The Effects Of Chloride Channel Inhibitor On BFGF-induced Proliferation And Migration Of Lens Epithelial Cells

ObjectiveTo study the role of chloride channel on proliferation of Human lens epithelialcells induced by basic-fibroblast growth factor (bFGF).MethodsHLE B-3at Logarithmic growth phase were cultured in DMEM wit-l highglucose and observed with inversion light microscope. Different concentrations ofChloride channel inhibitor:5-nitro-2-(3-phenylpropylamino) benzoicacid(50、100、150μmol/L)/4，4-diisothiocyanatostibene2,2disulphonicacid（10、50、100μmol/L）in the presence of10ug/L bFGF， were added in the culture plate to treat LECs withbFGF or without bFGF in20%serum group for24、48、72hours, respectively．TheCCK-8colorimetric assay was used to check the cell viability and evaluate the drugtoxicity．The effects of Chloride channel inhibitor on bFGF-induced proliferation ofLECs were analyzed to get all effective time and concentrations of LECs. The effects ofChloride channel inhibitor on the expression of proliferating cell nuclear antigen(KI-67) of Human lens epithelial cells induced by bFGF were observed by byimmunocytochemistry staining assay．The phase change of cell cycle was used toexamine by flow cytometry and cell proliferation index was calculated. The eggects ofNPPB/DIDS on bFGF-induced migration of HLE B-3cells were observed byscratch-wound assay and the Boyden chamber assay. Results1. CCK8assay showed that10μg/L bFGF can induce LECs proliferation, and theOD values were gradually declined with the increase of Chloride channel inhibitorconcentrations and time after Chloride channel inhibitor treatment with10μg/L bFGFfor24hours,48hours and72hours（bFGF+NPPB group：Ftime=305.282，Fconcentrations=18.76，P=0.000； bFGF+DIDS group：Ftime=94.436，Fconcentrations=24.415，P=0.000）. Different concentrations chloride channel inhibitor reduced the OD valueswith10μg/L bFGF in concentration and time-dependent manner.2. When added150μmol/L NPPB and100μmol/L DIDS and calculated with10μg/L bFGF in48hour,the expression of KI-67lowed, showing a significantdifference in comparison with the bFGF group (bFGF+NPPB group：F=580.3，P=0.000; bFGF+DIDS group：F=507.4，P=0.000). Compared with the bFGF group,the number of the positive expression of KI-67is decease respectively in（18.32±1.23）%�'�（11.21±1.02）%（P<0.01，P<0.01）．3. At the48th hour after treated with150μmol/L NPPB and100μmol/L DIDS,the rate of G1stage increased（(F=390.754，P=0.000）, the rate of S stage decreasedsignificantly（F=166.240，P=0.000）. Conclusions Chloride channel inhibitor play animportant role in modulating the proliferation of LECs induced by bFGF, and make thecell cycle staged at G1stage.4. Cell longitudinal migration by Boyden chamber assay showed thatbFGF-induced migration of HLE B-3cells was slow after NPPB/DIDS treated（F=56.039,578.746,P<0.001）, with the inhibitory rates35.44%,54.43%（F=56.039,P<0.001）. And the same results also appeared in Scratch-wound assay.Conclusions:1.10μg/L bFGF can induce LECs proliferation and migration by openNPPB/DIDS sensitive cl-channel.2. Chloride channel inhibitor can suppress bFGF-induced cell proliferation andmigration within definite concentration, which is time and concentration dependent. For 24h used150μmol/L NPPB及100μmol/L DIDS can inhibit Human lens epithelial cellsproliferation and migration effect.