Authors: Liu Hanruo, Yuan Bowei, An Ying, Wan Xiuhua
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Background
Sulforaphane (SFN) is an effective chemopreventive agent and can regulate the biological molecular mechanisms to inhibit the overgrowth of cells.Autophagy is a biological process of maintaining cellular internal environment.Understanding the affection of SFN to biological behavior of human lens epithelial cells (LECs) and the association of SFN with autophagy is helpful for the prevention and target treatment of posterior capsule opacification (PCO).
Objective
This study was to investigate the eradication effeccts of SFN on residual lens cell population in vitro posterior capsule opacification (PCO) model and evaluate the mechanism of SFN-induced cell death.
Methods
In vitro human capsular bag models were generated from fresh donor eyes by phacoemulsification and were cultured in EMEM containing 2% fetal bovine serum (FBS). Different concentrations of SFN (0, 1, 10 and 100 μmol) were added in the medium for 30 days respectively according to grouping, and the growth of LECs was observed by optical microscope and immunofluorescence technique.FHL124, a human LEC line, was cultured with EMEM containing 5%FBS and divided into 0, 1, 10, 30 and 100 μmol SFN groups.Lactate dehydrogenase (LDH) release rate in the medium was detected to evaluate cell damage/death.The migration of the cells on capsular bags was assessed by scratch test.The ultrastructure and number of autophagosomes were examined under the transmission electron microscope.The expression of LC3 in the cells were detected using Western blot in the presence or absence of autophagy inhibitors.
Results
The cell coverage rates on the capsular bags were significantly lower in the 10 and 100 μmol/L SFN groups than those in the 0 and 1 μmol/L SFN groups, with a statistically significant difference among the groups (F=48.57, P<0.01). Immunofluorescence showed that the density of F-actin-and Vimentin-positive cells was evidently decreased in the 10 and 100 μmol/L SFN groups compared with 0 and 1 μmol/L SFN groups.The releasing levels of LDH (absorbancy) were 0.19±0.03, 0.39±0.06, 0.56±0.07, 0.68±0.08 and 0.89±0.09 in the 0, 1, 10, 30 and 100 μmol/L SFN groups, respectively, and the releasing level of LDH was gradually increased in the 10 and 100 μmol/L SFN groups in comparison with the 1 μmol/L SFN group (all at P<0.01). With the increase of SFN concentration, the reduction rate of scratched area decreased with the increase of SFN concentration, and the decrease of scratch area was significantly lower than that of adjacent low mass concentration group and the differences were statistically significant (P<0.05). The relative expressions of LC3-Ⅱ protein were 0.423±0.003, 14.543±0.024, 0.668±0.024 and 0.576±0.056 in the blank control group, SFN group, SFN+ 3-MA group and 3-MA group, respectively, and the relative expressions of LC3-Ⅱ protein were significantly lower in the SFN+ 3-MA group and 3-MA group than those in the SFN group (all at P<0.01). The number of autophagosomes was 4.07±0.32, 4.13±0.34, 9.21±0.53 and 21.02±1.34 in the blank control group, and 1, 10, 100 μmol/L SFN groups, and the number of autophagosomes in the 10 and 100 μmol/L SFN groups was significantly higher than that in the blank control group and 1 μmol/L SFN group (all at P<0.01).
Conclusions
SFN mediates LECs death by promoting autophagy in ex vivo capsular bags, and SFN may be a novel agent of potential chemopreventive and target treatment for PCO.