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AIM: To investigate the changes of protein expressions in human lens epithelial cells(SRA01/04)undergoing oxidative damage, hoping to provide new protein target for the pathogenesis of age-related cataract(ARC).METHODS: SRA01/04 cells were divided into experimental group and control group. In the experimental group, cells were irradiated with ultraviolet-B(UVB)for 10min to establish the model of oxidative damage, whereas cells in the control group were untreated. Protein expression profile from the two groups was sequenced by isobaric tags for relative and absolute quantitation(iTRAQ). The filtering criteria that fold change >1.2 and p<0.05 was used to determine the differentially expressed proteins(DEPs). Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database were utilized for functional enrichment analysis of the top 50 DEPs with either up-regulated or down-regulated significance. Furthermore, Pathway commons software was used to establish the protein-protein interaction(PPI)network.RESULTS: Overall, 552 DEPs were screened out. A total of 176 DEPs were up-regulated in the experimental group compared with the control group, including HMGB1 and USP1, while 376 DEPs were down-regulated, including POLR2A and POLR2B. GO and KEGG enrichment analysis indicated that the top 50 DEPs with up-regulated or down-regulated significance were involved in various crucial biological processes and signaling pathways. PPI network revealed that oxidative damage repair(ODR)-related proteins might play a key role in UVB-induced oxidative damage.CONCLUSIONS: The expressions of multiple proteins, especially ODR-related proteins, can be altered in SRA01/04 cells via UVB irradiation. These findings may provide cellular-related insights into the pathogenesis of ARC and into proteins or pathways associated with therapeutic targets.
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Gigantol is a phenolic component of precious Chinese medicine Dendrobii Caulis, which has many pharmacological activities such as prevent tumor and diabetic cataract. This paper aimed to investigate the molecular mechanism of gigantol in transmembrane transport in human lens epithelial cells(HLECs). Immortalized HLECs were cultured in vitro and inoculated in the laser scanning confocal microscopy(LSCM) medium at 5 000 cells/mL. The fluorescence distribution and intensity of gigantol marked by fluorescence in HLECs were observed by LSCM, and the absorption and distribution of gigantol were expressed as fluorescence intensity. The transmembrane transport process of gigantol in HLECs were monitored. The effects of time, temperature, concentration, transport inhibitors, and different cell lines on the transmembrane absorption and transport of gigantol were compared. HLECs were inoculated on climbing plates of 6-well culture plates, and the ultrastructure of HLECs was detected by atomic force microscopy(AFM) during the transmembrane absorption of non-fluorescent labeled gigantol. The results showed that the transmembrane absorption of gigantol was in time and concentration-dependent manners, which was also able to specifically target HLECs. Energy and carrier transport inhibitors reduced gigantol absorption by HLECs. During transmembrane process of gigantol, the membrane surface of HLECs became rougher and presented different degrees of pits, indicating that the transmembrane transport of gigantol was achieved by active absorption of energy and carrier-mediated endocytosis.
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Humans , Lens, Crystalline/pathology , Cataract/prevention & control , Bibenzyls/pharmacology , Epithelial Cells , Cells, Cultured , ApoptosisABSTRACT
AIM: To investigate the mechanism of pyrrolidine dithiocarbamate(PDTC)on transforming growth factor-beta 2(TGF-β2)-induced epithelial-mesenchymal transition(EMT)in human lens epithelial cells(LECs).METHODS: LECs were treated with various doses of PDTC chemicals following TGF-β2 caused EMT on these cells. Cell proliferation and lateral migration were discovered using the CCK-8 and cell scratch test. The markers of EMT, including E-cadherin, α-SMA and nuclear factor-κB(NF-κB)signaling pathway-related expression, were tested by Western Blot as well as the changes in the expression of the apoptosis-related proteins BAX, BCL-2, Caspase-3, and Cyclin D1.RESULTS: The proliferation and migration viability of cells in the TGF-β2 treated group was increased compared to the group without TGF-β2, and the expression of α-SMA increased whereas the E-cadherin expression decreased. With the effect of TGF-β2, NF-κB p65 and phosphorylated NF-κB p65 expression increased, the concentration of TGF-β2 that had the greatest capacity for proliferation and migration was 10 ng/mL(P<0.05). Mechanism study of PDTC-induced EMT reversal and apoptosis showed that cell viability and migratory capability were both significantly reduced after PDTC intervention; PDTC prevents IκB phosphorylation, thus inhibiting NF-κB nuclear translocation. Protein associated to the NF-κB signaling pathway, and protein expression of NF-κB/IκBα/p-IκBα/Iκκ-α/p-Iκκ-α was decreased(P<0.05), PDTC increased the expression of the pro-apoptotic protein BAX/Caspase-3, expression of the inhibitor of apoptosis protein BCL-2 and the cell cycle protein Cyclin D1 was reduced. The expression of NF-κB/IκB mRNA was reduced, expression of the apoptosis-related mRNA BAX increased, while BCL-2 reduced.CONCLUSION: The EMT in LECs cells induced by TGF-β2 can be significantly reversed by PDTC, which may be related to the decreased expression of NF-κB p65/IκB/Iκκ-α and activation of apoptosis-related protein. PDTC can reverse EMT by inhibiting NF-κB signaling pathway and induce apoptosis of abnormally proliferated cells, which will provide new potential therapeutic agents for posterior capsular opacification(PCO)treatment.
