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@#AIM: To explore the effect of low expression of senescence marker protein 30(SMP30)on proliferation and oxidative stress of human lens epithelial cells(LECs)line SRA01/04 under high calcium conditions. <p>METHODS: Three RNAi sequences were designed to knock down SMP30 target gene RGN expression(KD1-3), and the blank-load sequence was used as the negative control group(NCKD), all of which were used to construct lentiviral vectors to infect SRA01/04 cells. Meanwhile, the uninfected SRA01/04 cells was used as the blank control group(CON). After transfecting SRA01/04 cells, the lentiviral vector with the highest knockdown efficiency was selected by RT-PCR for subsequent experiments. Cells were treated with 15mmol/L CaCl2 for 24h to simulate a high calcium conditions. BrdU-Elisa assay was used to measure cell proliferation, superoxide dismutase(SOD)assay kit and oxidized glutathione/total glutathione(GSSG/T-GSH)assay kit were used to detect the level of intracellular oxidative stress. <p>RESULTS: KD1-3 and NCKD lentiviral vectors were successfully constructed to infect SRA01/04 cells with an infection efficiency of about 80%. The knockdown efficiency of KD1-3 group was 93%, 60% and 74%, respectively, KD1 group was selected for follow-up experiment. Under the high calcium conditions, the activity of relative cell proliferation and SOD in KD1 group \〖(2.42±0.08)and(11.69±0.52U/mg)\〗 were significantly lower than that in NCKD group \〖(2.95±0.08)and(31.10±2.24U/mg)\〗 and CON group \〖(2.96±0.25)and(26.33±1.04U/mg)\〗, the ratio of GSSG/T-GSH in KD1 group(70.80±2.34)was significantly higher than that in NCKD group(15.93±3.47)and CON group(20.05±2.45)(<i>P</i><0.05); there was no significant difference between NCKD group and CON group(<i>P</i>>0.05).<p>CONCLUSION: Under high calcium conditions, SRA01/04 cells(HLECs)with low expression of SMP30 mediated by shRNA lentivirus resulted in the decrease of the proliferation activity and antioxidant capacity, suggesting that SMP30 may play a protective role in regulating cell proliferation and anti-oxidative stress in HLECs.
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@#【Objective】To explore the effects of overexpressed senescence marker protein 30 （SMP30） on cell proliferation and antioxidative activity in human lens epithelial cell（HLEC）line SRA01/04 under high-calcium mediated oxidative stress. 【Methods】There were 3 groups in this experiment：SMP30 overexpressed group （OE，experimental group），NCOE group （negative control group） and SRA01/04 group （blank control group）. OE and NCOE lentiviral vectors were used to transfect SRA01/04 respectively. A high- calcium- mediated- stress cell model was established by culturing cells with medium containing 15 mmol/L CaCl2 for 24 h. BrdU assay was used to measure cell proliferation. SOD assay kit and GSSG/T- GSH assay kit were used to detect the level of intracellular oxidative stress. 【Results】Green fluorescence protein could be observed in all transfected cell groups under fluorescence microscope and the transfection efficiency was close to 80% ，suggesting that OE cell model was constructed successfully. Under the high calcium culture conditions，the activity of relative cell proliferation and SOD in OE group［（3.89 ± 0.20）and（47.5 ± 4.3 U/mg）］were significantly higher than that in NCOE group［（2.82 ± 0.34）and（30.6 ± 4.2 U/mg）］and SRA01/04 group［（2.96 ± 0.25）and（26.8 ± 1.5 U/mg）］，the ratio of GSSG/T-GSH in OE group（2.36 ± 0.51）was significantly lower than that in NCOE group（16.36 ± 2.48）and SRA01/04 group（20.12 ± 2.54）（n=3，P<0.05）；there was no significant difference between NCOE group and SRA01/04 group （n=3，P>0.05）. 【Conclusions】Overexpression of SMP30 increased the activity of cell proliferation and SOD，but decreased the ratio of GSSG/T- GSH in SRA01/04 cell（HLEC），indicating that SMP30 may alleviate the progression of high-calcium-mediated oxidative cell damage and possess the cytoprotective functions in HLEC.
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@#AIM:To investigate the expression of glucose key metabolic enzymes in human lens epithelial cells(HLEB3)induced by high glucose.<p>METHODS:HLEB3 cells cultured <i>in vitro</i> were divided into normal control group(DMEM medium containing 5mmol/L of glucose), oxidative stress group(DMEM medium containing 5mmol/L of glucose and 200μmol/L of hydrogen peroxide), high glucose induction group(DMEM medium containing 30mmol/L of glucose). Apoptosis and cells were detected 24h after culture. Activity and expression of six key glucose metabolic enzymes(fructose-6-phosphate kinase-1, pyruvate kinase, hexokinase, citrate synthase, α-ketoglutarate dehydrogenase and 6-phosphate glucose dehydrogenase)were studied.<p>RESULTS:Apoptosis of HLEB3 cells was induced by high glucose. The cell viability of high glucose-induced group(63.43%±3.40%)was lower than that of normal control group(100.00%±0.00%)and oxidative stress group(91.90%±5.11%), and the expression levels of six key enzymes of glucose metabolism in high glucose-induced group were lower than that of normal control group and oxidative stress group(all <i>P</i><0.05).<p>CONCLUSION:High glucose can induce the expression of glucose-key metabolizing enzymes in HLEB3 cells to decrease, induce apoptosis and affect cell activity.
