August 2015
Volume 56, Issue 9
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Cornea  |   August 2015
The Antioxidant N-Acetylcysteine Inhibits Inflammatory and Apoptotic Processes in Human Conjunctival Epithelial Cells in a High-Glucose Environment
Author Affiliations & Notes
  • Jin Hyoung Park
    Department of Ophthalmology, University of Ulsan College of Medicine, Gangneng Asan Hospital, Gangneng, Republic of Korea
  • Soon-Suk Kang
    Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
  • Jae Yong Kim
    Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
    Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
  • Hungwon Tchah
    Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
    Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
  • Correspondence: Hungwon Tchah, Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul, Republic of Korea; hwtchah@amc.seoul.kr
Investigative Ophthalmology & Visual Science August 2015, Vol.56, 5614-5621. doi:10.1167/iovs.15-16909
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      Jin Hyoung Park, Soon-Suk Kang, Jae Yong Kim, Hungwon Tchah; The Antioxidant N-Acetylcysteine Inhibits Inflammatory and Apoptotic Processes in Human Conjunctival Epithelial Cells in a High-Glucose Environment. Invest. Ophthalmol. Vis. Sci. 2015;56(9):5614-5621. doi: 10.1167/iovs.15-16909.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We evaluated the effects of N-acetylcysteine (NAC), which is known to inhibit reactive oxygen species (ROS)–dependent apoptosis, on high glucose–induced ROS, apoptosis, inflammation, and delayed-wounding closure in primary cultured human conjunctival epithelial cells (pHCECs), and the regulatory effects of cleaved caspase-3, BAX, nuclear factor κB (NF-κB), IL-6, and TNF-α on these processes.

Methods: High glucose–induced ROS generation was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). The effects of NAC on high glucose–induced apoptosis were investigated in pHCECs using Annexin-V and PI staining, and cleaved caspase-3 and BAX expression levels using immunoblotting. To evaluate the inflammatory response, IL-6 and TNF-α expression levels were quantified by multiplex cytokine analysis, and NF-κB activation and IkB-α degradation were assessed by Western blot analysis. The effects of NAC on high glucose–delayed conjunctival epithelial wound healing were assessed by a scratch-induced directional wounding assay.

Results: Compared to the untreated control and normal glucose (5 mM), high glucose at 25 mM stimulated ROS generation, apoptosis, and release of inflammatory cytokines, and delayed wound healing in pHCECs. The addition of NAC markedly reduced the high glucose–induced ROS activation, Annexin-PI–positive cells, and levels of cleaved caspase-3, BAX, IL-6, and TNF-α. N-acetylcysteine also prevented high glucose–delayed wound healing.

Conclusions: High glucose levels promote apoptosis by affecting mitochondria-dependent caspase activity through elevation of ROS production, a process that can be reversed by the antioxidant NAC. These findings demonstrate that NAC has a beneficial effect on conjunctival epithelial cell wound healing, antiapoptosis, and anti-inflammation in the conjunctival epithelial cell.

Hyperglycemia has toxic effects on almost all cells in the body, including those of the eye. Although ophthalmic complications of hyperglycemia are most profound and sight-threatening in the retina, hyperglycemia also causes a diabetic corneal complication called diabetic keratopathy in the cornea. In the retina and choroid, directly or indirectly, a high glucose environment causes loss or damage of pericytes, vascular endothelial cells, and neuronal cells.1 In the cornea a high glucose environment causes epithelial cell apoptosis, stromal crosslinking, basal nerve plexus loss, and endothelial pump damage. Among these effects, a high glucose environment significantly alters the structure and function of an epithelium, resulting in basal cell degeneration25 and decreased6 or increased cell proliferation.7 These pathologic changes eventually appear as slit-lamp–identifiable clinical manifestations, including superficial punctate keratitis,8 recurrent erosions, and persistent epithelial defects,911 depending on the duration of diabetes. Although the described keratopathy can be attributed, in part, to defects in the epithelium–basement membrane adhesion complex and to corneal neuropathy,12,13 many epithelial abnormalities also may be related to the antioxidant defense system and inflammation. Moreover, mild types of diabetic ocular surface disease similar to dry eye syndrome, such as superficial punctate keratitis or even a patient's ocular discomfort, which is unidentifiable by slit-lamp, are likely to be related to the antioxidant defense system and inflammation. However, to our knowledge, there currently is no targeted therapy for diabetic ocular surface complications except for strict control of blood glucose. 
During the last few years, oxidative stress resulting from enhanced production of reactive oxygen species (ROS) and impaired antioxidant defense capabilities in response to hyperglycemia14,15 has been postulated as a unifying mechanism causing diabetic complications. In many tissues, including the retina and kidney, increasing ROS production is associated with the onset, progression, and pathological consequences of diabetes.16,17 Cells possess many naturally occurring antioxidants to detoxify the generation of damaging oxidizing agents. The antioxidant defense system includes antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and nonenzymatic antioxidants (glutathione and thioredoxin).18 Several artificial antioxidants are available that mimic the effects of natural antioxidants. One of them is N-acetylcysteine (NAC), which is known to inhibit ROS-dependent apoptosis.18 N-acetylcysteine is a synthetic precursor of intracellular cysteine and glutathione, and its anti-ROS activity results from its free radical scavenging properties either directly via the redox potential of thiols, or secondarily via increasing glutathione levels in cells.19 Reactive oxygen species can act as second messengers for several transcription factors, including nuclear factor kB (NF-κB), which has a critical role in the activation of multiple genes that contribute to the inflammatory response and end-organ damage in other disease states, such as hypertension.20 Indeed, a number of in vivo trials, including a combination of antioxidants (ascorbic acid, Trolox, NAC, β-carotene, and selenium),21 have been shown to reduce activation of NF-κB by reducing oxidative stress on the retina. 
The ocular surface (composed of the tear film, conjunctiva, and cornea) together with the aqueous humor forms the first physical and biochemical barrier to the eye and has a pivotal role in combating free radicals. Although Xu et al.22 reported hyperglycemia may cause oxidative stress by the generation of ROS and impairment of the intracellular antioxidant defense system in the cornea, little work has been done to demonstrate the role of conjunctival epithelial cells in the pathogenesis of hyperglycemia. Given that conjunctival epithelial cells cover more than 90% of the ocular surface, they could be important sources of ophthalmic complications of hyperglycemia and ROS. Thus, we have for the first time to our knowledge tested the hypothesis that high glucose in human primary conjunctival epithelial cells (pHCECs) enhances production of ROS, and that enhanced ROS induces apoptosis and inflammation, and delays wound healing. The other aim of our present study was to investigate the effects of NAC as a glucose-induced ROS scavenger on pHCECs. On the basis of our objectives, we determined that high glucose results in an increase in ROS-induced apoptotic cells and inflammatory cytokines in cultured pHCECs, and antioxidant NAC had beneficial effects on the conjunctival epithelial cell, enhancing wound healing, and reducing apoptosis and inflammation. 
Materials and Methods
pHCEC Isolation and Culture
Human conjunctivas were isolated from 12 eyes donated by six expired or brain-dead individuals. The cause of death was trauma (three of six donors), cerebrovascular disease (two of six donors), and cardiovascular disease (one of six donors). All conjunctivas were harvested completely within 2 hours after death of donor. The study protocol was approved by the institutional review board of the Asan Medical Center (IRB No. 2014-0665). The research followed the tenets of the Declaration of Helsinki, and informed consent was obtained from the appropriate family members. Conjunctival specimens were incubated for 1 hour at 37°C with 0.1% protease (Sigma-Aldrich Corp., St. Louis, MO, USA) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 nutrient mixture (DMEM/F12; Gibco, Life Technologies, Inc., Carlsbad, CA, USA) supplemented with 1% penicillin-streptomycin (Gibco). The loosened cells were scraped with a pipette, washed three times, and suspended in DMEM/F12 supplemented with 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (FBS; Gibco). Cells were pretreated on 60-mm plastic culture dishes for 1 hour at 37°C in a humidified, 5% CO2 atmosphere to eliminate fibroblasts by differential attachment. Suspended epithelial cells were seeded at 3 × 104 cells per dish onto 60-mm plastic culture dishes. Two cultures were seeded from each biopsy specimen. Bronchial epithelial growth medium and supplements (Clonetics Corp., Walkersville, MD, USA) were used as the culture medium as described previously. The culture medium was changed 1 day after seeding and every other day thereafter for 5 to 6 days until the cultures reached approximately 60% to 70% confluence, at which time they were dissociated with 0.25% trypsin-EDTA (Clonetics Corp.). 
Measuring Intracellular ROS
To examine whether high glucose treatment induces ROS production, we measured intracellular ROS levels in pHCECs using the redox-sensitive fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Life Technologies, Grand Island, NY, USA) and flow cytometry. The level of intracellular ROS was determined on the basis of the oxidative conversion of cell-permeable DCFH-DA to fluorescent DCF on reaction with hydroxyl radical, hydrogen peroxide, or peroxynitrite. The pHCECs at passage 3 were seeded onto 6-well plates. After cell attachment and growth for 1 day, the cells were treated with the indicated concentrations of glucose (5, 25 mM with or without 3 mM NAC; Sigma-Aldrich Corp.) or 20 mM mannitol as a osmolarity control for 24 hours. Cell then were incubated with DCFH-DA (10 μmol/L) at 37°C for 30 minutes in the dark, detached from culture wells by 0.25% trypsin-EDTA, and washed twice with ice-cold PBS. Flow cytometric measurements (BD Biosciences, Inc., San Diego, CA, USA) were performed six times for each treatment. The mean fluorescence intensity was quantified by CELLQuest software. Cells without DCFH-DA were used as the negative control. Experiments were repeated at least six times and demonstrated similar results. 
Assessing Apoptosis Using Flow Cytometry
To understand the effects of elevated glucose on the cellular response to apoptosis, analysis of apoptosis involved culturing pHCECs in medium containing high glucose in the presence of NAC for 24 hours, incubation with FITC-labeled Annexin V and propidium iodide, and analysis using flow cytometry. Human primary conjunctival epithelial cells were cultured in media with the indicated glucose concentrations with or without NAC for 24 hours. To assess apoptosis, cells were washed twice with ice-cold PBS, detached with 0.25% trypsin-EDTA, and harvested, and cell pellets were collected by centrifugation, resuspended in binding buffer, and incubated with FITC-labeled Annexin V and PE-labeled propidium iodide (BD Biosciences) at room temperature in the dark for 15 minutes. Cells then were analyzed using FACSCalibur flow cytometry (BD Biosciences). 
Western Blot Analysis
To evaluate whether NAC could reduce the activation of NF-κB in high glucose–induced pHCECs, Western blotting was used to quantify the nuclear translocation of the NF-κB protein subunit p65. To examine whether high glucose–induced ROS production inhibits wound closure via the PI3K/Akt pathway, we next measured pAkt and pERK levels using Western blot analysis. pHCECs were cultured in glucose media with or without NAC for 24 hours, collected, and lysed by the addition of a lysis solution to detect the protein levels of cleaved caspase-3, BAX, IkB-α, NFκB-p65, pAkt, pERK, and β-actin. The total protein concentrations in the supernatants were determined using the Bradford method. Aliquots of protein (30 μg) were boiled in equal volumes of Laemmli sample buffer and resolved using 10-12% SDS-PAGE. The blots were treated with antibodies against cleaved caspase-3 (catalog no. 9661; Cell Signaling Technology, Danvers, MA, USA), BAX (catalog no. 2772; Cell Signaling Technology), IkB-α (catalog no. 9247; Cell Signaling Technology), NF-κB-p65 (catalog no. sc-109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), pAkt (catalog no. 4060; Cell Signaling Technology), and pERK (catalog no. 9101; Cell Signaling Technology) using antibody against β-actin (Sigma-Aldrich Corp.) as the loading control. After three washes with Tris-buffered saline and 0.1% Tween-20 for 10 minutes each, the membranes were incubated with horseradish peroxidase-conjugated anti-IgGs (1:10,000). The target proteins that were specifically recognized by the antibodies were visualized using enhanced chemiluminescence reagents (Santa Cruz Biotechnology). All experiments were performed at least six times with each of six separate sets of cultures that were initiated from different donors. 
Scratch-Induced Directional Wounding Assay for Evaluating Cell Migration
To assess whether high glucose–induced ROS influences conjunctival epithelial wound healing and NAC enhances high glucose–exposed conjunctival epithelial wound closure, we cultured pHCECs in high glucose with and without NAC, using normal glucose as a control. For the cell migration assay, 70% confluent monolayers were seeded onto 6-well plates. When the cell density reached 90%, scratch wounds were created on the cell surface using a micropipette tip. Then, the cells were immediately washed twice and maintained for 24 hours in culture media treated with normal glucose (5 mM), high glucose (25 mM), high glucose with NAC (3 mM), or high mannitol (20 mM) until cell migration was photographed. Three different fields from each sample were quantitatively estimated and analyzed using Image-Pro Plus 6 (Olympus, Tokyo, Japan) according to the number of cells that migrated into the wounded areas. 
Multiplex Cytokine Analysis
Concentrations of the proinflammatory cytokines IL-6 and TNF-α in the supernatants of pHCECs cultured for 24 hours in normal or high glucose–added medium with or without NAC were determined using a custom designed cytokine/chemokine kit that uses multiplex bead technology (Luminex, Inc., Austin, TX, USA). Assays were conducted in accordance with the manufacturer's instructions. 
Statistical Analysis
All quantitative experiments were performed at least six times, and the data shown are the means ± SD of one representative experiment. Statistical significance was determined by using the Wilcoxon signed-rank test, and 95% confidence was defined as P < 0.05. 
Results
Generation of ROS in pHCECs Exposed to a High Glucose Environment
High glucose (25 mM) increased the number of cells with high intensity fluorescence compared to cells cultured in normal glucose (5 mM) or high mannitol (20 mM), indicating independent of osmotic changes (Fig. 1). High glucose caused an increase in fluorescence intensity over that in normal glucose, indicating oxidative stress in the high glucose treatment. In other words, high glucose–induced generation of ROS in cultured pHCECs was confirmed by fluorescent microscopy (data not shown). Furthermore, the production of ROS induced by high glucose was attenuated by NAC, a nonspecific ROS scavenger (Fig. 1). 
Figure 1
 
High glucose (HG) generates DCF-sensitive ROS in pHCECs, and NAC reduces HG-induced ROS. The pHCECs were cultured in normal medium (untreated control), medium containing normal glucose (NG, 5 mM), high mannitol (HM, 20 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours and then incubated with cell permeable DCFH-DA (10 μM) for 45 minutes. Cells were detached by trypsin digestion and washed with PBS. Fluorescence of DCFH-DA oxidized by intracellular ROS was detected by flow cytometry for quantitative measurement. The mean fluorescence intensity of the flow cytometry was quantified as the means ± SD. *P < 0.05 compared to NG or HN. **P < 0.05 compared to HG.
Figure 1
 
High glucose (HG) generates DCF-sensitive ROS in pHCECs, and NAC reduces HG-induced ROS. The pHCECs were cultured in normal medium (untreated control), medium containing normal glucose (NG, 5 mM), high mannitol (HM, 20 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours and then incubated with cell permeable DCFH-DA (10 μM) for 45 minutes. Cells were detached by trypsin digestion and washed with PBS. Fluorescence of DCFH-DA oxidized by intracellular ROS was detected by flow cytometry for quantitative measurement. The mean fluorescence intensity of the flow cytometry was quantified as the means ± SD. *P < 0.05 compared to NG or HN. **P < 0.05 compared to HG.
Effect of an Antioxidant on Apoptosis in pHCECs Exposed to or in a High Glucose Environment
The percentage of FITC-positive cells, which was 14.8% in normal glucose medium, increased to 41.1% in high glucose medium (Fig. 2). The apoptosis induced by high glucose was partially rescued (21.7%) by treatment with NAC (Fig. 2). The percentage of PI-positive cells, which was 18.1% in normal glucose medium, increased to 57.2% in high glucose medium. The necrosis induced by high glucose was partially rescued (9.8%) by treatment with NAC (Fig. 2). To investigate the possible mechanisms underlying the antiapoptotic effects of NAC, Western blotting was performed using cleaved caspase-3 and BAX antibodies. Western blot analysis revealed that the levels of cleaved caspase-3 and BAX were significantly increased upon exposure to high glucose (Fig. 3). However, the addition of NAC markedly reduced the high glucose–induced increase in levels of cleaved caspase-3 and BAX. 
Figure 2
 