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Objective:To explore the inhibitory effect of long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) by targeting microRNA-199a-5p (miR-199a-5p) on the apoptosis of human lens epithelial cells (LECs).Methods:The anterior lens capsule tissue of 23 age-related cataract patients who underwent cataract surgery in Xinxiang First People's Hospital from December 2018 to August 2019 was collected.At the same time, anterior lens capsules from 20 healthy donor were collected.The expressions of KCNQ1OT1 and miR-199a-5p in the tissues were detected by real-time fluorescence PCR.Human LECs SRA01/04 cultured in vitro were divided into blank control group, model control group, small interfering RNA-negative control (siR-NC) group, siR-KCNQ1OT1 group, miR-NC group, miR-199a-5p group, siR-KCNQ1OT1+ anti-miR-NC group and siR-KCNQ1OT1+ anti-miR-199a-5p group.No intervention was administered to blank control group.Cells in model control group were cultured with 100 μmol/L H 2O 2 for 24 hours to establish oxidative stress injured model, and cells in the other six groups were transfected with corresponding transfection reagents for 6 hours by liposome method according to grouping, and then treated with 100 μmol/L H 2O 2 for 24 hours.The expressions of KCNQ1OT1 and miR-199a-5p in lens anterior capsule tissue and LECs cells were determined by real-time fluorescent quantitative PCR.Cell viability was detected with thiazolyl blue (MTT). Cell apoptosis was analyzed by flow cytometry.The expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2 related X protein (Bax) proteins were assayed by Western blot.The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between KCNQ1OT1 and miR-199a-5p was verified by dual luciferase reporter experiment.The study protocol was approved by an Ethics Committee of Xinxiang First People's Hospital (No.2019-001). Written informed consent was obtained from relatives of patient. Results:The relative expression of KCNQ1OT1 in the anterior capsule of patients with age-related cataract was 2.41±0.42, which was significantly higher than 0.97±0.19 of normal people, and the relative expression of miR-199a-5p in the capsule of patients with age-related cataract was 0.36±0.12, which was lower than 1.04±0.15 of normal people, and the differences were statistically significant ( t=14.112, 16.507; both at P<0.001). Compared with blank control group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content were significantly increased, and the relative expressions of miR-199a-5p and bcl-2 protein, cell viability and SOD activity were significantly reduced in model control group, showing statistically significant differences (all at P<0.001). Compared with siR-NC group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content in cells of siR-KCNQ1OT1 group were decreased, while the relative expression of bcl-2 protein, cell survival rate and SOD activity were increased, and the differences were statistically significant (all at P<0.05). Compared with miR-NC group, the KCNQ1OT1-wild type (WT) luciferase activity in miR-199a-5p group was significantly decreased, with a statistically significant difference ( t=21.131, P<0.001). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly increased, and the relative expression of bax protein, cell apoptosis rate and MDA content were significantly decreased in miR-199a-5p group than those in miR-NC group, and the differences were statistically significant (all at P<0.05). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly lower, and the cell apoptosis rate, relative expression of bax protein and MDA content were significantly higher in siR-KCNQ1OT1+ anti-miR-199a-5p group than those in siR-KCNQ1OT1+ anti-miR-NC group, and the differences were statistically significant (all at P<0.05). Conclusions:The inhibition of KCNQ1OT1 can promote the cell viability of human LECs, inhibit H 2O 2-induced cell apoptosis and oxidative stress, and the mechanism may be related to the up-regulation of miR-199a-5p.
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@#AIM: To explore the relationship between the protective effect of 17β-estradiol(E<sub>2</sub>)on human lens epithelial cells and pyroptosis. <p>METHODS: Human lens epithelial cells were cultured <i>in vitro</i> and divided into blank control group, H<sub>2</sub>O<sub>2</sub> treatment group, and 17β-estradiol+H<sub>2</sub>O<sub>2</sub> treatment group. Scanning electron microscope to observe the cytological morphology; immunofluorescence technique to detect Gasdermin D(GSDMD)distribution and fluorescence intensity; CCK-8 to detect cell viability; TUNEL to detect cell pyroptosis; Western-blot to detect Cysteinylaspartate specific proteinase-1(Caspase-1), GSDMD, NOD-like receptor protein 3(NLRP3)protein expression level; ELISA to detect interleukin-1β(IL-1β)expression. <p>RESULTS: Compared with the control group, the cell viability of the H<sub>2</sub>O<sub>2</sub> treatment group was significantly decreased, the expression of Caspase-1, GSDMD, and NLRP3 protein were significantly up-regulated, and the secretion of IL-1β was significantly increased. Compared with the H<sub>2</sub>O<sub>2</sub> treatment group, the expression of Caspase-1, GSDMD, and NLRP3 protein in the 17β-estradiol+H<sub>2</sub>O<sub>2</sub> treatment group were down-regulated, and the secretion of IL-1β decreased, and it showed a decreasing trend with the increase of estrogen concentration. <p>CONCLUSION: 17β-estradiol has a protective effect on human lens epithelial cells, and its protective mechanism is related to the inhibition of the pyroptosis process of human lens epithelial cells, and the classical pyroptosis pathway is involved.
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@#AIM:To observe the expression pattern of Cyclin D1 in human lens epithelial cells(HLECs)after traumatic stimulation in high-glucose culture<p>METHODS: The activity of HLECs was detected by MTT method after incubate with differernt concentration glucose for 24h <i>in vitro </i>to determine the optimal glucose concentration. qRT-PCR and Western blot were used to detect the high glucose pretreatment group and the non-high glucose pretreatment group. The expression of Cyclin D1 in HLECs at different time points after traumatic stimulation was detected.<p>RESULTS: The viability of HLECs were increased when treatment with low concentration glucose, but the concentration should not exceed 25.5mmol/L, or it will inhibit the activity of HLECs; The reasult of high glucose pretreatment group reveal that the expression of Cyclin D1 is down-regulated in a time-dependent manner within a certain time range. While the expression of Cyclin D1 was irregular in the non-pretreatment group, it was increased at the time point of 12h and 48h. The score treatment can up-regulate the expression of Cyclin D1 in HLECs in a certain degreen.<p>CONCLUSION: The effects of glucose on HLECs activity and Cyclin D1 experssion are irrugular. Trauma treatment can stimulate the expression of Cyclin D1 in HLECs to some extent.
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@#AIM: To observe the effect and mechanism of MicroRNA-34a on senescence and apoptosis of human lens epithelial cell line SRA01/04.<p>METHODS: MicroRNA-34a expression levels in ARC lens and transparent lens epithelial cells were detected by qRT-PCR. MicroRNA-34a mimics, MicroRNA-34a inhibitors and empty liposome(control group)were transfected into SRA01/04 cells by liposome transfection kit. Annexin V-FITC/PI double staining was used to detect the effect of MicroRNA-34a on the apoptosis of human lens cell line SRA01/04. The expression of Cdc42 and Rac1 protein was detected by western blot. <p>RESULTS: The expression level of MicroRNA-34a in anterior capsular tissue of transparent lens was significantly lower than that in ARC anterior capsular tissue(<i>P</i><0.05). The positive rates of SA-β-gal in the MicroRNA-34a mimics group, the control group and the MicroRNA-34a inhibitors group were(87.56±2.34)%,(12.22±2.74)% and(3.45±0.45)%, respectively. The positive rates of SA-β-gal in the MicroRNA-34a mimics group was significantly higher than the control group, while the SA-β-gal positive rate in the MicroRNA-34a inhibitors group was significantly lower than that in the control group(<i>P</i><0.05). The apoptosis rate of the MicroRNA-34a inhibitors group, control group and MicroRNA-34a mimics group were(5.87±1.22)%,(12.26±2.14)% and(29.45±3.12)%, respectively. The apoptosis rate of the MicroRNA-34a mimics group was significantly higher than that of the control group, while that of the MicroRNA-34a inhibitors group was significantly lower than that of the control group(<i>P</i><0.05). The expressions of Cdc42 and Rac1 in the MicroRNA-34a mimics group were significantly higher than those in the control group(<i>P</i><0.05), while the expressions of Cdc42 and Rac1 in the MicroRNA-34a inhibitors group were significantly lower than those in the control group(<i>P</i><0.05).<p>CONCLUSION: MicroRNA-34a may promote the senescence and apoptosis of human lens epithelial cells by up-regulating Cdc42 and Rac1.
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@#AIM: To discuss the protective effects of betaine-homocysteine methyl transferase(BHMT)on oxidative damaged human lens epithelial cells(HLEC)induced by homocysteine.<p>METHODS: HLEC were cultured <i>in vitro</i> and then randomly divided into 3 groups. Normal group:normal cultured HLEC; control group: normal cultured HLEC transfected with empty vector; BHMT gene overexpression group(OE): HLEC transfected with BHMT gene overexpression. All groups were cultured in 10% FCS DMEM +5mmol/L Hcy for 16h. After cultured, BHMT mRNA expression was measured by qRT-PCR and Western blot, the cell proliferation was detected by EdU Assay Kit,The level of ROS and GSH of HLEC were measured by Flow Cytometer and Visible Spectrophotometers. The expression level of of protein(GRP78, Nrf2, Caspase-12)was measured by western blotting. <p>RESULTS: After cultured 16h, cell proliferation ability in OE group was increased by 30.0% compared with NC group(<i>P</i><0.05).The expression of ROS in normal group(89.2043±0.3511)% was obviously higher than OE group(49.5625±0.4502)%, <i>P</i><0.05, GSH activity in OE group was obviously higher than control group and normal group,(<i>P</i><0.05). The expression level of GRP78 in the normal group and the control group was significantly higher than overexpression group. The expression level of Nrf2 in the normal group and the control group was significantly lower than overexpression group. The expression level of Caspase-12 in the overexpression group was significantly lower than that in the control group.<p>CONCLUSION: BHMT <i>in vitro</i> can prevent the oxidative damage of HLEC by high homocysteine, clear the ROS and decrease the ER stress reaction. Apoptosis of lens epithelial cells was inhibited. BHMT plays an important protective role in oxidative damaged HLEC induced by Hcy.
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@#AIM:To study the effect of zinc on galactose-induced cell apoptosis in human lens epithelial cells(HLEC). <p>METHODS:HLEC cell line SRA01/04 cells were cultured in DMEM medium and divided into six groups: control group, galactose treatment group, zinc supplement group, zinc supplementation combined with galactose treatment group, zinc deficiency group, zinc deficiency combined with galactose treatment group. The cell viabilities were assayed by MTT, cell morphology and apoptosis were detected by fluorescence microscope and flow cytometry, respectively. <p>RESULTS:The cell viabilities induced by galactose(0, 25, 50, 75, 100, 125mmol/L)were(100.0±5.4)%,(97.5±3.2)%,(91.3±5.3)%,(93.4±0.6)%,(86.6±1.4)% and(83.5±0.4)%, respectively. When the concentration of galactose was 100 and 125mmol/L, cell viability was significantly decreased, compared with the untreated cells(<i>P</i><0.05). Fluorescence microscopy results showed that the cell nucleus remained uniformly stained in control group and zinc supplement group. Nuclear shrinkage, a typical apoptotic morphology, was visible in some cells in the galactose treatment group, zinc supplementation combined with galactose treatment group, zinc deficiency group and zinc deficiency combined with galactose treatment group. The cell apoptosis in the six groups were(1.5±0.1)%,(7.1±0.2)%,(1.4±0.1)%,(4.4±0.2)%,(5.5±0.2)% and(15.8±0.3)%, respectively. The cell apoptosis were significant increased in galactose treatment group, zinc supplementation combined with galactose treatment group, zinc deficiency group and zinc deficiency combined with galactose treatment group, compared with the control group(<i>P</i><0.05), and those of zinc supplementation combined with galactose treatment group were significant decreased, and zinc deficiency combined with galactose treatment group were increased(<i>P</i><0.05), compared with the galactose treatment group. <p>CONCLUSION:Zinc supplementation protects human lens epithelial cells against apoptosis induced by galactose and may have an inhibition effect on cataract formation.
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Objective To discuss the protective effects of thioltransferase (TTase) on oxidative damaged human lens epithelial cells (HLEC) induced by ultraviolet radiation.Methods HLEC were cultured in vitro and then randomly divided into 4 groups:Normal group:normal cultured HLEC;UV group:normal cultured HLEC + UV radiation (with 302 nm UV radiation irradiation intensity 55.56 μW · cm-2 for 15 minutes,totaling irradiation volume 500 J · m-2);TTase siRNA group:HLEC transfected with TTase siRNA;TTase siRNA + UV group:HLEC transfected with TTase siRNA + UV radiation(with 302 nm UV radiation irradiation intensity 55.56 μW · cm-2 for 15 minutes,totaling irradiation volume 500 J · m-2).TTase mRNA expression was measured by qRT-PCR,the cell proliferation was detected by LDH Assay Kit,and the TTase activity was measured.TTase expression was detected by Western blotting.The levels of TGSH,GSH and GSSG of HLEC were measured,and then GSSG/T-GSH ratio was calculated.Results Cell proliferation ability in UV group,TTase siRNA group and TTase siRNA + UV group were decreased by 21.0％,17.0％ and 29.0％ compared with normal group (all P ＜ 0.05).TTase activity in UV group was 2.1 times of the normal group,TTlase siRNA group was 67.0％ of the normal group,Tlase siRNA + UV group was 1.3 times of TTase siRNA group (all P ＜ 0.05).TTase expression in UV group was 3.9 times of the normal group,TTase siRNA group was 35.0％ of the normal group,TTase siRNA + UV group was 3.0 times of siRNA group (all P ＜ 0.05).GSH content in UV group,TTase siRNA group and TTase siRNA + UV group were 68.4％,79.0％,61.7％ of the normal group (all P ＜ 0.05).GSSG content in UV group,TTase siRNA group and TTase siRNA + UV group were 2.3 times,1.4 times,3.7 times of the normal group (all P ＜ 0.05).GSSG/T-GSH in UV group,TTase siRNA group and TTase siRNA + UV group were 3.1 times,1.7 times,5.2 times of the normal group (all P ＜ 0.05).Conclusion TTase plays an important protective role in oxidative damaged HLEC induced by ultraviolet radiation.
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Objective To investigate the protective effects of 17β-estradiol on human lens epithelial (HLE) cells in oxidative damage induced by H2 O2 and its involved mechanisms.Methods HLE cells cultured in vitro were collected and divided into 4 groups;cells in negative control group were cultured with normal medium,cells with 80％ fusion in n2O2 damage group was treated with 100 μmol · L-1 H2O2 for 12 h,cells in 17β-estradiol low dose group were incubated with 10 μmol · L-1 17β-estradiol for 24 h then subjected to 100 μmol · L-1 H2O2 for 12 h and ceEs in 17β-estradiol high dose group were cultured in 100 μmol · L-1 17β-estradiol for 24 h then treated with 100 μrnol · L-1 H2O2 for 12h.Next,cell viability was tested by CCK-8 colorimetric assay,while apoptotic rate was detected by flow cytometry,and intracellular reactive oxygen species (ROS) level was detected by H2 DCFDA fluorescence probe labeling method,as well as the contents of catalase (CAT),superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by 721 D spectrophotometer.Results When compared with the negative control group,the survival rate of HLE ceils in the H2 O2 damage group was significantly decreased,and there was significantly different between both groups (P ＜0.05).Moreover,the survival rate of the 17β-estradiol low dose group (67.44％) and high dose group (78.52％) was obviously higher than that of the H2O2 damage group (59.34％),and the difference was statistically significant (P ＜ 0.05).After H2O2-induced injury,there was significant difference in the apoptotic rate of HLE cells between the H2O2 damage group (41.30 ±3.21)％ and the negative control group (1.67+0.32)％.In addition,the apoptotic rates of the 17β-estradiol low dose group and high dose group was (20.97 + 1.13)％ and (14.27 + 0.90)％ respectively,which was statistically different from the H2 O2 damage group (all P ＜ 0.05).H2 DCFDA fluorescent labeling test results showed that ROS fluorescence signal intensity gradually weakened after treated with 17β-estradiol.Besides,17β-estradiol significantly increased the expression of CAT、SOD and GSH-Px in HLE cells.Conclusion 17β-estradiol has obvious protective effects on HLE cells from the damage induced by H2O2.
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Objective To investigate the protective effects of 17β-estradiol on human lens epithelial (HLE) cells in oxidative damage induced by H2 O2 and its involved mechanisms.Methods HLE cells cultured in vitro were collected and divided into 4 groups;cells in negative control group were cultured with normal medium,cells with 80％ fusion in n2O2 damage group was treated with 100 μmol · L-1 H2O2 for 12 h,cells in 17β-estradiol low dose group were incubated with 10 μmol · L-1 17β-estradiol for 24 h then subjected to 100 μmol · L-1 H2O2 for 12 h and ceEs in 17β-estradiol high dose group were cultured in 100 μmol · L-1 17β-estradiol for 24 h then treated with 100 μrnol · L-1 H2O2 for 12h.Next,cell viability was tested by CCK-8 colorimetric assay,while apoptotic rate was detected by flow cytometry,and intracellular reactive oxygen species (ROS) level was detected by H2 DCFDA fluorescence probe labeling method,as well as the contents of catalase (CAT),superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by 721 D spectrophotometer.Results When compared with the negative control group,the survival rate of HLE ceils in the H2 O2 damage group was significantly decreased,and there was significantly different between both groups (P ＜0.05).Moreover,the survival rate of the 17β-estradiol low dose group (67.44％) and high dose group (78.52％) was obviously higher than that of the H2O2 damage group (59.34％),and the difference was statistically significant (P ＜ 0.05).After H2O2-induced injury,there was significant difference in the apoptotic rate of HLE cells between the H2O2 damage group (41.30 ±3.21)％ and the negative control group (1.67+0.32)％.In addition,the apoptotic rates of the 17β-estradiol low dose group and high dose group was (20.97 + 1.13)％ and (14.27 + 0.90)％ respectively,which was statistically different from the H2 O2 damage group (all P ＜ 0.05).H2 DCFDA fluorescent labeling test results showed that ROS fluorescence signal intensity gradually weakened after treated with 17β-estradiol.Besides,17β-estradiol significantly increased the expression of CAT、SOD and GSH-Px in HLE cells.Conclusion 17β-estradiol has obvious protective effects on HLE cells from the damage induced by H2O2.
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AIM: To discuss the protective effects and possible mechanisms of 17β-estradiol on human retinal pigment epithelial ( RPE) cells induced by high glucose. ·METHODS: RPE cells were cultured and divided into four groups according to randomized controlled method:blank control group:the cells were treated with 5. 5mmol/L routine glucose medium for processing; high glucose group: cells were treated with 100mmol/L glucose for 12h;17β-estradiol low concentration group: after treated with 10 μmol/L 17β-estradiol, cells were treated with 100mmol/L glucose for 12h; 17β-estradiol high concentration group: after treated with 100 μmol/L 17β-estradiol, cells were treated with 100mmol/L glucose for 12h. Cell viability were tested by MTT colorimetric detection. Cells apoptosis were detected by Hochest33258 staining. Intracellular reactive oxygen species( ROS) level were detected by H2 DCFDA staining. Expression of CAT, SOD and MDA were tested by colorimetric detection. · RESULTS: RPE cell activity decreased with the concentration of glucose increased; 17β-estradiol inhibited high glucose-induced cell viability decrease in RPE cells, decreased the apoptosis rate of RPE cells and intracellular ROS generation; besides, 17β-estradiol significantly increased the expression of CAT, SOD and decreased the expression of MDA in RPE cells. ·CONCLUSION: The 17β-estradiol effectively inhibited high glucose -induced RPE cells damage, which provide reliable experimental basis for the treatment of injuries in RPE cells.
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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.
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Objective To investigate the effect of ZnO nanoparticles on the expressions of plasma membrane calcium ATPasel (PMCA1) of human lens epithelial cell B-3 (HLEB-3) at both mRNA and protein levels in the presence and absence of ultraviolet B (UVB) irradiation.Methods HLEB-3 was cultured in RPMI 1640 medium,and the cytotoxic effect of different concentrations of ZnO (0 μg · mL-1,2.5 μg · mL-1,5.0μg · mL-1,10.0 μg · mL-1) on HLEB-3 was investigated in the presence and absence of UVB irradiation.DAPI staining was used to monitor the effect of ZnO on HLECB-3 nuclei,and cell apoptosis was evaluated using annexin V-FITC/PI staining in the presence and absence of UVB irradiation.In addition,the intracellular calcium ion (Ca2 +)levels were assayed using Fluo-3/AM staining,and the expression levels of both PMCA1 mRNA and protein within HLEB-3 were detected by real-time PCR and Western blot,respectively.Results DAPI staining showed that the ZnO-treated HLEB-3 displayed a concentration-dependent apoptosis,and UVB irradiation could further aggravate the cytotoxic effect of ZnO on HLEB-3.In addition,in the presence of UVB irradiation,concentration gradient of ZnO (2.5 μg · mL-1,5.0 μg · mL-1,10.0 μg · mL-1) increased the intracellular calcium ion levels [from (156.34 ±4.59) nmol · L-1 to (173.88 ±7.17)umol · L-1,(289.02 ± 9.09) nmol · L-1,(488.36 ± 48.16) nmol · L 1,respectively] and upregulated HLEB-3 apoptosis,with statistical difference (all P ＜ 0.05).Moreover,the expression level of PMCA1 in the 2.5 μg · mL-1,5.0 μg · mL-1,10.0 μg · mL-1 ZnO-treated epithelial cells was accordingly 0.75,0.57 and 0.41 as much as that in the 0μg · mL-1 ZnO-treated cells in the absence of UVB irradiation (all P ＜ 0.05),and was accordingly 0.64,0.24 and 0.09 in the present of UVB irradiation,with significant difference (all P ＜ 0.05).Conclusion Both ZnO nanoparticle and UVB irradiation can exert cosuppression effect on HLEB-3 via calcium-mediated signaling pathway,indicating it has great potential for the treatment of posterior capsular opacification with UVB irradiation.
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Objective To explore the effects of PP242 on the expression of apoptosis protein Bcl?2 in human lens epithelial cells(HLECs). Meth?ods Immortal HLECs SRA01/04 were cultured and treated with different concentrations of PP242. The cell growth was examined by MTT assay at 24 h and 48 h after PP242 treatment. Real?time quantitative polymerase chain reaction(RT?PCR)and Western blot were adopted to detect the mRNA and protein expression of Bcl?2 respectively. Results The cultured HLECs SRA01/04 were collected and treated with different concentra?tions(100,250,500,750,1 000,1 500 nmol/L)of PP242. After treatment for 24 h and 48 h,the inhibitory rate in each well was determined by MTT assay. The inhibition rate was as below,24 h:7.55%,9.43%,16.98%,22.64%,26.42%,30.19%;48 h:11.11%,23.81%,36.51%,42.86%, 49.21%,63.49%. Compared with the control group,the difference was statistically significant(P<0.05). Compared with the control group(0 nmol/L),the expression of Bcl?2 protein was significantly decreased with the increasing concentrations of PP242(P<0.05),while the expression of Bcl?2 mRNA was inhibited by PP242 based on the results of RT?PCR. Compared with the control group(0 nmol/L),the RT?PCR results of Bcl?2 mRNA were 0.723±0.039,0.517±0.028,0.353±0.052,0.167±0.046,respectively(P<0.05). Conclusion PP242 could inhibit cell proliferation of HLECs in vitro with a concentration and time depended manner. Bcl?2 protein is expressed in HLECs,which could be down regulated by PP242 treatment.
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?AlM: To investigate theprotective effect of melatonin against hydrogen peroxide ( H2 O2 )-induced oxidative damage to human lens epithelial cells. ?METHODS: Sub-culture human lens epithelial cells preprocessed with different concentrations of melatonin for 12h and then 100 μmol/L H2 O2 for 24h. The impact of melatonin on H2 O2-induced lens epithelial cell viability was detected by MTT assay, rate of apoptosis was detected by flow cytometry instrument and activity of apoptosis-related factors, Caspase-3 and Caspase-9, were detected by colorimetric method. ?RESULTS: MTT assay showed that melatonin had no effect on the activity of lens epithelial cells, and the drug can inhibit the decrease of H2 O2-induced cell activity, as well as flow cytometry showed that melatonin can inhibit H2 O2-induced apoptosis. ln addition, melatonin can also reduce H2 O2-induced Caspase-3 and Caspase-9 activity in lens epithelial cells, and their activity decreased with effect of melatonin along with extending time. ?CONCLUSlON: Melatonin can obviously inhibit H2 O2 -induced apoptosis of human lens epithelial cells, which provide reliable experimental basis for drug on treatment of cataract.
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Objective To explore whether PI3K/ AKT signaling pathway participates in the inhibiting effect of Ru-tin on H2 O2-induced apoptosis in human lens epithelial cells( HLEC). Methods HLEC were divided into four groups: control group,H2 O2 group,rutin group,LY294002 group. Cell survival rates were determined by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay; cell apoptosis rates were monitored by flow cytometry with Annexin V-FITC and propidiun iodide(PI) staining. Western blot was used to measure the expres-sion levels of AKT and p-AKT. Results H2 O2 induced HLEC apoptosis. Compared with H2 O2 group,rutin group not only increased the expression lever of p-AKT,but also reduced cell apoptosis rate(P < 0. 01). In LY294002 group, LY294002, an inhibitor of PI3K/ AKT signaling pathway,could significantly block the change of these inde-xes produced by rutin group(P < 0. 01), but no significant change compared with H2 O2 group. Conclusion Rutin inhibits H2 O2-induced cell apoptosis and may be associated with PI3K/ AKT signaling pathway.
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Objective To observe the expression of small ubiquitin?related modifiers(SUMO)protein in normal cultured human lens epithelial cells(SRA01/04)and discuss regulation effects of SUMO protein on oxidative stress induced by high glucose. Methods The expression and local?ization of SUMO 1,2/3,4 was detected in normal cultured SRA01/04 cells through immunocytochemistry. The mRNA expression levels of SUMO 1?4 were examined by RT?PCR after the SRA01/04 cells treated with high glucose media at different concentrations and time points. Samples were grouped by medium concentrations(glucoses 5.5 mmol/L,12.5 mmol/L,25 mmol/L,50 mmol/L respectively for 24 h)and by treatment time(0 h, 6 h,12 h and 24 h respectively). After highly efficient transfection of GFP?SUMO2 into SRA01/04 cells,the survival and apoptotic rates of transfect?ed and un?transfected cells treated with high glucose was detected by CCK8 method and AV/PI double staining flow cytometry. Results The immu?nocytochemistry results showed that SUMO1,2/3,4 proteins were mainly located in the nucleus of SRA01/04 cells and part of SUMO2/3 was in the cytoplasm. RT?PCR results showed that compared with the low?glucose group,the mRNA expression of SUMO1?4 was increased along the increas?ing glucose concentration in the high?glucose group(P<0.05). Compared with 0 h,the mRNA expression of SUMO1?4 was enhanced at 6 h,12 h and 24 h(P<0.05)in the high?glucose group treated at 50 mmol/L concentration. Compared with the un?transfected cells,the survival rate was in?creased and the apoptotic rate was decreased in GFP?SUMO2 transfected cells in oxidative stress induced by high glucose(P<0.05). Conclusion SUMO protein was positively expressed in SRA01/04 cells and the expression of SUMO mRNA was affected by oxidative stress induced by high glu?cose.
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Background Recombinant hirudin variant Ⅲ(rHV3) can effectively prevent galactose-induced human lens epithelial cells LECs injury,but little is known about the molecular mechanism of its action.Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose.Methods The rHV3 was extracted by our research group,and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt's method.Human LECs (SRA01/04) were cultured using 125×10-3 mol/L D-galactose+10% FBS+D/F12 medium to establish the damaged human LECs model.rHV3 was added into the medium of the damaged human LECs model.Human LECs were cultured in D/F12 medium containing 10% FBS as normal control.The expression of apoptosis-related genes,such as aldose reductase (AR),bax,bcl2 and p53,in LECs at the mRNA level was detected using RT-PCR.The abundance ratio of target genes was presented with the absorbance (A) of gene mRNA/GAPDH mRNA.Results Compared to the normal control group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group (t=3.90E-06,t=8.44E-04,t=5.15E-08,P＜0.01).However,in the rHV3-treated group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group (t=5.90E-06,t=1.51E-04,t=3.42E-06,P＜0.01).The bcl2 mRNA/GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group (t=1.86E-05,P＜0.01);while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group (t=8.56E-05,P＜0.01).Conclusion 125×10-3mol/L D-galactose induces the damage and apoptosis of human LECs.rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway.