Anti-inflammatory and anti-apoptotic effects of N-acetylcysteine in diabetic rat corneal epithelium

Abstract

Aim: To characterize the anti-inflammatory and anti-apoptotic effects of N-acetylcysteine (NAC) in streptozotocin (STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells (HCECs) exposed to a high-glucose environment.

Methods: HCECs were incubated in 0, 5, 50 mmol/L glucose medium, or 50 mmol/L glucose medium with NAC for 24h. Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group. We characterized receptor for advanced glycation end-products (RAGE) expression using immunofluorescence, and interleukin (IL)-1β and cleaved caspase-3 (CCAP-3) expression using immunohistochemistry. Circulating tumor necrosis factor (TNF)-α concentration was measured by ELISA and cleaved poly-ADP ribose polymerase (PARP) concentration was quantified by Western blotting. Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining.

Results: Diabetic rats had higher expression of RAGE (2.46±0.13 fold), IL-1β, and CCAP-3 in apoptotic cells of their corneas than control rats. The expression of RAGE (1.83±0.11 fold), IL-1β, and CCAP-3, and the number of apoptotic cells, were reduced by topical NAC treatment. HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α (310±2.00 pg/mL) and cleaved PARP (7.43±0.56 fold), and more extensive apoptosis than cells in 50 mmol/L glucose medium. However, the addition of NAC reduced the concentrations of TNF-α (153.67±2.31 pg/mL) and cleaved PARP (5.55±0.31 fold) and the number of apoptotic cells.

Conclusion: NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

Keywords: N-acetylcysteine; apoptosis; corneal epithelium; diabetes; inflammation; rat.

Figure 1. NAC reduces the expression of RAGE, an AGE receptor that is associated with pro-inflammatory gene activation, in the corneal epithelium of diabetic rats

RAGE expression was detected by immunofluorescence in the STZ diabetic rats of DM+NAC group were topically applied with 10 µL of 200 µg/mL NAC for 4wk. A: Immunofluorescent staining was performed using an anti-RAGE antibody (green). The nuclei were counterstained using DAPI (blue). Representative data of one of three mice are shown. White scale bar: 50 µm. B: Relative RAGE expression was semi-quantified. The error bars represent the standard deviation of separate sample. ^aP<0.05; DM: Diabetes mellitus; NAC: N-acetylcysteine; DAPI: 4′-6-diamidino-2-phenylindole; RAGE: Receptor for advanced glycation end-products.

Figure 2. NAC inhibits inflammatory cytokine expression in the corneal epithelium of diabetic rats and in high glucose treated HCECs

Immunohistochemistry analysis for IL-1β was performed on diabetic rats that were topically administrated with NAC (10 µL of 200 µg/mL). And cells were treated with glucose (0, 5, 50 mmol/L) or glucose with NAC (3 mmol/L) for 24h. A: Representative images of the immunohistochemical staining for IL-1β in the corneal epithelium of diabetic rats that were or were not treated with NAC (n=3). Black scale bar: 50 µm. B: The concentration of TNF-α in the media surrounding HCECs that were exposed to high-glucose concentrations and treated with or without NAC was quantified using an ELISA. Representative data from three independent experiments are shown. The error bars represent the standard deviation of three independent experiments. ^aP<0.05; DM: Diabetes mellitus; NAC: N-acetylcysteine; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α.

Figure 3. NAC reduces the levels of extrinsic apoptotic factors in diabetic corneal epithelium and HCECs

In the corneal epithelium of the STZ induced-diabetic rats that is topically applied with or without 10 µL of 200 µg/mL NAC for 4wk, immunohistochemistry analysis for CCAP-3 was performed. A: Representative immunohistochemistry for CCAP-3 in the corneal epithelium of diabetic rats that were or were not treated with NAC. Representative data of one of three mice are shown. Black scale bar: 50 µm. B: Cleaved PARP levels were measured by Western blotting in HCECs subjected to glucose (0, 5, 50 mmol/L) stress±co-treated with NAC (3 mmol/L) for 24h. β-actin was used as a loading control. Representative data for three independent experiments and the quantitative densitometry results are shown. ^aP<0.05; DM: Diabetes mellitus; NAC: N-acetylcysteine; CCAP-3: cleaved caspase-3; PARP: Poly-ADP ribose polymerase.

Figure 4. NAC reduces apoptosis in diabetic corneal epithelium

Few TUNEL-positive nuclei were detected in corneal epithelial cells of STZ induced-diabetic rats of DM+NAC group were topically applied with 10 µL of 200 µg/mL NAC for 4wk. A: TUNEL assay (green) of corneal epithelium from diabetic rats that were or were not treated with NAC. The nuclei were counterstained using DAPI (blue). Representative images of one of three mice are shown. White scale bar: 50 µm. B: The number of TUNEL-positive cells in epithelium was counted. The field is 0.03 mm². ^aP<0.05. DAPI: 4′-6-diamidino-2-phenylindole; DM: Diabetes mellitus; NAC: N-acetylcysteine.

Figure 5. NAC reduces apoptosis in HCECs exposed to high-glucose concentrations and treated with or without NAC

Apoptosis analysis involved culturing HCECs in the presence of glucose media (0, 5, 50 mmol/L) with or without NAC (3 mmol/L). A: The level of apoptosis was determined using flow cytometry. B: The percentage of FITC-labeled cells was calculated as the sum of the values in quadrants Q2 and Q4. The percentage of PI-labeled cells was calculated as the sum of the values in quadrants Q1 and Q2. Representative data for three independent experiments are shown. ^aP<0.05 for PI-positivity; ^bP<0.05 for FITC-positivity; NS: Not significant; NAC: N-acetylcysteine.