ABSTRACT
Background Human LECs can express telomerase activity,which participates in the formation of cataract.It is reported that estrogen can increase the expression of telomerase activity in human endometrial cancer and breast cancer cells and play an important role in promoting proliferation and anti-apoptosis,but whether estrogen exerts its role on human LECs is still unclear.Objective This study aimed to investigate whether β-estradiol (β-E2) can increase the telomerase activity of human LECs and the influence of β-E2 on proliferation and apoptosis of human LECs.Method Human LECs line was cultured and passaged in vitro,and 1×10-6 mol/L β-E2 was added in the medium for 0,6,12,24 and 48 hours,and reverse transcription PCR was used to determine the optimal time of the expression of human telomerase reverse transcriptase (hTERT) mRNA in the cells.Cultured cells were divided into five groups.The cells in the blank contol group were cultured in the routin medium.Ethanol of 0.1％ was added in the solvent control group,and 1 × 10-8,1 × 10-7 or 1 × 10-6 mol/L β-E2 was added in the medium in different contents of β-E2 groups,respectively.The relative expression level of hTERT mRNA in different groups was detected by reverse transcription PCR.Telomere repeat amplification protocol (TRAP)-ELISA was employed to determine the telomerase activity.The proliferative value of the cells was assayed by cell counting kit-8,and the apoptosis rate of the cells was examined by Hoechst33258 staining.Results The optimal time of β-E2 to rise the expression of hTERT mRNA (absorbance) was at 24 hours under the 1×10-6 mol/L.The relative expression levels of hTERT mRNA in the cells were 0.477±0.015,0.712±0.013 and 0.914±0.031 in the 1 ×10-8,1 ×10-7 and 1 ×10-6 mol/L β-E2 group,which were signifincatly higher than 0.428±0.010 in the blank control group and 0.426±0.010 in the solvent control group (all at P＜0.05).The telomerase activity values (absorbance) were 0.711 ±0.015,0.941±0.010 and 1.249±0.047 in the 1×10-8,1×10-7and 1×10-6 mol/L β-E2 group,which were higher than 0.535±0.013 in the blank control group and 0.543 ±0.013 in the solvent control group (all at P =0.000).The proliferantive values of the cells (absorbance) were significantly raised in the 1 × 10-8 mol/L β-E2 group compared with l × 10-7 and 1 × 10-6 mol/L β-E2 group (both at P =0.000),and no significnant difference was found in the proliferetive values between the blank control group and the solvent control group (P =0.718,0.856).The apoptosis rates of the cells in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L β-E2 group were lower than those in the the blank control group and the solvent control group (all at P=0.000),and there was no significant difference between the blank control group and the solvent control group (P =0.777).No obvious correlation was found between the HLECs preliferative values and hTERT mRNA expression levels or telomerase activity values (r=-0.299,P=0.278;r=-0.157,P=0.576).However,significantly negative correlations were seen between apoptosis rates and hTERT mRNA expression levels or telomerase activity values (r =-0.975,P=0.000;r=-0.981,P=0.000).Conclusions β-E2can increase the activity of telomerase in human LECs,and high dose of β-E2can inhibit apoptosis,but it dose not promote proliferation.
ABSTRACT
Background Age-related cataract is a common cause of blindness.However,its cause and pathogenic mechanism have not been fully understood.Recent studies revealed that aquaporin 1 (AQP1) and AQP0 are closely related to the pathogenesis of cataract.Objective This study was to investigate the differential distribution and expression of AQP0 and AQP1 in lenses with age-related cataract and explore its effect on pathogenesis of age-related cataract.Methods Seventeen anterior capsular membrane samples and nucleus samples of lenses were collected from age-related cataract patients during the small incision nonphacoemulsification cataract extraction,and 6 normal lens samples were obtained from health donors in the First Affiliated Hospital of Fujian Medical University.The expression and distribution of AQP1 and AQP0 in the lenses were detected by immunohistochemistry,and the relative expression levels of AQP1 and AQP0 proteins in the lenses were assayed by using Western blot assay.This study protocol was approved by Ethic Committee of this hospital,and written informed consent was obtained from each patient.Results Immunohistochemistry showed that in the normal lenses,AQP1 expressed mainly in LECs;while AQP0 primarily expressed in fiber cells of the lens cortex and nucleus.The relative expression levels of AQP1 and AQP0 in the lenses with age-related cataract (absorbance) were 0.223±0.008 and 0.118±0.015,which were significantly lower than 0.246±0.007 and 0.149±0.007 in the normal lenses (t =-4.508,-3.291,both at P＜0.01).Western blot revealed that the relative expression levels of AQP1 and AQP0 in the lenses with age-related cataract (absorbance) were 0.663 ± 0.012 and 0.599 ± 0.015,which were significantly reduced in comparison with 0.844±0.041 and 0.955 ±0.064 in the normal lenses (t =-7.492,P＜0.05;t =-9.570,P＜0.01).Conclusions AQP1 and AQP0 distribute in different sites of lenses.The expressions of AQP1 and AQP0 are obviously down-regulated in lenses with age-related cataract,suggesting that AQP1 and AQP0 probably play different roles in the pathogenesis of age-related cataract.
ABSTRACT
Background Age-related cataract is one of the common causes of blindness.Although the pathophysiology of age-related cataract is far from clearly understood,it is well accepted that DNA damage plays an important role in the disease pathogenesis.Objective The purpose of this study was to quantitatively evaluate the DNA damage in peripheral lymphocytes of age-related cataract.Methods A cross-sectional study was carried out.This study complied Declaration of Helsinki and approved by Ethic Committee of Affiliated Hospital of Nantong University.Written informed consent was obtained from each subject.Two hundred and eleven patients with agerelated cataract and 147 normal subjects were enrolled from a “ Jiangsu Eye Study:Funing 2011 Eye Disease Epidemic Survey”.All the subjects aged from 50 through 80 years with matched age and gender between the two groups.The percentage of tail DNA and Olive tail moment (OTM) were detected by comet assay to assess the extent of DNA damage in peripheral lymphocytes.Statistical analyses were performed with SPSS 17.0 software,and the differences of the percentage of tail DNA and OTM were compared between the age-related cataract group and normal control group by independent sample t test as well as among the 50-59 years group,60-69 years group and ≥70 years group by one-way analysis of variance.Results Comet assay showed a round lymph cell with the clear border in the normal group;while in the age-related cataract group,the cell was bigger with a comet-like tail.The percentage of tail DNA and OTM in peripheral lymphocytes were (21.75 ± 3.51) ％ and 6.54 ± 1.65 in the age-related cataract group,and those in the normal control group were (9.31 ±3.60)％ and 2.18 ± 1.10,respectively,with significant differences between them (t =32.67,P =0.00 ; t =28.02,P =O.00).In the 50-59 years subgroup of the age-related cataract group,the percentage of tail DNA and OTM in peripheral lymphocytes were (20.04±2.86) ％ and 5.92± 1.14,and in the 60-69 years subgroup of the age-related cataract group,the percentage of tail DNA and OTM in peripheral lymphocytes were (20.77 ±2.93) ％ and 6.13 ± 1.14,which were significantly reduced in comparison with (22.79 ± 3.67)％ and 6.95±1.91 of the ≥70years subgroup(TailDNA％:q=2.75,P=0.00; q=2.02,P=0.00;OTM:q=1.03,P =0.02 ; q =0.82,P =0.00).Conclusions The pathogenesis and development of age-related cataract probably is associated with DNA damage.