| Objective : To investigate the establishment of a posterior cataract model with TGF-β2 acting on HLECs cells to stimulate epithelial-mesenchymal transition,Then pyrrolidine dithiocarbamate was intervened to explore the reversal of EMT by PDTC and its relationship with NF-κB/apoptotic signaling pathway,providing a theoretical basis for the application of PDTC in the treatment of PCO.Methods:(1)Posterior cataract model established: HLECs cells were stimulated with different concentrations of TGF-β2(0,1,5,10,15 and 20 ng/m L)for 24,48 and 72 h and TGF-β2 10 ng/m L at different time points(24,48 and 72 h),respectively,and the migration ability of the cells after scratching was assessed by scratching assay and detected by Western blot assay was used to detect the expression of EMT-related marker pathway-related proteins.Thus,the effective concentration and time of action were screened to determine the most suitable conditions for establishing the PCO model.(2)Reversal of EMT and induction of apoptosis in HLECs cells by PDTC: The optimal conditions for the modeling of PCO model were selected by stimulating HLECs cells with TGF-β2 10ng/m L,after giving pretreatment with PDTC.Cell migration ability was determined by scratch assay,cell viability was assessed by MTT method,cell morphology and number were recorded by cell phenotype observation,intracellular mitochondrial damage level was detected by JC-1 kit,apoptosis was detected by TUNEL kit,and the expression of NF-κB signaling pathway and apoptosis pathway-related proteins was detected by Western blot method.Results:(1)The first part of the study on the mechanism of TGF-β2-induced MET in LECs cells,with the increase of TGF-β2 concentration and the extension of stimulation time,the morphological changes and the number of cells gradually increased,showing a concentration and time dependent trend,compared with the normal group Control,when the TGF-β2 concentration kept increasing,the migration distance of cells at the scratch gradually decreased,in TGF-β2 10ng/m L was the most obvious(P<0.05),and after TGF-β2 10ng/m L stimulated cells for 0,24,48 and 72 h,cells had the strongest migration ability at 48h(P<0.05),and the expression of epithelial mesenchymal transition marker Ecadherin gradually decreased compared with Control group;the expression of mesenchymal marker α-SMA gradually increased;NF-κB p65 and phosphorylated NF-κB p65 activated expression were elevated(P<0.05).(2)Mechanistic study of the reversal effect of PDTC on EMT and induction of apoptosis.When TGF-β2 was given to stimulate the cells it was obvious that the cell growth rate was accelerated and the number was significantly increased.Then after pretreatment with different concentrations of PDTC,cell viability was significantly inhibited and showed concentration dependence.Compared with the model group,after treatment with PDTC 25,50,75 and 100 μM,cell viability was significantly reduced,intracellular mitochondrial damage was increased,apoptosis rate was significantly increased,and p-NF-κB/IκB/p-IκB/Iκκ-α/p-Iκκ-α protein expression were decreased(P<0.05),apoptosis pathway-related protein BAX/caspase-3expression was significantly increased(P<0.05),while BCL-2/Cyclin D1 expression was significantly decreased(P<0.05).Conclusion:PDTC has a reversal effect on TGF-β2-induced EMT,and its mechanism of action may be related to the inhibition of NF-κB and induction of apoptotic signaling pathways for the treatment and prevention of PCO. |
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