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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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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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@#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 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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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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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.
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Humanos , Angina Pectoris , Apoptose , Fibrilação Atrial , Bisbenzimidazol , Caspase 3 , Técnicas de Cultura de Células , Morte Celular , Células Epiteliais , Citometria de Fluxo , Glutationa , Hipertensão , Imuno-Histoquímica , Disco Óptico , Estresse Oxidativo , RNA Mensageiro , VerapamilRESUMO
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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Background Epithelial-myofibroblast transition (EMT) of human lens epithelial cells (LECs) induced by transforming growth factor-β2 (TGF-β2) is the main mechanism in the pathogenesis of posterior capsular opacification(PCO).Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO.Objective The purpose of this study was to investigate the inhibitory effect of rapamycin (RAPA)on the proliferation of human LECs and TGF-β2-induced EMT.Methods Human LEC strain(SRA01/04)was cultured in DMEM with high glucose and 10％ fetal bovine serum.The cells were consequently cultured in serumfree DMEM with 5 mg/L TGF-β2,TGF-β2+10 mg/L RAPA,TGF-β2 + 100 mg/L RAPA,TGF-β2 + 1000 mg/L RAPA or TGF-β2 +10 000 mg/L RAPA for 72 hours,and SRA01/04 cultured in serum-free DMEM were used as control.The proliferation rate(A490)of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated.The expressions of the α-smooth muscle actin(α-SMA) and E-cadherin(E-cad)mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-β2 +400 mg/L RAPA treatment.Results The A490 value of SRA01/04 was 0.680±0.020,0.550±0.013,0.480±0.014,0.400±0.011 and 0.200±0.019 in the control group,TGF-β2 group,TGF-β2 + 10 mg/LRAPA group,TGF-β2 + 100 mg/L RAPA group,TGF-β2 + 1000 mg/L RAPA and TGF-β2 + 10 000 mg/L RAPA group,respectively,showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations (F =101.920,P =0.000).RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA (α-SMA mRNA/β-actin mRNA)and protein (α-SMA/β-actin)in the cells were significantly increased in the TGF-β2 treatment group.However,with exposure to RAPA,the relative expression levels of α-SMA mRNA and protein were significantly lowered with increasing RAPA concentrations,but the expression levels of E-cad mRNA and protein were raised (α-SMA mRNA:F =294.660,P =0.000 ; α-SMA protein:F =346.950,P =0.000 ; E-cad mRNA:F =264.250,P =0.000 ; E-cad protein:F =317.327,P =0.000).In addition,after exposure to 400 mg/L RAPA,the expression levels of α-SMA protein gradually reduced and those of E-cad protein gradually increased with increasing treatment durations,showing significant differences among the different time points (α-SMA:F =693.864,P =0.000 ;E-cad:F=369.286,P =0.000).Conclusions RAPA can inhibit the proliferation of SRA01/04 in vitro and arrest EMT of SRA01/04 induced by TGF-β2 in a dose-and time-dependent manner.
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Background The pathogenesis and development of cataract is associated with oxidative stress-induced apoptosis of human lens epithelial cells(LECs).BH3-only protein is a factor that can initiate apoptosis,and thus the apoptotic process is probably related to the activation of the c-Jun N-terminal kinase(JNK).However,the relationship between oxidative stress-induced apoptosis of human LECs and the JNK pathway remains to be illuminated.Objective This study was to investigate the effects of the JNK/c-Jun pathway and its target gene,Bim (Bcl-2 interacting mediator of cell death)and PU M A(p53 up-regulated modulator of apoptosis),on oxidative stressinduced apoptosis of human LECs.Methods The human LECs cell line(HLEC-B3)was cultured and passaged in DMEM with 10％ fetal bovine serum in vitro.Confluent cells were incubated in 24 well plates and divided into 4 groups.Hydrogen peroxide(H2O2)(50 μmol/L)was used to treat the cells for 4,8 or 12 hours,and cells without H2O2 treatment served as the control group.Apoptosis was detected using Hoechst 33258 staining and quantified by counting the number of cells with pyknotic nuclei.In addition,confluent cells were seeded in 6 well plates,and Western blot and RT-PCR were used to detect the expression of the caspase-3,c-Jun,Bim and PUMA proteins and their mRNA in HLEC-B3,respectively.The JNK/c-Jun pathway inhibitors,CEP11004 or SP600125,were added into cultured media with H2O2,and cells treated with DMSO or H2O2 only served as negative and positive control groups.The expression of the p-JNK,JNK,p-c-Jun,c-Jun,Bim,PUMA proteins was detected by Western blot and apoptosis was assayed using Hoechst 33258 staining.200 pmoL/L of Bim or PUMA small interference RNA(siBim or siPUMA)fragments were transfected into the cells for 24 hours,respectively,and H2O2 was then used to treat the cells for 8 hours.The expression of the Bim and PUMA protein and their mRNA in the cells was detected by Western blot and RT-PCR,respectively.Results After H2O2 treatment in HLEC-B3 cells for 4,8,or 12 hours,the rates of apoptosis were 4.30％±1.15％,27.08％±0.74％ and 46.59％±0.91％,showing a significant difference among them (F=1909.433,P=0.000),and those of the 4,8,12 hour groups were significantly increased in comparison to the control group(P =0.049,0.000,0.000).Compared to untreated cells,the levels of expression of the JNK,Bim,PUMA proteins and their mRNA in HLEC-B3 cells were significantly elevated.After the addition of CEP11004 or SP600125,the expression of these protein and mRNA in HLEC-B3 cells in the presence of H2O2 was significantly weaker than that in the DMSO control group(P =0.000,0.000).After the tranfection of siBim or siPUMA,the apoptosis rates of the H2O2 treated groups were significantly higher than those in the Bim-/-or PIMA-/-group (P＜0.05).Conclusions H2O2 can activate the JNK/c-Jun pathway and up-regulate the expression of its target genes Bim and PUMA in human LECs in a time-dependent manner.Inhibiting the JNK/c-Jun pathway and interfering with the expression of Bim and PUMA can protect human LECs against oxidative stress-induced apoptosis.
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Background Epithelial-mesenchymaltransition (EMT)isamajorcontributortothe pathogenesis of posterior capsular opacification(PCO).Kruppel-like factor 6 (KLF6) is a zinc finger protein,which can be stimulated by high glucose in proximal tubule cells and involved in transforming growth factor beta (TGF-β)induced EMT of diabetic nephropathy.ObjectiveThis study was designed to investigate the effect of high glucose on the expression of KLF6 and its target genes( TGFB1,TGFBR1,COLIA1,HSP47) in human lens epithelial cells (LECs).MethodsHuman LECs(SRA01/04) were cultured and exposed to different concentration of glucose.The expressions of KLF6 mRNA and protein were analyzed by real time polymerase chain reaction( real time PCR) and western blot after treatment with high glucose.The expressions of KLF6 target genes were analyzed by real time PCR to evaluate the EMT of SRA01/04 cells.ResultsCompared with the control group(5.5 mmol/L),the relative mRNA levels of t-KLF6 and wt-KLF6 in SRA01/04 treated with high glucose(22.2,44.4,66.6 mmol/L) increased obviously (F =72.53,42.02,P＜0.01 ).Then,the concentration of 22.2 mmol/L was used in the next experiments.The relative mRNA levels of t-KLF6 and wt-KLF6 increased to the peaks after treatment with high glucose for 12 h,and began to decrease after 24 h until lower levels after 48 h ( F =100.12,125.52,P ＜ 0.01 ).Western blot showed that the expression of KLF6 protein was also upregulated by high glucose treatment.With the promotion of the expression of KLF6 gene,the relative mRNA levels of TGFB1,TGFBR1,COLlAl and HSP47 of treated cells also respectively increased after treatment for 12 h,and began to decrease after 24 h until nearly at the levels of the control groups after 48 h( F=6.73,162.35,64.39,12.05,P＜0.05 ).ConclusionsIt was concluded that high glucose induced the expression of KLF6 in human LECs,and KLF6 transiently stimulated the expression of target genes TGFB1,TGFBRl,COLlAl and HSP47 which were mainly involved in the mechanism of EMT.
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The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells (LECs) and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations (20,40,60 and 80 ng/mL) of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance (A) values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR) and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time- and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.
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Summary: The effects of NO-Fluvastatin on proliferation of human lens epithelial cells (HLECs) and the action mechanism were investigated. Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. The expression of cell cycle regulatory proteins CyclinE mRNA and P21wafl mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). MTT staining colorimetry showed that HLECs proliferation was markedly inhibited by NO-Fluvastatin and the effect was dependently related to time (24, 48 and 72 h) and dosage (1, 5 and 20 μmol/L). Flow cytometry revealed that NO-Fluvastatin could significantly block HLECs in the G0/G1 phase, resulting in the increased cells in the G0G1 phase and decreased in the S phase (P<0.05). RT-PCR showed that NO-Fluvastatin could obviously inhibit the CyclinE mRNA expression and induce the P21wafl mRNA expression as compared with the negative control groups (P<0.05). This experiment suggested that NO-Fluvastatin could suppress the proliferation of HLECs by regulating cell cycle regulatory proteins (inhibiting the expression of CyclinE mRNA and inducing the expression of P21wafl mRNA), resulting in the arrest of HLECs in the G0/G1 phase, which can offer theory basis for NO-Fluvastatin in treating posterior capsular opacification in clinic practice.