N-acetylcysteine prevents HG-induced apoptosis in pHCECs. Cells were treated with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours. (A) Cellular apoptosis was determined by a flow cytometry analysis. The percentage of the total cells in quadrants corresponded to early apoptotic cells (Annexin V-positive cells, Q4-1, lower right), late apoptotic cells (Annexin V-positive and PI-positive cells, Q2-1, upper right), and necrotic cells (Annexin V-negative and PI-positive cells, Q1-1, upper left). Fluorescein isothiocyanate–positive rates were calculated as the sum of the values of quadrants Q2-1 and Q4-1, while PI-positive rates were calculated as the sum of the values of quadrants Q1-1 and Q2-1.
Figure 2
 
N-acetylcysteine prevents HG-induced apoptosis in pHCECs. Cells were treated with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours. (A) Cellular apoptosis was determined by a flow cytometry analysis. The percentage of the total cells in quadrants corresponded to early apoptotic cells (Annexin V-positive cells, Q4-1, lower right), late apoptotic cells (Annexin V-positive and PI-positive cells, Q2-1, upper right), and necrotic cells (Annexin V-negative and PI-positive cells, Q1-1, upper left). Fluorescein isothiocyanate–positive rates were calculated as the sum of the values of quadrants Q2-1 and Q4-1, while PI-positive rates were calculated as the sum of the values of quadrants Q1-1 and Q2-1.
Figure 3
 
The expression of cleaved caspase-3 and BAX, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 3
 
The expression of cleaved caspase-3 and BAX, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Anti-Inflammatory Effect of NAC Against High Glucose–Induced Inflammation in pHCECs
Levels of p65 were much greater for high glucose than for normal glucose, while NAC considerably reduced expression of this protein in pHCECs (Fig. 4A). To determine the molecular mechanisms of NF-κB activation in pHCECs, we determined cytoplasmic IkB-α protein levels by Western blot analysis. In high glucose medium IkB-α was markedly decreased, while NAC addition significantly prevented the degradation of IkB alpha (Fig. 4A). Interleukin-6 and TNF-α promoters both contain a binding site or sites for NF-κB; therefore, we next determined whether would inhibit production of secreted IL-6 and TNF-α cytokine proteins in supernatants of pHCECs cultured in normal or high glucose with or without NAC. There was a significant increase in TNF-α and IL-6 secretion from pHCECs exposed to high glucose compared to normal glucose media, while there was a significant reduction in the TNF-α and IL-6 levels measured in high glucose pHCECs treated with NAC (Fig. 4B). 
Figure 4
 
N-acetylcysteine inhibits HG-activated NF-κB and inflammatory cytokines in pHCECs. (A) The expression of NF-κB p65 (nuclear fraction) and IkB-α, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. (B) After an identical treatment, IL-6, and TNF-α expression were quantified by multiplex cytokine analysis. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 4
 
N-acetylcysteine inhibits HG-activated NF-κB and inflammatory cytokines in pHCECs. (A) The expression of NF-κB p65 (nuclear fraction) and IkB-α, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. (B) After an identical treatment, IL-6, and TNF-α expression were quantified by multiplex cytokine analysis. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 5
 
N-acetylcyisteine diminishes HG-delayed wound closure in pHCECs. Cells were grown to confluence on 6-well plates. A scratch wound on the monolayer was created by a sterile plastic pipette tip, and then treated with NG (5 mM), HM (20 mM), HG (25 mM), or HG with NAC (3 mM). Micrographs of cell migration into the scratch-wound region were recorded at 24 hours under a phase-contrast microscope. The number of cells that migrated toward the wounded area was quantitatively analyzed at 24 hours. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG or HM. **P < 0.05 compared to HG.
Figure 5
 
