Mechanism Of Chloride Channel On The Volume Change Of Lens Epithelial Cell Induced By Oxidative Stress

Objective:Using H2O2-induced lens epithelial cell damage as the model of cells damage inducedby oxidative stress, to investigate the role of chloride channels in oxidativestress-induced apoptotic volume changes and apoptosis in lens epithelial cells (HLEB-3).Methods:1. The volume changes of living cells (HLE B-3) folloing exposure to hypotonicsolution was measuredby lag-time microphotograph acquisition and analysissystem.Iron replacement and block of chloride channels were also applied in thepresent study to investigate the role of chloride channels in regulatory volumedecrease.2. H2O2was used to induce HLE B-3cells and the protective effect of chloridechannel blockers were clarified. HLE B-3cells were exposed to H2O2of500μmol/Land the change of cell shape and nuclear morphology were observed by lightmicroscope with HE staining and nuclear staining with AO/EB after H2O2-inducedfor12hours.The cell viability of HLE B-3cells was detected by3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.the apoptosis of HLE B-3cells was detected by flow cytometry (FCM) with annexin and propidium iodide (PI)stain, 3. To observe the role of chloride channels blocker in H2O2-induced apoptosis andapoptotic volume decrease in lens epithelial cell, H2O2was used to induce HLE B-3cells apoptosis. During6h,The cell viability of HLE B-3cells was detected by3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.theexpression of apoptosis-related proteins (Caspase-3) was assessed by western blot andELISA. The cell apoptosis of HLE B-3cells was detected by DNA Ladder assasy,.thevolume changes of living cells (HLE B-3) folloing exposure to hypotonic solutionwas measuredby lag-time microphotograph acquisition and analysis system.Result:1. Extracelluar hypotonic treatment made the cells swell and induced RVD,and RVDwas correlated positively to the swelling.Substitution of nitrate for Cl in perfusingsolutions to deplete cellular Cl to markedly abolishe,and chloride channel blockersinhibited RVD.2.5-uitro-2-(3-phenylpropylamino) benzoic acid (NPPB,100mmol/L) and4,4’-diisothioeyano-2,2’-disulfonic acid stilbene (DIDS,100mmol/L) show protective effects onH2O2-induced apoptosis after12h of treatment,inhibited the H2O2-induced apoptosisby（20.78士2.62）%and（15.83士2.57）%3. Treatment for6hours with500mol/L H2O2resulted in significantly decrease in cellvolume．However，the early-phase alterations in cell volumewere markedy abolishedby pretreatment with chloride channels blockers．An early apoptosis-related volumedecrease was induced significantly by H2O2in HLE B-3cells．The ceil shrunkgradually in the period observed and the volume decreased by (26.98士2.35)%in6h.The chloride channel blocker，5-uitro-2-(3-phenylpropylamino)benzoic acid (NPPB,100mo1/L) and4,4’-diisothioeyano-2,2’-disulfonic acid stilbene(DIDS,100mo1/L)inhibited largely the H2O2-induced ceil volume decrease to (6.81士0.57)%and (7.03士0.67)％.Conclusion: 1. HLE B-3cells are capable of RVD, Cl efflux through Cl channels is the keymechanism of RVD.2. Chloride channels blockers NPPB and DIDS show protective effects on H2O2-induced apoptosis.3. Apoptosis-related cell volume decrease is an early event before Caspase-3activityand DNA fragmentation, Chloride channels may mediate Apoptosis-related volumedecrease through inducing H2O2-induced HLE B-3cell apoptosis．...