Cell Viability Change And Molecular Mechanism Of Distilled Water On Human Lens Epithelial Cells

Part Ⅰ: Cell viability change and clearing effect of distilled water on human lens epithelial cells ex vivoPurpose: To evaluate the effective and most effective application time of anterior lens epithelial cells(LECs)from age-related cataract patients death caused by distilled water(DW)or detachment resulted from DW combined with rinse ex vivo.To observe and analyze the histopathological changes of LECs after DW treatment.Methods: 156 anterior lens capsules were collected carefully from uneventful cataract surgery and cut in half for 312 small pieces.157 small pieces used for cell viability study were divided into 4 groups,negative control,positive control,balanced salt solution(BSS)group and DW group(capsules were immersed in BSS or DW for 1,2 or 3 minutes respectively).Statistical analysis was did for cell viability indicators.125 capsule small pieces were separated into 2 groups,BSS with rinse group and DW with rinse group(capsules were immersed in BSS or DW for 1,2,or 3 minutes and then rinsed by BSS with height of 70 cm respectively).Then we judged the clearing effect by calculating detached LECs’ percentage.Regarding the analysis of histopathological changes,15 capsule small pieces were analyzed by light microscope with hematoxylineosin(HE)staining and 15 capsule small pieces were studied via transmission electron microscopy(TEM).Results:(1)Cell viability change of DW on LECs: LECs treated by DW for 2 or 3minutes had significantly lower LECs density,higher number and rate of dead LECs compared with negative control group.DW each group had significantly higher number and rate of dead LECs than BSS each group with the same action time.LECs treated with DW for 3 minutes had the maximum dead LECs’ number and rate.(2)Clearing effect of DW on LECs: Per DW group with or without rinse had a significantly higher percentage of exfoliated LECs than negative control group.DW combined with rinse caused obviously higher fallen LECs’ percentages than using DW only at any action time point.DW 3 minutes with rinse group had the maximum detached LECs’ percentage.(3)Microstructural changes of LECs after DW treatment: HE staining results showed that LECs destruction could be observed after DW treatment at 1,2 or 3 minute and partial LECs detachment after DW immersion for 2 or 3 minutes.(4)Ultrastructural variations of LECs triggered by DW: In DW 1,2 and 3 minutes group,we noticed that cytolysis,swelling organelles,cell conjunctions damage.Furthermore,the loosening junction between LECs and the lens capsule could be found in DW 1 or 2 minutes group,and DW 3 minutes group showed that cell detachment from the capsule partially.Conclusions:(1)Either DW 2 or 3 minutes treatment caused obvious reduction of LECs’ viability,leading to cell death,with the phenomenon most obvious in DW 3minutes group.(2)With the combination of BSS rinse,DW treatment for 1,2 or 3 minutes could detach LECs from the anterior capsule effectively ex vivo,with the best clearing effect was in DW 3 minutes with rinse group.(3)LECs changes in histopathology reminded that DW led to necrosis.Part Ⅱ:Molecular mechanism of distilled water on human lens epithelial cellsObjective:To determine whether lens epithelial cells(LECs)apoptosis occur in response to distilled water(DW),and identify the valid and most valid action time of apoptosis occurence after DW treatment.To find the effective and most effective action time for apoptosis associated proteins change,which included increased Bax,Cleaved caspase 3,Cleaved caspase 9 and decreased Bcl-2.Methods:The SRA01/04 human LECs were cultivated and there were 4 groups:negative control,positive control,balanced salt solution(BSS)group and DW group(capsules were treated by BSS or DW for 30 seconds,1,2 or 3 minutes separately).The detection of cell apoptosis was based on flow cytometry and statistical analysis of cell apoptosis rates was did subsequently.Western blotting was then used to measure the change of apoptosis related proteins,Bax,Bcl-2,Cleaved caspase 3 and Cleaved caspase9.Statistical analysis of four protein levels among different groups was also did.Results:(1)Flow cytometry: Both DW 2 and 3 minutes group could induce obviously higher apoptosis rates and late apoptosis rates than negative control group;DW3 minutes group triggered significantly higher early apoptosis rate than negative control group.DW raised higher apoptosis rate and late apoptosis rate than BSS for 2 or 3minutes.DW 3 minutes group had the most significant increased apoptosis rate.(2)Western blotting: DW 2 and 3 minnutes treatment had the ability of obviously increasing Bax,Cleaved caspase 3,Cleaved caspase 9 protein levels and decreasing Bcl-2 protein level compared to negative control group.DW could induced significantly higher Bax,Cleaved caspase 3 and Cleaved caspase 9 protein levels and lower Bcl-2protein level than BSS with the same action time of 1,2 or 3 minutes.The most obvious change of apoptosis associated proteins was in DW 3 minutes group.Conclusions:(1)DW 2 or 3 minutes treatment could induce LECs apoptosis,and mainly caused late apoptosis.DW 3 minute treatment was the most effective method to trigger LECs apoptosis.(2)The significantly higher Bax,Cleaved caspase 3 and Cleaved caspase 9 protein levels and lower Bcl-2 protein level were found in DW 2 or 3 minutes group,with the most obvious change in DW 3 minutes group.