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Objective To investigate the effects of decorin (DCN) on apoptosis and oxidative stress in human lens epithelial cells (LECs) under high glucose condition.Methods HLE-B3 cells were cultured in vitro and the effect of DCN with different concentrations on HLE-B3 viability was determined by using cell counting kit-8 (CCK-8).The cultured cells were divided into normal control group,DCN group,high glucose group and DCN + high glucose group.Flow cytometry was used to detect the apoptosis rate and the expression of reactive oxygen species (ROS) in the cells.Microplate spectrophotometer was used to measure total superoxide dismutase (SOD) enzyme activity and the radio of glutathione (GSH)/glutathione disulfide (GSSG).Western blot was used to detect the expressions of bax and bcl-2 proteins.Results HLE-B3 cells were spindle shaped,with centered and clearly visible nuclei and neatly cell arrangment.According to CCK-8 method,survival rates of HLE-B3 in all groups were more than 90％.Different concentrations of DCN showed no significant effect on HLEoB3 survival rate (all at P＞0.05).After 48 hours of cell culture,the apoptosis rate of high glucose group was significantly higher than that of normal control group,and the apoptosis rate of DCN+high glucose group was significantly lower than that of high glucose group (both at P =0.000).The mean fluorescence intensity of intracellular ROS in the high-glucose group was significantly higher than that in the normal control group,and the mean fluorescence intensity of ROS in the DCN group was significantly higher than that in the high glucose group (both at P=0.000).The total SOD activity in the high glucose group was significantly lower than that in the normal control group and DCN group (P =0.007,0.004).The GSH/GSSG ratio of the high-glucose group was significantly lower than that of the normal control group and DCN group (both at P=0.000).Conclusions DCN can inhibit the apoptosis and oxidative stress of HLE-B3 under high glucose,which provides the basis for the treatment of diabetic cataract.
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Objective To investigate whether poly arginine as the carrier can carry foreign proteins to penetrate the cell membrane and even penetrate the eyeball barrier.Methods Poly-Args (R9) was used as a CPP in this study.R9-green fluorescent protein (GFP) and GFP were constructed.In vitro,human lens epithelial cells were treated with these two proteins.Then,MTT assay were used to detect whether the protein could affect the proliferation of the cells.Flow cytometry and laser confocal microscopy were used to detect the penetrability of CPPs on the cells.In vivo,eyes of mice were treated with protein in eye drops way for 7 days.Then total protein were extracted,ELISA were used to detect the penetrability of CPPs.Results The results of MTT,flow cytometry and laser confocal microscopy showed that CPPs could carry protein into cells in a dose dependent manner without affecting cell proliferation.In vivo,slit lamp showed that the mice eyeballs had no any abnormal after treated by GFP,R9-GFP,and ELISA results also showed that R9 could effectively get foreign protein into the eyeball.Conelusion R9 can carry foreign protein into the cell membrane and eyeball barrier.This study provides the basis for the eye medication and dosing mode improvement.
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Objective To investigate the influence of calcium elevation on oxida tive stress in human lens epithelial cells (HLEC) SRA01/04.Method The cells (2 x 103 cells/well) which in the period of logarithmic phase were seeded into 96-well plates with three replicates for the two groups;and in the experimental group,SRA01/04 cells were exposed to a CaCI2 concentration gradient (3 mmol · L-1,5 mmol · L-1,7 mmol · L-1,9 mmol · L-1,11 mmol · L-1,13 mmol · L-1,15 mmol · L-1,17 mmol · L-1,19 mmol · L-1) for 0 h,12 h,24 h,36 h;while the cells in the control group were cultured in complete 1640 medium.Cell counting kit-8 (CCK-8) assay was used to measure cell viability.The levels of intracellular superoxide dismutase (SOD),glutathione (GSH) content and oxidized glutathione (GSSG) / total glutathione (T-GSH) were determined by using the microplate-reader method with the commercial total/oxidized glutathione and sod quantification kit.Results At first,the survival rate of SRA01/04 cells treated with 3 mmol · L-1,5 mmol · L-1,7 mmol· L-1 CaCL2 for 24 h showed a significant decrease with the increase of CaCl2 concentration by CCK-8 assays,but gradually increased when the concentration increased to 9 mmol · L-1,and the difference approached statistical significance (P ＜ 0.05).Meanwhile,there was significant difference in the viability of the control group (0.592 + 0.055) and cells exposed to 15 mmol · L-1 CaCI2 (0.293 + 0.02) (t =7.811,P ＜0.05).Cell treatment with 15 mmol· L-1 CaC12 for 24 h was the most appropriate condition for HLEC apoptosis,followed by the appearance of nuclear fragmentation and dissolution,enhanced intracellular SOD viability (t =-6.417,P ＜ 0.05),decreased T-GSH content (t =13.816,P ＜ 0.05),and increased ratio of GSSG/T-GSH (t =-4.396,P ＜ 0.05) when compared with the control group,and the differences were statistically significant.Conclusion Intracellular calcium elevation can inhibit the cell viability and increase the levels of SOD and GSSG in HLEC to aggravate the intracellular oxidative damage.
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Objective To investigate the influence of calcium elevation on oxida tive stress in human lens epithelial cells (HLEC) SRA01/04.Method The cells (2 x 103 cells/well) which in the period of logarithmic phase were seeded into 96-well plates with three replicates for the two groups;and in the experimental group,SRA01/04 cells were exposed to a CaCI2 concentration gradient (3 mmol · L-1,5 mmol · L-1,7 mmol · L-1,9 mmol · L-1,11 mmol · L-1,13 mmol · L-1,15 mmol · L-1,17 mmol · L-1,19 mmol · L-1) for 0 h,12 h,24 h,36 h;while the cells in the control group were cultured in complete 1640 medium.Cell counting kit-8 (CCK-8) assay was used to measure cell viability.The levels of intracellular superoxide dismutase (SOD),glutathione (GSH) content and oxidized glutathione (GSSG) / total glutathione (T-GSH) were determined by using the microplate-reader method with the commercial total/oxidized glutathione and sod quantification kit.Results At first,the survival rate of SRA01/04 cells treated with 3 mmol · L-1,5 mmol · L-1,7 mmol· L-1 CaCL2 for 24 h showed a significant decrease with the increase of CaCl2 concentration by CCK-8 assays,but gradually increased when the concentration increased to 9 mmol · L-1,and the difference approached statistical significance (P ＜ 0.05).Meanwhile,there was significant difference in the viability of the control group (0.592 + 0.055) and cells exposed to 15 mmol · L-1 CaCI2 (0.293 + 0.02) (t =7.811,P ＜0.05).Cell treatment with 15 mmol· L-1 CaC12 for 24 h was the most appropriate condition for HLEC apoptosis,followed by the appearance of nuclear fragmentation and dissolution,enhanced intracellular SOD viability (t =-6.417,P ＜ 0.05),decreased T-GSH content (t =13.816,P ＜ 0.05),and increased ratio of GSSG/T-GSH (t =-4.396,P ＜ 0.05) when compared with the control group,and the differences were statistically significant.Conclusion Intracellular calcium elevation can inhibit the cell viability and increase the levels of SOD and GSSG in HLEC to aggravate the intracellular oxidative damage.
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@#AIM: To observe the effect of applied direct electric field(EF)on the migration, cell growth, apoptosis and cell cycle of human lens epithelial cells(hLECs). <p>METHODS: HLE-B3 cells were exposed to EF at 100mV/mm, 200mV/mm and 400mV/mm, respectively. Cells without EF-exposure were treated as normal controls. Photos of HLE-B3 cells were captured before and after EF-exposure, and the cell numbers were calculated. Apoptosis and cell cycle were detected with flow cytometry after EF-exposure for 24h. <p>RESULTS: After exposure to EF at 400mV/mm for 3h, HLE-B3 cells showed directed migration to the cathode. The cell number of HLE-B3 cells decreased gradually with continuous EF-exposure, and decreased by 12.6% at 6h and by 18.6% at 12h, respectively(<i>P</i><0.05). After exposure to EF at 100mV/mm, 200mV/mm and 400mV/mm for 24h, the apoptosis rates of HLE-B3 cells increased dramatically compared to that of the normal control, which were(9.2±1.9)%,(23.9±2.6)% and(54±2.5)%, respectively(<i>P</i><0.05). Accordingly, the results of flow cytometry showed that cell numbers in G2/M phase were increased after EF-exposure, which were(13.8±2.2)% and(15.6±2.5)% at 200mV/mm and 400mV/mm, respectively(<i>P</i><0.05). <p>CONCLUSION: Applied direct EF induces directed migration of HLE-B3 cells and inhibits the cell growth with the prolongation of the EF-exposure time. EF also induces cell apoptosis and G2/M phase inhibition of the cell cycle in HLE-B3 cells.
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Objective To investigate the expression of microRNA-34a (miR-34a) and silent information regulator 1 (SIRT1) in human lens epithelial cells under H2O2-induced oxidative stress.Methods Different concentrations of H2O2 (0 μmol · L-1,100 μ mol· L-1,200 μmol · L-1,300 μmol · L-1,and 400 μmol · L-1) were used to stimulate SRA01/04 cells for 24 hours.Cell viability was measured using cell counting kit-8 (CCK-8) assay.Cell apoptosis was detected by flow cytometry.Expression levels of miR-34a/SIRT1 were measured by RT-PCR.Results CCK-8 assay showed that a certain concentration range of H2O2 had a proliferation inhibition on SRA01/04 cells.There was a dose response relationship between 100 μmol · L-1 and 400 μmol · L-1.Compared with 0 μmol · L-1 H2O2 group,the difference was statistically significant (all P ＜ 0.01).According to flow cytometry results,apoptotic rate of SRA01/04 cells in control group and H2O2(100-300 μmol · L-1) groups were (6.1 ± 1.2)％,(26.3 ± 1.8)％,(32.5 ± 2.2) ％,and (64.7 ± 5.3) ％.Compared with 0 μmol · L-1 H2 O2 group,the differences were statistically significant (all P ＜ 0.01).RT-PCR test results showed that the expression of miR-34a increased significantly in a dose-dependent manner after the SRA01/04 cells treated with different concentrations of H2O2,while SIRT1 expression level was decreased,there were significant differences compared with control group (all P ＜ 0.001).Conclusion There is a significantly increase of miR-34a and decrease of SIRT1 in human lens epithelial cells under the oxidative stress of a certain concentration of H2O2.Down-regulated expression of miR-34a can increase the survival rate of human lens epithelial cells under H2O2-induced oxidative stress.
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Objecttve To explore the roles of p53,Bax and Bcl-2 in rat lens epithelial cells apoptosis induced by ultraviolet radiation.Methods Healthy SpragueDawley rats (40,6-weeks old,150 g) were selected and divided into 5 groups randomly.After SD rats were injected 100 g · L-1 chloral hydrate (0.35 mL/100 g) intraperitoneally and pupil dilation,the eyes of SD rats were radiated 15 minutes by using UV-B (300-320 nm).The exposed animals were sacrificed at 1 day,3 days,5 days and 7 days after exposure.Both lenses from all animals were extracted.The apoptosis of rats lens epithelial cells was performed by Hoechst 33258 staining.The expression of p53,Bax and Bcl-2 in rat lens epithelial cells at mRNA level was detected by real-time PCR.The distribution and expression of p53,Bax and Bcl-2 were observed by immunohlstochemistry.Results The apoptosis of lens epithelial cells was aggravated after exposure.The expression of p53 and Bax at mRNA and protein level was increased after UV exposure (P ＜ 0.01).But for Bcl-2 the expression both at mRNA and protein level was decreased after a 1 day-exposure(P ＜0.01) and was increased after UV exposure of 5 days and 7 days(P ＜ 0.01).The mRNA expression level of p53 was positively correlated with the Bax (r =0.952,P ＜ 0.01).The expression of p53 and Bax protein were increased after UV exposure,and transferred from cytoplasm to nucleus;While the expression of Bc1-2 protein was decreased at 1 day after UV exposure,and increased gradually at 3 days,5 days and 7 days.Conclusion The apoptotic process of rat lens epithelial cells induced by ultraviolet may be related with p53 regulating the expression of Bax/Bcl-2.
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PURPOSE: To evaluate the histopathological changes of anterior capsule and lens epithelial cells in various types of cataract. METHODS: Patients scheduled for cataract surgery of phacoemulsification with intraocular lens implantation were enrolled in this study. Anterior capsule tissues sized 5 mm were obtained at the time of continuous curvilinear capsulorhexis during surgery. Histological examination of the obtained tissue was performed by transmission electron microscope. RESULTS: Nuclear cataract showed a uniform cuboidal monolayer of epithelial cells firmly attached to the anterior capsule. But, the mitochondria, Golgi apparatus, and endoplasmic reticulum were damaged and replaced with vacuoles. Anterior subcapsular cataract showed multilayers of epithelial cells with irregular intracellular structures. Epithelial cells of mature cataract were severely damaged and detached from the anterior capsule, accompanied by expansion of intra-cellular space and a large amount of vacuoles. Epithelial cells were irregular and severely damaged, and intracellular structures were hardly observed in traumatic cataract. Deposition of pseudoexfoliation materials on the anterior capsule was observed in pseudoexfoliation cataract. CONCLUSIONS: Changes in epithelial cells caused by fluid accumulation and electrolyte imbalance in the lens attributes more to cataract formation than do changes the in lens capsule.
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Humans , Capsulorhexis , Cataract , Endoplasmic Reticulum , Epithelial Cells , Golgi Apparatus , Lens Implantation, Intraocular , Mitochondria , Phacoemulsification , VacuolesABSTRACT
AlM:To explore the primary culture conditions for four kinds of lens epithelial cells ( LECs) of rat, rabbit, dog, and human, and measure their growth characteristics.METHODS:The lens capsule or anterior capsular tissue of rat, rabbit, dog and patient were removed by different methods, and they were cut into tiny pieces for primary culture by modified tissue adherent method. The morphological features of four kinds of LECs were observed under an inverted microscope.RESULTS: Four kinds of LECs of rat, rabbit, dog and human could be cultured primarily by tissue adherent method. With the evolution of tissue source, the adherent capacity of LECs gradually strengthened, cells form were changed from irregular polygon to oval, nucleus rounded and cytoplasm enriched gradually. Four kinds of LECs had fibrotic changes after several passages.CONCLUSlON: LECs of rat, rabbit, dog and human can be primarily cultured. This method lays the foundation for the mechanism research of caratact and related fields on the cellular and molecular levels.
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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.
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Background The primary pathologic mechanism of posterior capsular opacification (PCO) is proliferation,migration and epithelial-mesenchymal transition (EMT) of residuary lens epithelial cells (LECs) after cataract extract surgery.Researches showed that MG132,a proteasome inhibitor,can attenuate the proliferation of bovine LECs,but its effect on human LECs remains unclear.Objective This study was to investigate the inhibitory effect of MG132 on proliferation,migration and differentiation of human LECs in vitro.Methods Human lens capsule were collected during the surgery.Human LECs were primarily cultured by explant method and passaged.The second or third generation of cells were incubated to 96-well plates at the density of 5×105/ml (200 μl/well) for 24 hours.Fibroblast growth factor-2 (FGF-2,10 mg/L),MG132 (10 μmol/L) or MG132+FGF-2 was added into the culture medium for 24 hours separately,and regular cultured cells served as the control group.The proliferation value (absorbance,A490) of the cells was assayed by MTT colorimetric method.A bare area was made by a sterile cotton swab in the cell layer,and migrated cell number in the blank zone was counted to evaluate the migration ability of the cells after 24 hours.Transforming growth factor-32(TGF-β2),MG132 or MG132+TGF-β2 was added into the culture medium for 24 hours separately,and the expression of fibronectin (FN) in the cells was detected using immunochemistry.Results The proliferation values (A490) of the cells were 0.582±0.020,0.723±0.010,0.434± 0.011 and 0.465±0.008 in the control group,FGF-2 group,MG132 group and MG132 + FGF-2 group,respectively,showing a significant difference among the groups (F =110.482,P＜0.01).The A value was significantly higher in the FGF-2 group and lower in the MG132 group and MG132+FGF-2 group than that of the control group (all at P＜ 0.05).The migrated cell number was 8.67 ± 1.08,11.58 ± 1.59,2.67 ± 0.09 and 2.75 ± 0.09 in the control group,FGF-2 group,MG132 group and MG132+FGF-2 group,respectively,with a significant difference among the groups (F=34.301,P＜0.01),and more cells in the blank zone were seen in the FGF-2 group and less cells were in the MG132 group and MG132+FGF-2 group in comparison with the control group (all at P＜0.05).Compared with the control group,the proliferative rate and migrating rate of the cells declined by 25.4％ and 75.0％ in the MG132 group as well as 20.1％ and 68.3％ in the MG132+FGF-2 group,but in the FGF-2 group,they increased by 24.2％ and 33.6％.The expressing levels (A value) of FN in the LECs were 1.242±0.023,2.329±0.113,1.043 ±0.021 and 1.163±0.018 in the control group,TGF-β2 group,MG132 group and MG132 +TGF-β2 group,respectively,with a significant difference among the groups (F =113.752,P＜0.01),a considerably increased expressing value was seen in the TGF-β2 group and decreased value was in the MG132 group and MG132+TGF-β2 group when compared with the control group (all at P＜0.05).Conclusions MG132 can effectively inhibit the proliferation,migration and differentiation of human LECs in vitro.
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Background Posterior capsular opacification (PCO) following the extracapsular extract of cataract is associated with the proliferation and migration of residual lens epithelial cells (LECs).Study showed that the incidence of PCO is higher in diabetic patients than those of non-diabetes.So if insulin-like growth factor-1 (IGF-1) participates in the pathogenesis of PCO deserve research.Objective This study was to explore the active mechanism of IGF-1/IGF-1 receptor (IGF-1 R) system in the migration of LECs and offer theoretical basis for clinical prevention and treatment of PCO.Methods Human lens epithelial cell lines (HLEC-B3) were cultured and passaged in DMEM.The cells were identified using fluorescence immunocytometry.IGF-1 with the concentrations of 0,30,90 μg/L were added into the medium separately for 48 hours.The numbers of migrated cells were calculated by Transwell test.The cells were cultured in DMEM containing 0,1.5,30,60,90 μg/L IGF-1,and the expressions of IGF-1 Rα and IGF-1Rβ in the cells were assayed and compared by Western bolt.Results The cultured showed the positive response for α-crystallin anibody with red fluorescence in the cellular membrane.Twelve hours after Transwell incubation,the number of migrated cells (Median) was 0(0,1),10(10,11) and 29(27,31) in the 0 μg/L IGF-1 group,30 μg/L IGF-1 group and 90 μg/L IGF-1 group,respectively,showing a significant difference among the 3 groups (Z=12.610,P=0.002).The number of migrated cells in the 30 μg/L IGF-1 group and 90 μg/L IGF-1 group was significantly more than that of the 0 μg/L IGF-1 group (both at P =0.008),and the number of migrated cells in the 90 μg/L IGF-1 group was significantly more than that of the 30 μg/L IGF-1group (P =0.009).Western blot assay showed that the expressions of IGF-1Rα and IGF-1Rβ in the cells were significantly different among the 0,1.5,30,60,90 μg/L IGF-1 groups (F=63.700,130.530,both P =0.000).The expressions of IGF-1 Rα and IGF-1Rβ were gradually elevated as increase of IGF-1 doses when then concentration of IGF-1 was ＞ 30 μg/L,with significant differences among the different concentrations groups (all at P＜0.05).Conclusions IGF-1 can upregulate the expressions of IGF-1R in HLEC-B3 cells in vitro in a dose-dependent manner.Also,IGF-1 enhances the migration ability of HLEC-B3 cells.These results suggest that activation of IGF-1/IGF-1R system may be associated with the pathogenesis of PCO.
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Background Plasma membrane calcium ATPase 3 (PMCA3) participates in the regulation of Ca2+ level in lens epithelial cells (LECs),which may be associated with the pathogenesis of cataract.It has been proved that ultraviolet B (UVB) is one of causing-factors of cataract.However,the effect of UVB on the expression of PMCA3 in LECs is unclear.Objective This study was to investigate the effects of UVB irradiation on the expression of PMCA3 in human LECs B-3.Methods HLE B-3 cells were cultured and passaged.The cells were exposed to 0,5,10 and 20 mJ · s/cm2 UVB for 0,20,40,80 s,respectively and further cultured for 24,48 and 72 hours.MTT assay was used to detect the cell proliferation rate.JC-1 staining was used to detect the changes of mitochondrial membrane potential (△ψm) in the cells.The intracellular reactive oxygen species (ROS) level was detected by DCFH-DA staining,and cell apoptosis was evaluated using annexin V-FITC/PI staining.In addition,the intracellular calcium ion (Ca2+) concentration in the cells was assayed with Fluo-3/AM staining.The expression levels of PMCA3 mRNA and PMCA protein in the HLE B-3 cells were detected by real-time PCR and Western blot,respectively.Results The survival rates of the cells were significantly reduced with the increase of irradiated intensity of UVB and the lapse of time (Fgroup =72.411,P =0.000 ; Ftime =36.588,P =0.000),and the survival rates of the cells in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB for 24 hours were (75.3 ± 2.2) ％ and (48.7 ±4.5) ％,respectively,which were significantly lower than those in the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P=0.001,0.000).The survival rates of the cells in the 5,10,20 mJ · s/cm2 UVB for 48 hours were (84.9± 1.2) ％,(69.3±17.4)％ and (32.8±4.5)％,showing significant declines in comparison with the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P =0.047,0.000,0.000).In 72 hours following 5,10,20 mJ · s/cm2 UVB irradiation,the survival rates of the cells were (55.1 ± 3.0) ％,(42.1 ± 1.9) ％ and (26.1 ±4.7) ％,respectively,with significant differences in comparison with the 0 mJ · s/cm2 UVB group ([100.0 ± 0.0] ％) (P =0.000,0.000,0.000).JC-1staining exhibited the intracellular red fluorescence in the normal cells group.However,in the 5 mJ · s/cm2 UVB group,weak green fluorescence was seen,and the green fluorescence was enhanced in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB groups.After irradiated by 5,10 and 20 mJ · s/cm2 UVB,the ROS levels in the cells increased from 0.4％ to 35.8％,51.9％ and 76.7％,respectively.The apoptosis and necrosis rate of the cells was 2.0％ in the 0 mJ · s/cm2 UVB and 4.2％,7.6％,15.1％ in the 5,10,20 mJ · s/cm2 UVB groups,respectively.The Ca2+ level raised by (1.2±0.1) and (1.3±0.1) folds in the 10 and 20 mJ · s/cm2 UVB groups more than that in the 0 mJ · s/cm2 group (P =0.039,0.004).The expression levels of PMCA3 mRNA in the cells were significantly reduced (P=0.001,0.004,0.000),and the expression levels of the PMCA protein were declined in the 5,10 and 20 mJ · s/cm2 UVB groups compared with the 0 mJ · s/cm2 UVB group (P=0.000,0.000,0.001).Conclusions UVB irradiation causes cataract probably through downregulating the expression of PMCA3 in human LECs and inducing apoptosis of LECs in a dose-and time-dependent manner.
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Verapamil is used in the treatment of hypertension, angina pectoris, and atrial fibrillation. Recently, several studies have demonstrated that verapamil increased the optic nerve head blood flow and improved the retrobulbar circulation. All these show that verapamil is potentially useful for ophthalmic treatment. Thus, the aim of this study is to investigate whether verapamil could protect human lens epithelial cell (HLEC) from oxidative stress induced by H2O2 and the cellular mechanism underlying this protective function. The viability of HLEC was determined by the MTT assay and apoptotic cell death was analyzed by Hoechst 33258 staining. Moreover, Caspase-3 expression was detected by immunocytochemistry and flow cytometry analysis. We also detected Caspase-3 mRNA expression by reverse-transcription-polymerase chain reaction and the GSH content in cell culture. The results showed that oxidative stress produced significant cell apoptotic death and it was reduced by previous treatment with the verapamil. Verapamil was effective in reducing HLEC death mainly through reducing the expression level of apoptosis-related proteins, caspase-3, and increasing glutathione content. Therefore, it was suggested that verapamil was effective in reducing HLEC apoptosis induced by H2O2.
Subject(s)
Humans , Angina Pectoris , Apoptosis , Atrial Fibrillation , Bisbenzimidazole , Caspase 3 , Cell Culture Techniques , Cell Death , Epithelial Cells , Flow Cytometry , Glutathione , Hypertension , Immunohistochemistry , Optic Disk , Oxidative Stress , RNA, Messenger , VerapamilABSTRACT
Background Protein kinase B (Akt) is the center of multiple cellular signaling pathways,and it participates in the regulation of cell function,such as proliferation,migration,and metabolism of cells.Phosphoinositide 3-kinase (PI3K) promotes activation of Akt and therefore triggers many signal pathways.PI3K inhibitor can silent Akt,but whether it can affect the biological behavior of lens epithelial cells (LECs) during the posterior capsular opacity (PCO) is worthy of investigation.Objective This study was to explore the effect of LY294002,a PI3K inhibitor,on Akt activation in human LECs.Methods Human LECs strain,HLEC-B3 cells,were cultured and passaged.The cells were incubated to 96-well plate for 24 hours,then LY294002 was added with the final concentration at 0,10,20,30,40,50,60,70,80 μmol/L,respectively.After incubation for 24 hours,cell counting kit-8 (CCK-8) was used to detect the inhibitory rate of cell proliferation.Transforming growth factor-β2 (TGF-β2) of 10 μg/L was added in the medium of cells as TGF-β2 group,TGF-β2 + LY294002 (20 μmol/L) was used to coculture the other group of cells,and the cells without TGF-β2 and LY294002 served as the control group.Phosphorylation of Akt (p-Akt) in the cells was detected by laser scan confocal immunofluorescence microscope.The expression level of p-Akt was evaluated using Western blot.Results CCK-8 assay showed that the A value of the cells was gradually reduced with the increase of LY294002 concentration (Fgroup =9.72,P =0.00),but the A value was significant raised along with the lapse of time (Ftime =1737.54,P=0.00).Confocal immunofluorescence revealed that a little of p-Akt was expressed in the control cells.After induced by TGF-β2,lots of red fluorescence of p-Akt was seen in cells around the cell membranes.But in the TGF-β2+LY294002 co-culture cells,the fluorescence of Akt was much weaker.Western blot showed that the expression level of p-Akt was 0.91±0.08,1.48±0.13 and 0.95±0.19 in the control group,TGF-β2 group and TGF-β2 +LY294002 group,respectively,with a significant difference among the three groups (F =15.04,P =0.00).Conclusions LY294002 can inhibit the activation of Akt induced by TGF-β2.LY294002 may have utility in the prevention and treatment of PCO.
