Effect Of Dynamic Concentration Of Intracellular Free Calcium On Aquaporin-1Expression And Cell Viability In Human Lens Epithelial Cells In Vitro

ObjectiveIn order to investigate the effect of dynamic concentration of intracellular free calcium onthe expression of Aquaporin-1(AQP1) in human lens epithelial cells(LECs) Cultured inVitro.To study the effect of dynamic concentration of intracellular free calcium on cellviability of human LECs in vitro.To evaluating the relationship between concentration ofintracellular free calcium and AQP1and the possible pathogenesis of Age-relatedcataract.Methods1. Human LECs were cultured in DMEM medium and DMEM medium with additionalof0.5,1.0,5.0,10.0mmol/L of calcium for2and6days. Human LECs concentration ofintracellular free calcium levels was detected by calcium fluorescent probe Fluo-3andlaser scanning confocal microscope.2. Immunohistochemical technique and Western blottechniques were used to detect the effect of dynamic concentration of intracellular freecalcium on the expression of AQP1.2. Human LECs were cultured in DMEM medium and DMEM medium with additionalof1.0,5.0,10.0mmol/L of calcium for72h,CCK-8and TUNEL were used to detect cellactivity and cell apoptosis. Human LECs ultrastructural was observed and compared byTransmission electron microscopy.Results1. Laser scanning confocal microscope images showed human LECs fluorescenceintensity increased in the calcium-treated group,the fluorescence intensity graduallyincreased with the calcium concentration increased in the DMEM medium (P<0.01). Theimmunohistochemistry and Western blot showed that the expression of AQP1in thecalcium-treated group decreased compared to normal control group in human LECs cultured in vitro (P<0.01), the expression level of AQP1was gradually reduced withconcentration of intracellular free calcium increased(P<0.01).2.The human LECs activity and apoptosis rate has no significant changes in DMEMmedium with additional of1.0mmol/L calcium compared to normal controlgroup(P>0.05).When the DMEM medium with additional of5.0mmol/L calcium,thehuman LECs activity decreased and cell apoptosis rate increased, the difference wasstatistically significant (P <0.05),the human LECs activity continued to decline with theconcentration of intracellular free calcium increased,while apoptosis rate increased((P<0.05).3. Transmission electron microscopy showed the human LECs ultrastructural has nochange when cultured in DMEM medium with additional of1.0mmol/L calcium.whileincreased the calcium with the DMEM medium with additional of5.0mmol/Lcalcium,different stages of apoptotic cells were seen,showing chromatin condensationpyknosis and margination,with mitochondria swollen and endoplasmic reticulum edema.Conclusions1. The expression of AQP1decline with the concentration of intracellular free calciumincreased in human LECs cultured in vitro.2. The human LECs activity and apoptosis rate has no effect when concentration ofintracellular free calcium increased slightly.While concentration of intracellular freecalcium increased abnormal levels,the cell avtivity decreased and cell apoptosis appeared.3.When concentration of intracellular free calcium increase,it may reduce the AQP1andinduce the human LECs apoptosis.This may be one of the Age-related cataractpathogenesis.