The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells / 中华实验眼科杂志

ABSTRACT

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation,migration and epithelialmesenchymal transition (EMT) of human lens epithelial cells (LECs).To explore the target treatment of PCO is very important.Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs.Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured,and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor-β2 (TGF-β2) (5,10,20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group,TGF-β2 group and Smac-hyperexpression +TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group,H2 O2 group and siRNA-Smac+H2 O2 group.The expressions of Smac,caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot.Results The GFP+ cells were≥ 80％ 12 hours after siRNA-Smac3 transfection,with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32 ± 2.31)％ in the siRNA-Smac3 transfection group,which was significantly higher than that in the blank plasmid group ([4.91 ±0.24] ％) (t=116.342,P＜0.001).The relevant expression levels of Smac was 35.21 ±4.11 in the Smachyperexpression group,and that in the blank plasmid group was 15.24±2.48,with a significant difference between them (t =215.47,P＜0.05).The cell viability of 20 ng/ml TGF-β2 affected PBS group,TGF-β2 group and Smachyperepression+TGF-β2 group was (98.4 ± 1.7) ％,(98.9 ± 0.1) ％ and (64.2 ± 3.1) ％,and the cell viability of Smac-hyperepression+TGF-β2 group was significantly lower in the Smac-hyperepression+TGF-β2 group than that in the TGF-β2 group (P＜0.05).The apoptotic rate in the PBS group,H2 O2 group and siRNA-Smac+H2 O2 group were (2.9 ± 1.2) ％,(45.1 ±4.5) ％ and (27.5 ± 1.8) ％,and the apoptotic rate was evidently lower in the siRNA-Smac +H2O2 group than that in the H2O2 group (P＜0.05).RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group,H2 O2 group and siRNA-Smac + H2 O2 group were 0.321 ± 0.103,0.715 ± 0.112 and 0.479 ±0.209,respectively.Compared with the H2 O2 group,the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased,the difference was statistically significant (P＜ 0.05).The PCNA mRNA expression levels in PBS group,TGF-β2 group and Smac-hyperepression+TGF-β2 group were 0.299±0.013,0.645± 0.102 and 0.490±0.209,respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051,with significant difference between the two groups (t =132.52,P＜0.05).The relative expression of PCNA protein in Smac-hyperepression+TGF-β2 group was 0.782±0.212,which was lower than 1.126±0.251 in the TGF-β2 group (P＜0.05).Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs.