ABSTRACT
Objective To investigate the effects of acute oxidative stress induced by H2O2 on expression of senescence marker protein30 (SMP30) and morphology,survival rate of human lens epithelial cells (HLECs).Methods HLECs were treated with H2O2(0 μmol · L-1,100 μmol · L-1,200 μmol · L-1,300 μmol · L-1) for 24 hours,the acute oxidative stress models were established,the changes of cell morphology was observed,and MTT was used to analyze the cells state,the expressions of SMP30 were measured by Western blot.Results The cell density decreased,morphological changed and viability of cells significant decreased in 100 μmol · L-1 and 200 μmol ·L-1 treated group,the large and round cells appeared,the cell body stretched with unclear boundary.With the H2O2 concentration increased,the viability of cells were gradually decreased in treated group,there were statistical differences compared with 0 μmol · L-1 treated group (all P ＜ 0.05).The relative expression of SMP30 in control group and 100 μmol · L-1 and 200 μmol · L-1 treated group were 0.273 ±0.055,0.464 ± 0.058,0.442 ± 0.050,respectively.There were significant differences between 100 μmol · L-1,200 μmol · L-1 treated group and control group (all P ＜ 0.05),and there was no statistical difference between 100 μmol · L-1 and 200 μmol · L-1 treated group (P ＞ 0.05).Conclusion SMP30 is up-regulated in HLECs under acute oxidative stress induced by H2O2,the cell morphology is changed,the viability of cells is decreased,and SMP30 may be involved in the process of acute oxidative stress in HLECs.
ABSTRACT
Background Senescence marker protein 30 (SMP30) is a new calcium regulatory protein,which plays anti-oxidation,stable calcium and anti-apoptosis roles in vivo.Researches determined that the expression of SMP30 in human cells gradually decreased as ageing.However,the study on the relationship between SMP30 and age-related cataract is rarely.Objective The aim of this study was to investigate the expression of SMP30 in lens epithelial cells (LECs) of different ages of cataract patients.Methods This study was approved by Ethic Commission of the First Affiliated Hospital of Guangxi Medical University.A non-randomized controlled trail was designed.The samples of the anterior capsular membrane of lens were collected during the cataract surgery from the children group (1-18 years),youth group (19-45 years),mid adult group (46-60 years) and elder group (＞60 years) and 12 pieces of capsular membrane for each group.In addition,12 pieces of lens capsular membrane from normal donors aged 19-45 years were obtained as normal control group.The expression of SMP30 in the samples was detected by indirect immunofluorescence method.The average fluorescent values were calculated as absorbance (A) / area.Results SMP30 was expressed in LECs of different groups with the green fluorescence primarily in the cytoplasm.The mean fluorescence intensity was 0.185±0.020,0.181±0.034,0.207±0.018 and 0.126±0.027 in the children group,youth group,mid adult group and elder group,respectively,which were significantly enhanced than 0.087±0.007 of the normal control group(q=3.96,3.82,4.01,3.55,all at P＜0.01).No significant differences in the expression of SMP30 among the children group,youth group and mid adult group (all at P＞0.05).However,the expression of SMP30 in LECs in the elder group was significantly lower than that in the children group,youth group and mid adult group (q =3.42,3.21,3.80,all at P＜ 0.05).Conclusions Expression of SMP30 in LECs dramatically increases in cataract patients,suggesting that SMP30 may be a protective factor for LECs.But SMP30 contents are lower in age-related cataract patients,which may be one of causes of senior cataract.
ABSTRACT
Background Posterior capsular opacification (PCO) is a primary cause of blurred vision after extra-capsular cataract extraction (ECCE),and there is a higher incidence of PCO in the patients with diabetes mellitus.Echistatin (Ecs) can suppress the proliferation of lens epithelial cells (LECs) and thereby inhibit the formation of PCO.However,its mechanism and safe dose deserve to study.Objective The aim of this study was to observe the inhibitory effect of different concentrations of Ecs on LECs proliferation in the early stage of PCO in diabetic rabbits and explore the safe dose of Ecs.Methods Diabetic mellitus was induced by injection of 90 mg/kg alloxan via ear vein in 15 New Zealand white rabbis.ECCE was performed in the right eyes of the rabbits.The rabbits were randomized to the control group and 5.0,7.5,10.0 and 15.0 μg/ml Ecs group according to randomized number table method.Ecs of 0.2 ml in above doses was injected into the anterior chamber after ECCE in different concentrations of Ecs groups,and 0.2 ml distilled water was used in the same way in the only diabetic rabbits as the control group.Postopeartive response of ocular anterior segment was observed and PCO was graded under the slit lamp microscope.The corneal and retinal specimens were prepared 10 days after operation for the assay of preliferative cell nuclear antigen (PCNA) expression in LECs by immunochemistry to evaluate the effective dose of Ecs.Regular histopathological examination was performed,and apoptosis of corneal endothelial cells and retinal cells was detected by TUNEL method to assess the safe concentration of Ecs.Results Different degrees of corneal edema and exudation in anterior chamber were seen in the eyes of different groups.The inflammatory response disappeared 3-5 days in the control group and 5.0 μg/ml Ecs group and 7 days in the ≥7.5 μg/ml Ecs groups.PCO was 1-2 grade in the control group and 5.0 μg/ml Ecs group and 0 grade in the ≥ 7.5 μg/ml Ecs groups.The difference in the positive expression level (absorbance,A) for PCNA in LECs was significantly different among the control group and various Ecs groups (F=18.006,P=0.001),and the positive expression level of PCNA in the ≥ 7.5 μg/ml Ecs groups was markedly reduced in comparison with that in the control group (P =0.010,0.001).Hematoxylin and eosin staining showed an normal morphology and order arrangement in corneal endothelial cells and intact structure in retinal internal limiting membrane in the groups.TUNEL assay revealed that the apoptosis values (mean A value) of corneal endothelial ceils and retinal cells in the ≤ 10.0 μg/ml Ecs groups were not significantly changed in comparison with the control group (all at P＞0.05),but the apoptosis values in the 15.0 μg/ml Ecs group were markedly higher than those in the control group (P=0.004,0.018).Conclusions Ecs can inhibit the early PCO in diabetic rabbits and show the optimal effect at the concentrations of 7.5 and 10.0 μg/ml without visible eytotoxicity to eye other tissues.Therefore,these two doses of Ees might be used for the study of long-term therapeutic effectiveness.