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Hungwon Tchah, Soon-Suk Kang, Eun-Soon Kim, Jae-yong Kim, ; Effect of Nerve Growth Factor on in vitro Human Primary Conjuntival Epithelial Cell Apoptosis Induced by Hyperosmolar Stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2127.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effect of nerve growth factor (NGF) known to be activated during inflammatory episodes of ocular diseases on apoptosis of cultured human primary conjunctival epithelial cells (pHCEC). Conjunctival epithelial cells cover more than 90% of the ocular surface. However, the role of conjunctival epithelial cells in dry eye pathogenesis, where hyperosmolar stress may be important.
For NGF transcription and production levels, RT-PCR and ELISA were assessed with pHCECs in normal osmolar medium (307 mOsm/L) or higher-osmolarity media, 350, 400, and 450 mOsm/L. For apoptosis analysis, pHCEC were cultured in normal- or 400 mOsm/L osmolar medium with NGF neutralizing antibody or recombinant human NGF for 6 hours and analysed by FACSCalibur flow cytometer. Bcl-xL, Bax, phospho-JNK, and cleaved caspase-3 expression were detected by Western blotting. Inflammatory cytokine, IL-6 was analyzed using ELISA.
NGF levels in cultured pHCECs were up-regulated during hyperosmolar conditions. Positive apoptotic cells significantly were increased in hyperosmolar-induced cells and NGF neutralizing antibody-added cells. When recombinant human NGF is added in 400 mOsm/L osmolar medium, apoptotic cells were decreased and phospho-JNK, Bax, and cleaved caspase-3 expression were down-regulated but Bcl-xL expression was increased. Upon hyperosmotic stress, IL-6 level increased and recombinant human NGF reduced IL-6 level.
Our results suggest that hyperosmolarity induces apoptosis of pHCECs by JNK signaling pathway. Up-regulated NGF under hyperosmolar condition may contribute, at least in part, to reduce apoptosis of pHCEC and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.
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