Authors: Kong Deqian, Zheng Guangying, Li Li, Liu Yue, Cai Fangfei
Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). To explore the target treatment of PCO is very important.
This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs.
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, H2O2 group and siRNA-Smac+ H2O2 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.
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 Smac-hyperexpression 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 Smac-hyperepression+ 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-β2group than that in the TGF-β2 group (P<0.05). The apoptotic rate in the PBS group, H2O2 group and siRNA-Smac+ H2O2 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, H2O2 group and siRNA-Smac+ H2O2 group were 0.321±0.103, 0.715±0.112 and 0.479±0.209, respectively.Compared with the H2O2 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).
Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs.