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Soon-Suk Kang, Jin Hyoung Park; Effect of antioxidant on conjunctiva exposed to high glucose. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3044.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effects of N-acetylcystein (NAC), which is known to inhibit ROS-dependent apoptosis, on high glucose-induced ROS, apoptosis, inflammation, and delayed-wounding closure in primary cultured human conjunctival epithelial cells (pHCECs) and regulatory effects of cleaved caspase-3, Bax, NF-kB, and IL-6 and TNF-α on this process.
High glucose-induced ROS generation was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). Effects of NAC for 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 inflammatory response, IL-6 and TNF-α expression levels were quantified by multiplex cytokine analysis and NF-kB activation and IkB-α degradation were assessed by Western blot. The effects of NAC on high glucose-delayed conjunctival epithelial wound healing were assessed by Scratch-induced directional wounding assay.
Compared with untreated control and normal glucose, 5 mM, high glucose at 25 mM stimulated ROS generation, apoptosis, inflammatory cytokines, and delayed wound healing in pHCECs. The addition of NAC markedly abolished the high glucose-induced ROS, Annexin-PI-positive cells, levels of cleaved caspase-3 and Bax, and IL-6 and TNF-α. Also, NAC prevented high glucose-delayed wound healing.
High glucose promotes apoptosis by affecting mitochondria-dependent caspase activity, through elevated ROS, a process that can be reversed by the antioxidant NAC. These findings demonstrated that NAC have a beneficial effect on conjunctival epithelial cell wound healing, anti-apoptosis, and anti-inflammation.
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