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Y. Kwon, S. Nam, J. Han, S. Kim, J. Lee; Prevention of Lens Eithelial Cell Death After H2O2 Exposure Using Jak Inhibitor AG490 in Human Cataract. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1464. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether lens epithelial cell death caused by H2O2 might be related to JAK/STAT pathway, using Jak inhibitor AG490.
The Human lens epithelial(HLE) B-3 cell cultures were incubated in modified essential medium(MEM) supplemented with 0.5% fetal bovine serum (FBS) for 20 hours and then treated with 1 mM H2O2, (Sigma) in serum free MEM for 45 minutes. Quantification of H2O2 induced apoptotic and/or necrotic HLE was determined by fluorescein isothiocyanate(FITC)-conjugated annexin V and propidium iodide (PI). To see the effect of AG490 (Calbiochem) , 40, 4, or 0.4 µM AG490 was treated for 1 hour before treatment of 1 mM H2O2. 100 µL cell suspension in binding buffer was stained with 10 µL annexin V-FITC and 5 µL PI. Then, the samples were immediately analyzed by flow cytometry. Cells were treated for 45 minutes with H2O2 alone, or after 1 hour pretreatment of 40 µM AG490 and observed using confocal microscope, then phosphorylated Stat3 was surveyed with Western blot analysis.
After 1mM H2O2, 50.95% of cells were dead predominantly showing apoptosis. However, if AG490 was pretreated, the proportion of cell death reduced to 4.05% after 40µM AG490, 22.34% after 4µM AG490, and 23.51% after 0.4µM AG490, respectively. Pretreated AG490 suppressed phosphorylation of Tyr-705 Stat3, Jak2. On confocal microscpic analysis, AG490 enhanced cell survival.
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