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Background Epithelial-mesenchymal transition (EMT) is a major event in the pathogenesis of posterior capsular opacification (PCO),and the expressions of α-smooth muscle actin (α-SMA) is the marker of EMT.Previous studies showed that breviscapine plays an important role in anti-fibrosis and suppression EMT,however,the mechanism of its effect on EMT in LECs is unclear.Objective Present study was to investigate the effect of breviscapine on the expression of α-SMA and fibronectin (FN) in human LECs induced by transforming growth factor-β2 (TGF-β2).Methods Human LECs strain,HLE-B3,was cultured and passaged in DMEM containing 10％ fetal bovine serum.Different concentrations of breviscapine (6.75,12.75,25.00,50.00 and 100.00 mg/L)were added into the medium for 24,48 and 72 hours respectively,and then cell cunting kit-8 (CCK-8) was used to evaluate the half maximal inhibitory concentration (IC50) of breviscapine to HLE-B3.In addition,HLE-B3 was subcultured at the density of 1 × 106/hole and divided into 4 groups.The cells were in free-serum medium as the normal control;the cells were exposed in 10 μg/L TGF-β2 as TGF-β2 group;while in the breviscapine group,10 mg/L of breviscapine was added into the culture medium and another group was the combination of 10 mg/L breviscapine and 10 μg/L of TGF-β2 treatment.Real-time PCR and Western blot were used to detect the mRNA expressions of α-SMA and FN as well as their protein in HLE-B3 72 hours after cultured.Results The inhibitory rate of breviscapine to HLE-B3 proliferation was gradually elevated with the increase of concentration of breviscapine,showing a significant inhibition of the cell proliferation among the different groups and at various time points (F =292.851,P=0.000;F =65.037,P=0.000).IC50 of HLE-B3 at 72 hours was 22 mg/L,and therefore,in rest of experiment 10 mg/L of breviscapine was used which was 1/2 only half of the IC50.α-SMA and FN were expressed in cultured normal HLE-B3.The expressing level of α-SMA mRNA and FN mRNA in the HLE-B3 was significantly different among the normal group,TGF-β22 group,breviscapine treatment group and combination group as well as at various time points (F =105.490,P =0.000 ; F =1041.414,P =0.000).Similarly,the protein expressions of α-SM A and FN in the HLE-B3 was significantly different among the four groups and different time points (F=136.872,P=0.000;F=119.820,P=0.000).The expression levels of α-SMA and FN mRNA and their proteins in HLE-B3 were remarkably increased in the 10 μg/L TGF-β2 group compared with the normal control group (at all P=0.000),and those in the combination group were obviously declined in comparison to the TGF-β2 group (P =0.001,0.001,0.001,0.010).No significant difference was found in the expressions of α-SMA and FN in the HLE-B3 between the breviscapine group and normal control group in both transcriptional level and protein level (P =0.551,0.292,0.551,0.360).Conclusions 10 mg/L breviscapine can arrest the proliferation and EMT of human LECs.This result suggests that using breviscapine may be a potential prophylactic approach in the prevention of PCO.
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Background The pathogenesis of age-related cataract is associated with the apoptosis of lens epithelial cells (LECs) caused by oxidative stress.Previous studies showed that intracellular focal adhesion kinase (FAK) pathway can be activated by H2O2 in vitro,which induced apoptosis of cells.To investigate the effect of oxidative on FAK expression in LECs is one of important studies in the prevention of age-related cataract.Objective This study was to investigate the expression and function of FAK in human LECs treated by H2O2.Methods Human LECs strain (HLECs-B3) were cultured in vitro in the low glucose DMEM with 10％ fetal bovine serum.Different concentrations (0,30,50,70,100,300,500,700,1000 μmol/L) of H2O2 were added into the culture medium for 24 hours.The survival rate of the cells was detected by Cell Counting Kit-8 (CCK-8) assay.Cell morphology as well as the expression and distribution of FAK in the cells were observed by immunofluorescent staining under the laser confocal microscope.Apoptosis was observed by hoechst33258 staining,and Western blot assay was used to quantitatively detect the expression and phosphorylation of FAK.Results The survival rate of the cells was (1.00±0.03) ％,(1.24±0.03)％,(1.36±0.24) ％,(0.93±0.02)％,(1.75±0.19)％,(1.37±0.18) ％,(0.64±0.01)％,(0.59±0.11)％,(0.14±0.05)％ in 0,30,50,70,100,300,500,700,1000 μmol/L H2O2 groups,with a significant difference among them (F =95.30,P =0.00).The survival rates of the cells in the below 300 μmol/L H2O2 groups were significantly higher than those in the 0 μmol/L H2O2 group,and survival rates of the cells in the above 500 μmol/L H2O2 groups were significantly lower than those in the 0 μmol/L H2O2 group(all at P＜0.05).After H2 O2 treatment for 24 hours,HLECs-B3 cells transformed from polygon shape to spindle shape and extended pseudopodiums,meanwhile the green fluorescence for FAK exhibited in the cytoplasm.Cell apoptosis was found in the 1000 μ mol/L H2O2 group.Western blot assay revealed that the expressing levels (grey scale) were significantly different among the various groups (F=28.08,P=0.00),and FAK expressing levels in the below 300 μmol/L H2O2 groups were significantly higher than those of the 0 μmol/L H2O2 group; while the expressing levels in the above 500 μmol/L H2O2 groups were lower than those of the control 0 μmol/L H202 group (all at P＜0.05).After treated by different concentrations of H2O2,the phosphorylation level of intracellular FAK (p-FAK) was significantly higher in 3 hours group than that in 30 minutes group (all at P＜0.05).Conclusions H2 O2 can affect the survival,proliferation and morphology of human LECs by activating the intracellular FAK pathway,indicating that FAK may play roles in the regulation process of cell biological behavior.