HCECs. This suggests that using diquafosol for the improvement of dry eye syndrome could be effective in the treatment of inflammation-related corneal and conjunctival diseases.

Keyword

Diquafosol; Human corneal epithelial cells; Inflammation; Interleukin-6; Tumor necrosis factor-alpha

MeSH Terms

Blotting, Western
Conjunctival Diseases
Dry Eye Syndromes
Epithelial Cells*
Fluorescent Antibody Technique
Humans*
Immunohistochemistry
Inflammation
Interleukin-6*
Necrosis*
Phosphorylation
Real-Time Polymerase Chain Reaction
RNA, Messenger
Tears
Tumor Necrosis Factor-alpha
Wounds and Injuries
Interleukin-6
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 Effects of diquafosol (DQF) on the (A) viability and (B) apoptosis of human corneal epithelial cells. Cells were treated with different concentrations of DQF solution. After 20 hours, the apoptosis rate was assessed using the CCK-8 assay kit or the annexin V and dead cell assay kit. Results are expressed as the percentage of surviving cells over control cells. Data are expressed as the mean ± standard deviation from three separate experiments (p < 0.01 and p < 0.001, significantly different from the control).

  • Fig. 2 Diquafosol downregulates mRNA and protein expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in human corneal epithelial cells. (A) Total RNA was extracted from human corneal epithelial cells. RNA levels were measured by real-time polymerase chain reaction. The cells were exposed to hyperosmotic media (500 mOsm/kg DMEM/F12, serum-free) for 30 minutes, followed by diquafosol (0.6–6 mg/mL) for 4 hours. The graph of multiple analyses shows the relative mRNA levels of TNF-α and IL-6. Glyceraldehyde-3-phosphate dehydrogenase was used as the reference gene. (B) Representative western blots of TNF-α and IL-6. Cells were exposed to hyperosmotic media (500 mOsm/kg DMEM/F12, serum-free) for 30 minutes, followed by diquafosol (0.6–6 mg/mL) for 24 hours. Expression levels of TNF-α and IL-6 were determined using β-actin as a control. The densities of bands relative to those of β-actin were measured using ImageJ software. Data are expressed as the mean ± standard deviation from three separate experiments (*p < 0.01 and **p < 0.01, significantly different from the control; #p < 0.05, ##p < 0.01, and ###p < 0.001, significantly different from the hyperosmotic group). HOS = hyperosmotic stress.

  • Fig. 3 Fluorescence immunocytochemistry for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in human corneal epithelial cells. Cells were exposed to hyperosmotic media (500 mOsm/kg DMEM/F12, serum-free) for 30 minutes, followed by diquafosol (DQF, 0.6–6 mg/mL) for 24 hours. Changes in the expression of cytoplasmic TNF-α and IL-6 were confirmed using fluorescence immunocytochemistry. Cells were counterstained with DAPI. The fluorescence intensity from green to blue was measured in ImageJ software using a color histogram. White scale bars: 100 µm (***p < 0.001, significantly different from the control; ###p < 0.001, significantly different from the hyperosmotic group). HOS = hyperosmotic stress.

  • Fig. 4 Effects of diquafosol on the nuclear factor-kappa B (NF-κB) signaling pathway in human corneal epithelial cells. (A) Expression levels of NF-κB, p-NF-κB, I-κB, and p-I-κB proteins in nuclear and cytosolic fractions were assessed using western blotting. The cells were exposed to hyperosmotic media (500 mOsm/kg DMEM/F12, serum-free) for 30 minutes, followed by diquafosol (1.5–6 mg/mL) for 30 minutes. Lamin B1 and ß-actin were used as standard proteins for quantitating the levels of the proteins of interest. (B) Expression of p-NF-κB in the nuclear fraction was confirmed using fluorescence immunocytochemistry. Cells were counterstained with DAPI. Fluorescence intensity and the density of bands were measured using ImageJ software. (C) Schematic diagram showing the inhibitory effects of diquafosol on hyperosmotic stress-induced inflammatory cytokine production via the NF-κB signaling pathway in human corneal epithelial cells. Data are expressed as the mean ± standard deviation from three separate experiments (***p < 0.001, significantly different from the control; #p < 0.05, ##p < 0.01 and ###p < 0.001, significantly different from the hyperosmotic group). HOS = hyperosmotic stress.


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