Effects Of Specific Inhibitor Of Src-family Tyrosine Kinase On Calcium Influx Induced By Hydrogen Peroxide In Lens Epithelial Cells

BackgroundOur previous study proved that cortical cataract formed in chick embryo lens mechanically removed from the eye, the opacities began in the equatorial region and developed gradually toward the inner part of the lens. The cortical cataract formed in this model was involed with the activation of SFK, then the inhibition of SFK in the cultured lens would block the formation of cataract.These results suggest that the activation of SFK play a vital role in the cataract formation. The activation of SFK by high H202 concentration has been suggested in different cell types, and was required for a series of signal transduction mechanisms, especially those stimulated by oxidant stress. Ca2+ homeostasis is highly significant in terms of lens transparency, and Ca2+ increseases sharply in cortial cataract. The studies on H202-induced cataract demonstrated that Ca2+-activated protease calpain induced opacification. Ca2+ have fumdamental importance as a second messenger in cell signalling mechanisms, the signalling path moduated by Ca2+ must be interacted with that initiated from H202-induced-SFK-activation.So we focus on whether the formation of cortical cataract is involed with the increasement of Ca2+ in lens that caused by SFK activation.AimTo investigate the effects of specific inhibitor of Src-family tyrosin kinase (SFK) on calcium increasement induced by hydrogen peroxide in human lens.MethodsThe isolated LECs were loaded with Fluo-3/AM and then treated with H2O2 at various concentrations. The alteration of intracellular calcium was observed by confocal laser scanning microscope.To investigate the effects of PP1 on intracellular calcium, we employed three sets of cells uploaded with Fluo-3/AM and pretreated with 0.1nmol/L PP1, 0.3u/L catalase, and DMSO. These three different sets of cells were then treated with 0.1mM H2O2 and cultured in medium DMEM with normal, low, or high concentration of calcium, respectively. The intracellular free calcium was determined by confocal laser scanning microscope.The embryonic lenses were cultured in medium 199,containing 10% fetal bovine serum with the condition of vitaminC,catalase and PP1,the control was added the vehicle DMSO.Culture media containing inhibitor or control media were renewed every day throughout the 10-day culture period.Lens were observed in a dissectiong microscope and images captured daily.The total area of each lens and the area of the lenses that were opaque were also determined;The calcium concentration in culture medium of lens respectively cultured for 1,5,10 days was detected by colotimetric method.After cultued for 10 days, the expression of p-src in lens capsule was determined by Western blot.ResultsThe fluorescent intensity of calcium in LECs increased with the concentration of H2O2 . In culture with normal DMEM, after treatment with 0.1mM H2O2, the increased amplitudes of fluorescent intensity in PP1 pretreated cells and catalase pretreated cells, were reduced 28.5%±4.2% and 33.8%±3.7% compared with DMSO-pretreated cells (P<0.01). While in the cells cultured with DMEM with low calcium, there were no obvious changes of fluorescent intensity of calcium in all three sets of cells after treatment with H2O2. In contrast with them, in the cells cultured in medium with high calcium, the fluorescent intensity increased dramatically induced by H2O2 in all three sets of cells, but the increased amplitudes in PP1 and catalase pretreated cells decreased 13.5%±1.8% and 21.3%±2.4% compared with DMSO-pretreated cells P<0.01), respectively. There were no significant differences between PP1 and catalase pretreated cells at reduced amplitudes when cultured in normal and high calcium medium (P>0.05).; Almost all lens in the control group developed cortical opacities covering approximately 52% of the lens area by day 10,while that of vitamin C,catalase and PP1-treated groups was 10%,19%,13%(P<0.01). After cultued for 10 days in vitro,the calcium concentration in culture mdium of four groups all raised, vitamin C,catalase and PP1-treated group was remarkably decreased(P<0.01),and the PP1- treated group had a most remarkable decreasing effect.The expressin of p-src in lens capsule was significantly low after treatment with vitamin C,catalase and PP1( P<0.01), and the effect of vitamin C,PP1 was superior to catalase( P<0.01). ConclusionsThe specific inhibitor of SFK, PP1 can block the calcium influx induced by oxidative stress, PP1 may prevent formation of cortical cataract by inhibiting Ca-dependent signaling pathways involved in cataractgenise.