| ObjectiveTo study how the glutathione (GSH), catalase (CAT) influence the transforming growth factor(TGF-β2)-induced the lens epithelial cell (LECs) apoptosis, expression of mRNA ofα-Smooth muscle actin (α-SMA) and proliferation in vitro rabbit lens epithelial cell. To investigate the mechanism of antioxidants affect TGF-β2-induced subcapsular cataract.Materials and methodsThe 2～4 weeks' healthy rabbits were randomly selected (from Zhengzhou University Laboratory Animal Center) without limit of sex and weight and were killed by injecting air in ear vein. LECs were cultured in vivo form the lens of killed rabbits. Cells were transferred when they nearly merged. The 2～3 generation cells were choosed to experiment and divided into 6 groups. Every group was added to differernt reagents. Group A: 75pg/mlTGF-β2, B:75pg/mlTGF-β2 +10 mMGSH, C:75pg/ml+TGF-β2300U/mLCAT, D:10mMGSH, E:300U/mLCAT and F:cntrol group. Cell morphology was observed by inverted microscope, inhibition rate of LECs' proliferation was assayed by MTT at 12,24,48h, cell apoptosis was assayed by TUNEL. Reverse transcriptase PCR (RT-PCR) cells were detected byα-SMA mRNA content and were all the results were compared with each other.Results1.The primary cells growed from the capsule edge after three days by inverted microscope, translucent, polygonal shape, extend pseudopods rich in cytoplasm. After adding 75pg/mlTGF-β2, the gap increased and cells showed net, they began to swell and appeared spindle gradually,but antioxidants could reduce the cellular changes2.Inhibition rate of LECs' proliferation were detected by MTT at 12,24,48h respectively. Group A: the rate was 14.6±4.62%,28.8±3.83%,34.2±2.49%; Group B:15.4±5.13%,28.6±3.78%,31.0±4.18%; Group C:14.6±3.36%,27.2±3.96%,33.6±2.50%. Comparing group A to B and C respectively, showing the value of P> 0.05, no statistically significant.3.TUNEL detect apoptosis:apoptotic cells were observed showing green fluorescence by fluorescence microscopy. Group D, E, F showed green fluorescent cells occasionally, in contrast, group A showed more green fluorescent cells. The number of fluorescent cells were significantly reduced in group B, C. Three horizons were randomly selected from every treatment group, counting the number of apoptotic cells and calculating the apoptosis rate (the number of apoptotic cells/the total number of cells in one field). F=142.873 P<0.05, the apoptosis rate between all the groups were different. The apoptosis rate of group A was 30.78±3.17%. Group A was compared to group B and C respectively, showing the value of P> 0.05, no statistically significant.4.The expression ofα-SMA mRNA was detected in LECs by RT-PCR. the experimental group appears specificα-SMA bands at 516bp. Gray value ofα-SMA mRNA in group A was 0.863±0.020. Compared to the gray value of group B and C respectively, the value of P> 0.05, no statistically significant.Conclusion1.TGF-β2 inhibited LECs proliferation and induced apoptosis and epithelial mesenchymal transition(EMT).2. GSH or CAT can reduce the LECs apoptosis, inhibite EMT, and have little effect on cell proliferation.Reactive oxygen species may involved in cell apoptosis and EMT induced by TGF-β2 as a cell signaling molecule.
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