Role of chloride channel inhibitors in oxidative stress-induced lens epithelial cell apoptosis

Objective To observe the effect of chloride channels in cell damage induced by oxidative stress and to probe into its possible mechanisms.Methods 500 μmol/L tert-butyl hydroperoxide (t-BHP) was used to induce HLE B-3 cells apoptosis,the cells treated by only t-BHP were used as t-BHP-induced group,and the cells cultured by regular method were used as control group,chloride channel blockers (NPPB or DIDS) with the concentrations of 5,100 and 200 μmol/L were added into the medium for 12h respectively and with or without 500 μmol/L t-BHP-induced the cells at the same time to induce the apoptosis in experimental groups.MTT method was used to observe the cell viability.Apoptosis was measured via the Annexin V-FITC and PI staining,the intracellular free Ca2+ concentration was determined by Fluo-3 fluorometry.AO/EB double fluorescent staining was observed under the fluorescent microscope,and the cells were collected respectively after different treatments for measuring MDA level and SOD with the corresponding detection kit according to the manufactures instructions.Results Compared with t-BHP-induced group,apoptosis rate and the intracellular free Ca2+ concentration in culture medium decreased after the treatment of NPPB or DIDS (100 and 200μmol/L),cell viability in these group were significantly higher than ones of t-BHP-induced group (P ＜0.01).Chloride channel blockers relieved cell injury caused by t-BHP,MDA content were lower than those in t-BHP-induced group (P ＜0.01),and SOD activity and ATP content were higher than that in t-BHP-induced group (P ＜0.01).Conclusions Blockage of chloride channels by DIDS and NPPB rescued HLE B-3 cells from t-BHP-induced apoptosis,the mechanism might be related to inhibiting the calcium overload and improve the imbalance of redox homeostasis. Key words: Apoptosis;  Chloride channels;  Cell volume;  Lens epithelial cells;  Blockers