ABSTRACT
Background Diabetic retinopathy (DR) is one of the common complications of diabetes.Retinal pigment epithelium (RPE) cells,as important constituent cells of the retina,play important roles in the development and progression of DR.Objective This study was to investigate the suppressive effects of autophagy inhibitor 3-MA on the proliferation of human retinal pigment epithelium cells (hRPECs) following high glucose culture.Methods HRPECs were divided into control group,high-glucose group and 3-MA+high glucose group.The cells were cultured by the DMEM/F12 with 5 mmol/L glucose in the control group,and by the DMEM/F12 with 5 mmol/L glucose in the high glucose group and by the DMEM/F12 with 10 mmol/L 3-MA (for 1 hour firstly) and 30 mmol/L glucose in the 3-MA+high glucose group.The cells were inoculated into 24-well plate with the content of 1 ×105/well,and then the cells were consecutively cultured for 24 hours with DMEM/F12 containing 0.5％ fetal bovine serum after achieved attached 80％ confluence.The morphology and uhrastructure of the cells were examined by optics microscope and transmission electronic microscope.The proliferative rate of the cells was assayed by CCK-8 kit.The expression of autophagy-related gene microtubule associated protein light chain 3B (LC3B) in the cells was detected by Western blot and LC3B-Ⅱ/LC3B-Ⅰ value was quantitatively evaluated among the groups.Results The cells grew well with uniform size and regulatory arrangement in the control group.The cells in the high glucose group enlarged and the number of cells evidently increased.In the 3-MA+high glucose group,the cells decreased with a disorder arrangement.Under the transmission electron microscope,the cells were normal with the round-or oval-like nucleus and normal organelles in the control group,and autolysosome could be seen in the cells in the high glucose group.In the 3-MA+high glucose group,some autophagic bodies were found.The proliferative rate of the cells was (100.0±2.0) ％,(116.9-±5.2)％ and (103.7 ±4.7)％ in the control group,high glucose group and 3-MA+high glucose group respectively,showing a significant difference among the groups (F =13.526,P =0.006).The proliferative rate was considerably raised in the high glucose group compared with the control group and 3-MA+high glucose group (both at P＜0.05),but there was no significant difference in the proliferative rate of the cells between the control group and 3-MA+high glucose group (P＞0.05).Compared with the control group,the expressing intensity of LC3B-Ⅰ protein was weakened and that of LC3-Ⅱ protein was enhanced,and the expression intensity of LC3B-Ⅰ and LC3B-Ⅱ proteins in the 3-MA+high glucose group was similar to that in the control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.131 ±0.065,2.504±0.097 and 0.274±0.007 in the control group,high glucose group and 3-MA+high glucose group,respectively,with significant differences among the groups (F =1 694.676,P =0.000),and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in the high glucose group in comparison with the control group and the 3-MA+high glucose group (all at P＜0.05).No significant difference was found in the LC3B-Ⅱ/LC3B-Ⅰ ratio between the control group and 3-MA + high glucose group (P ＞ 0.05).Conclusions High glucose culture of hRPECs can activate autophagy process and promote cell proliferation.3-MA,an autophagy inhibitor,suppresses the high glucoseinduced growth of HRPECs by inhibiting autophagy process.
ABSTRACT
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 μ mol) 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 μ mol/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 μ mol/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 μ mol/L SFN groups,respectively,and the releasing level of LDH was gradually increased in the 10 and 100 μ mol/L SFN groups in comparison with the 1 μmol/LSFN 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 μ mol/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.