N-acetylcyisteine diminishes HG-delayed wound closure in pHCECs. Cells were grown to confluence on 6-well plates. A scratch wound on the monolayer was created by a sterile plastic pipette tip, and then treated with NG (5 mM), HM (20 mM), HG (25 mM), or HG with NAC (3 mM). Micrographs of cell migration into the scratch-wound region were recorded at 24 hours under a phase-contrast microscope. The number of cells that migrated toward the wounded area was quantitatively analyzed at 24 hours. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG or HM. **P < 0.05 compared to HG.
High Glucose Significantly Attenuates Wound Healing Through the Downregulation of pAkt and pERK
High glucose alone (25 mM) significantly impaired conjunctival epithelial wound closure in cultured pHCECs, but NAC (3 mM) treatment had an effect on wound healing of pHCECs cultured in high glucose (Fig. 5). Levels of pAkt and pERK decreased significantly under high glucose in wounded pHCECs, but this effect was reversed by the addition of NAC (Fig. 6). 
Figure 6
 
The expression level of pAkt and pERK using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 6
 
The expression level of pAkt and pERK using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Discussion
N-acetylcysteine (NAC), an antioxidant sulfhydryl substance, is a precursor in the formation of glutathione (GSH) in the body. During the process of wound healing, various inflammatory cells, such as neutrophils, macrophages, endothelial cells, and fibroblasts, produce ROS.23 Glutathione is an antioxidant that prevents damage to important cellular components caused by ROS,24 and nuclear GSH is a key regulator of epigenetic events that may be critical in the regulation of cell proliferation, a vital process in wound healing.25 In addition, NAC has been used clinically to treat a variety of conditions, including acetaminophen toxicity, acquired immune deficiency syndrome, cystic fibrosis, chronic obstructive pulmonary disease, diabetes,26 hearing loss,27 perioperative atrial fibrillation,28 acute cholestasis-induced renal failure, and acute smoke inhalation injury.29,30 N-acetylcysteine, in its oral and injectable forms, is a convenient, safe, and inexpensive medicine for burn wounds. Toon et al.30 and Demir et al.31 reported that intraperitoneally or orally administered NAC improved wound healing in irradiated rats. Topical 8% NAC eye drops also have been shown to be effective in the early treatment of experimental alkali corneal burns.32 However, the potential benefits and action mechanisms of NAC administration are little documented in the high glucose–induced pathologic process of pHCECs, which mimics hyperglycemia-induced conjunctival complications. 
Several reports have indicated that a high level (20% or 1.34 M) of NAC has been considered toxic to the cornea.3335 Xu et al.22 showed that 25 μM NAC alone in high glucose–exposed corneal epithelial cells had no effect on corneal wound healing. Halasi et al.36 reported that NAC inhibits ROS and ROS-induced apoptosis in MDA-MB-231 breast and MIA PaCa-2 pancreatic cancer cells pretreated with 3 mM NAC for 2 hours and then treated with H2O2. Thus, in our present study pretreatment for 2 hours with 3 mM NAC was selected for our experiments. 
In the literature, in vitro and animal models of type 1 and 2 diabetes have been used to study diabetic corneal complications and wound healing, and a wealth of information has been obtained from these studies.4,6,15,3742 For example, hyperglycemia was found to decrease cell proliferation, but to have no effects on apoptosis or necrosis43 in the corneal epithelium of streptozotocin-induced diabetic rats, leading to decreased cell density and increased intercellular spaces,44 characteristics of diabetic keratopathy. However, the limited tissue size of the disease animals, such as rats, has hampered the biochemical analysis of hyperglycemia-induced alterations in the cornea.22 Khalfaoui et al.45 analyzed the expression of apoptotic factors BAX and Bcl-2 in the bulbar conjunctiva of diabetic patients without retinopathy and compared the levels to the expression of these factors in nondiabetic patients. They suggested that the diabetic human conjunctiva, with its inflammatory and cicatricial phenomena, is a privileged target for apoptotic cell death. Therefore, first, we assessed ROS levels in pHCECs cultured in high glucose with or without NAC and found that NAC efficiently quenched the ROS associated with high glucose. Next, we found that NAC partially abolished ROS-dependent cell death induced by high glucose. Taken together, to the best of our knowledge, our current study is the first to demonstrate that high glucose may affect mitochondria-dependent caspase activity, leading to apoptosis, through elevated ROS, a process that can be reversed by the antioxidant NAC in human conjunctival epithelial cells. 
Activation of NF-κB is caused by a variety of cellular stimuli, including ROS. On activation, NF-κB translocates to the nucleus and regulates transcription of many genes. Among the genes regulated by NF-κB, a number have been shown to be involved in the dystrophic disease process, including proinflammatory cytokines.46 Hyperglycemia related diseases, such as diabetes and its complications, also are characterized by high levels of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α.47,48 Our current results showing that NAC treatment significantly reduced nuclear NF-κB p65 protein expression and proinflammatory cytokines IL-6 and TNF-α suggest that ROS are important mediators of NF-κB activation and translocation to the nucleus in pHCECs as well as other cell types. 
In diabetic glomeruli and mesangial cells treated with high glucose, elevated Akt phosphorylation, through the EGFR/phosphatidylinositol 3-kinase pathway, was shown to mediate high glucose–induced collagen I upregulation.49 On the other hand, high glucose specifically downregulates Akt activities in endothelial cells50,51 and progenitor cells,52,53 and disruption of the Akt pathway has been linked to endothelial dysfunction and diabetic complications, macro- and microvascular.54 Thus, the effects of hyperglycemia on the PI3-kinase/Akt pathway may be tissue-/cell type–specific. Because PI3-kinase and Akt have been shown to control diverse cellular activities, including cell survival, growth, proliferation, metabolism, and migration,55 low Akt activity induced by hyperglycemia may be a contributing factor to abnormalities, such as basal cell degeneration,5 decreased cell proliferation,9 and, more importantly, the delayed epithelial wound healing3,6,56 observed in the diabetic cornea. Therefore, high glucose–mediated impairment of the PI3-kinase/Akt pathway may contribute to delayed wound healing in human conjunctival epithelial cells cultured in high glucose. Our present results show that high glucose impairs the Akt pathway, resulting in delayed conjunctival epithelial wound healing. To our knowledge, we have here shown for the first time that high glucose specifically targets PI3-kinase in human conjunctival epithelial cells. 
In summary, we have provided evidence that a 3 mM level of NAC can inhibit high glucose–induced apoptosis and inflammation, and delay wound healing in primary human conjunctival epithelial cells. If topical NAC proves to be a successful therapeutic modality for the treatment of hyperglycemia-induced ocular surface change, including that in the cornea and conjunctiva, this antioxidant may significantly decrease the cost of treatment and, thus, substantially broaden the scope of patients eligible for treatment. Therefore, further studies are needed to determine the optimal doses of NAC in these ocular disease patients affected by hyperglycemia. The main limitation of our current study was its in vitro design and further in vivo animal studies or clinical trials are needed to confirm our findings. 
Acknowledgments
Supported by Grant W15-049 from the Asan Institute for Life Sciences, Seoul, Korea. 
Disclosure: J.H. Park, None; S.-S. Kang, None; J.Y. Kim, None; H. Tchah, None 
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Figure 1
 
High glucose (HG) generates DCF-sensitive ROS in pHCECs, and NAC reduces HG-induced ROS. The pHCECs were cultured in normal medium (untreated control), medium containing normal glucose (NG, 5 mM), high mannitol (HM, 20 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours and then incubated with cell permeable DCFH-DA (10 μM) for 45 minutes. Cells were detached by trypsin digestion and washed with PBS. Fluorescence of DCFH-DA oxidized by intracellular ROS was detected by flow cytometry for quantitative measurement. The mean fluorescence intensity of the flow cytometry was quantified as the means ± SD. *P < 0.05 compared to NG or HN. **P < 0.05 compared to HG.
Figure 1
 
High glucose (HG) generates DCF-sensitive ROS in pHCECs, and NAC reduces HG-induced ROS. The pHCECs were cultured in normal medium (untreated control), medium containing normal glucose (NG, 5 mM), high mannitol (HM, 20 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours and then incubated with cell permeable DCFH-DA (10 μM) for 45 minutes. Cells were detached by trypsin digestion and washed with PBS. Fluorescence of DCFH-DA oxidized by intracellular ROS was detected by flow cytometry for quantitative measurement. The mean fluorescence intensity of the flow cytometry was quantified as the means ± SD. *P < 0.05 compared to NG or HN. **P < 0.05 compared to HG.
Figure 2
 
N-acetylcysteine prevents HG-induced apoptosis in pHCECs. Cells were treated with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours. (A) Cellular apoptosis was determined by a flow cytometry analysis. The percentage of the total cells in quadrants corresponded to early apoptotic cells (Annexin V-positive cells, Q4-1, lower right), late apoptotic cells (Annexin V-positive and PI-positive cells, Q2-1, upper right), and necrotic cells (Annexin V-negative and PI-positive cells, Q1-1, upper left). Fluorescein isothiocyanate–positive rates were calculated as the sum of the values of quadrants Q2-1 and Q4-1, while PI-positive rates were calculated as the sum of the values of quadrants Q1-1 and Q2-1.
Figure 2
 
N-acetylcysteine prevents HG-induced apoptosis in pHCECs. Cells were treated with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours. (A) Cellular apoptosis was determined by a flow cytometry analysis. The percentage of the total cells in quadrants corresponded to early apoptotic cells (Annexin V-positive cells, Q4-1, lower right), late apoptotic cells (Annexin V-positive and PI-positive cells, Q2-1, upper right), and necrotic cells (Annexin V-negative and PI-positive cells, Q1-1, upper left). Fluorescein isothiocyanate–positive rates were calculated as the sum of the values of quadrants Q2-1 and Q4-1, while PI-positive rates were calculated as the sum of the values of quadrants Q1-1 and Q2-1.
Figure 3
 
The expression of cleaved caspase-3 and BAX, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 3
 
The expression of cleaved caspase-3 and BAX, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 4
 
N-acetylcysteine inhibits HG-activated NF-κB and inflammatory cytokines in pHCECs. (A) The expression of NF-κB p65 (nuclear fraction) and IkB-α, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. (B) After an identical treatment, IL-6, and TNF-α expression were quantified by multiplex cytokine analysis. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 4
 
N-acetylcysteine inhibits HG-activated NF-κB and inflammatory cytokines in pHCECs. (A) The expression of NF-κB p65 (nuclear fraction) and IkB-α, using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. (B) After an identical treatment, IL-6, and TNF-α expression were quantified by multiplex cytokine analysis. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 5
 
N-acetylcyisteine diminishes HG-delayed wound closure in pHCECs. Cells were grown to confluence on 6-well plates. A scratch wound on the monolayer was created by a sterile plastic pipette tip, and then treated with NG (5 mM), HM (20 mM), HG (25 mM), or HG with NAC (3 mM). Micrographs of cell migration into the scratch-wound region were recorded at 24 hours under a phase-contrast microscope. The number of cells that migrated toward the wounded area was quantitatively analyzed at 24 hours. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG or HM. **P < 0.05 compared to HG.
Figure 5
 
N-acetylcyisteine diminishes HG-delayed wound closure in pHCECs. Cells were grown to confluence on 6-well plates. A scratch wound on the monolayer was created by a sterile plastic pipette tip, and then treated with NG (5 mM), HM (20 mM), HG (25 mM), or HG with NAC (3 mM). Micrographs of cell migration into the scratch-wound region were recorded at 24 hours under a phase-contrast microscope. The number of cells that migrated toward the wounded area was quantitatively analyzed at 24 hours. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG or HM. **P < 0.05 compared to HG.
Figure 6
 
The expression level of pAkt and pERK using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
Figure 6
 
The expression level of pAkt and pERK using β-actin as a control after treatment with NG (5 mM), HG (25 mM), or HG with NAC (3 mM) for 24 hours was analyzed and quantified by Western blotting. The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared to NG. **P < 0.05 compared to HG.
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