HLE-B3 Cells Research Articles

MDM2 phosphorylation mediates H2O2-induced lens epithelial cells apoptosis and age-related cataract

Lens epithelial cells (LECs) apoptosis induced by oxidative stress is a major factor in age-related-cataract (ARC) pathogenesis, but there are still many blind nodes in this progress. This study aimed to investigate the effects of MDM2 phosphorylation in ARC and H2O2-induced lens epithelial cells apoptosis. Our results confirmed that the levels of p-MDM2 (Ser166) and p-MDM2 (Ser186) in the anterior lens capsules of human cataracts were reduced compared to that in normal capsules. Similarly, in naturally aging cataract mice, the level of MDM2 phosphorylation also decreased. Oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with 200 μM H2O2. It was confirmed that MDM2 could regulate lens epithelial cell apoptosis, and MDM2 inhibitors could partly inhibited AKT’s role in suppressing apoptosis induced by H2O2. Besides, we examed the decreased level of p-AKT(Ser473) in apoptosis of lens epithelial cells and ARC. Our study revealed that MDM2 phosphorylation mediated H2O2-induced lens epithelial cells apoptosis and ARC, which could provide new ideas for the clinical treatment of ARC.

MicroRNA-184 is a key molecule responsible for the transforming growth factor-β2 -induced epithelial-mesenchymal transition in human lens epithelial-B3 cells.

TGF-β2-induced epithelial-mesenchymal transition (EMT) is an important mechanism for posterior capsule opacity (PCO) in lens epithelial cells (LECs). This study aimed to investigate if MicroRNA-184 (miR-184) plays a role in the TGF-β2-induced EMT in LECs. Human LECs (HLE-B3 cells) were used in this study. Quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) was performed to analyse miR-184 expressions in HLE-B3 treated with TGF-β2 at different concentrations (0-15 ng/mL) and different time (10 ng/mL, 0-48 hours). After transfection of miR-184 mimics or miR-184 inhibitor, cells were treated with 10 ng/mL TGF-β2 for 24 hours, and the expression levels of miR-184, E-cadherin, vimentin, zinc finger E-box binding homeobox 2 (ZEB2), α-Smooth muscle actin (α-SMA), Collagen 1 and bin3 were determined by qRT-PCR and Western blot, respectively. TGF-β2 treatment significantly downregulated E-cadherin and upregulated vimentin generally in a dose-dependent and time-dependent manner. TGF-β2 treatment significantly elevated the level of miR-184 in both dose- and time-dependent manners. In addition, transfection of miR-184 inhibitor RNA significantly attenuated TGF-β2-induced downregulation of E-cadherin as well as upregulation of vimentin, ZEB2, α-SMA and Collagen 1, whereas transfection of miR-184 mimic further enhanced the effects of TGF-β2 on the expressions of these markers. Furthermore, TGF-β2 treatment significantly downregulated bin3, and transfection of miR-184 mimic and miR-184 inhibitor significantly enhanced and attenuated the inhibition effect of TGF-β2 on bin3, respectively. miR-184 plays a key role in the TGF-β2-induced EMT in LECs, and bin3 may be a downstream protein.

Decorin inhibits glucose-induced lens epithelial cell apoptosis via suppressing p22phox-p38 MAPK signaling pathway.

PurposeTo determine the effect of decorin on oxidative stress and apoptosis of human lens epithelial (HLE) cells under high glucose condition.MethodsHLE cell line (HLEB3) was incubated in normal glucose (5.5 mM) or high glucose (60 mM) medium. Decorin (50 nM) was applied 2 hours before high glucose medium was added. Apoptosis detection was executed by flow cytometry and western blotting (analysis of bcl-2 and bax). Oxidative stress level was measured by the generation of reactive oxygen species (ROS), glutathione peroxidase (GSH) and superoxide dismutase (SOD). P38 mitogen-activated protein kinase (MAPK) phosphorylation, the expression of p22phox of HLE cells and human lens anterior capsules were detected by western blotting. Small interfering RNA transfection to p22phox and p38 MAPK was also carried out on HLEB3.ResultsHigh glucose caused HLE cells oxidative stress and apoptosis exhibiting the increase of apoptotic cells and ROS production and decrease of bcl-2/bax ratio, GSH/GSSG ration and SOD activity. P22phox and phospho-p38 MAPK were upregulated in high glucose treated HLEB3 cells. Knocking down p22phox or p38 by siRNAs can reduce high glucose induced cell apoptosis and oxidative stress level. Silencing p22phox by siRNA can downregulate the phosphorylation of p38 MAPK. Decorin can inhibit the apoptosis, oxidative stress level and the induction of p22phox and phospho-p38 of HLEB3 induced by high glucose. Furthermore, the expression of p22phox and p38 were found significantly increased in lens anterior capsules of diabetic cataract patients compared to that of normal age-related cataract patients.ConclusionsResults showed that p22phox-p38 pathway may be participated in high glucose induced lens epithelial cell injury, decorin may inhibit the high glucose induced apoptosis and oxidative stress injury by suppressing this pathway in part.

Ginsenosides induce extensive changes in gene expression and inhibit oxidative stress-induced apoptosis in human lens epithelial cells

BackgroundThe effect of ginsenosides on the growth and apoptosis of human lens epithelial (HLE) B3 cells exposed to H2O2 was investigated. In addition, the effect of ginsenosides on gene expression in HLE-B3 cells was analyzed using microarray assays to determine its molecular mechanism.MethodsHLE-B3 cells were treated with 1.75 M H2O2 in the presence or absence of 5, 10 or 20 μM ginsenosides. Cell viability and apoptosis were examined by MTT assays and flow cytometry, respectively, at 24 to 120 h after the treatment. Furthermore, HLE-B3 cells were treated with 20 μM ginsenosides for 8 days and total RNA was isolated and analyzed using the Affymetrix GeneChip Array. Principal component analysis was performed to visualize the microarray data.ResultsAddition of ginsenosides significantly alleviated the growth inhibitory effect of H2O2 on HLE-B3 cells and the percentage of viable cells was increased by more than 3 folds. Flow cytometric analysis showed that 6.16 ± 0.29% of H2O2-treated HLE-B3 cells were early apoptotic cells, and the percentage was reduced to 4.78 ± 0.16% (P < 0.05) in the presence of 20 μM ginsenosides. Principal component analysis revealed that ginsenoside caused extensive changes in gene expression in HLE-B3 cells. A total of 6219 genes showed significant differential expression in HLE-B3 cells treated with ginsenoside; among them, 2552 (41.0%) genes were significantly upregulated, whereas 3667 (59.0%) genes were significantly downregulated. FOXN2, APP and RAD23B were the top three upregulated genes while WSB1, PSME4 and DCAF7 were the top three downregulated genes in HLE-B3 cells treated with ginsenosides.ConclusionGinsenosides induce extensive changes in the expression of genes involved in multiple signaling pathways, including apoptotic signaling pathway and DNA damage response signaling pathway. Ginsenosides alleviate H2O2-induced suppression of the growth of HLB cells and inhibit H2O2-induced apoptosis of HLB cells.

Corrigendum: Contribution of Connexin Hemichannels to the Decreases in Cell Viability Induced by Linoleic Acid in the Human Lens Epithelial Cells (HLE-B3).

[This corrects the article DOI: 10.3389/fphys.2019.01574.].

Contribution of Connexin Hemichannels to the Decreases in Cell Viability Induced by Linoleic Acid in the Human Lens Epithelial Cells (HLE-B3).

Connexin (Cx) proteins form gap junction channels (GJC) and hemichannels that a allow bidirectional flow of ions and metabolites between the cytoplasm and extracellular space, respectively. Under physiological conditions, hemichannels have a very low probability of opening, but in certain pathologies, hemichannels activity can increase and induce and/or accelerate cell death. Several mechanisms control hemichannels activity, including phosphorylation and oxidation (i.e., S-nitrosylation). Recently, the effect of polyunsaturated fatty acids (PUFAs) such as linoleic acid (LA), were found to modulate Cxs. It has been seen that LA increase cell death in bovine and human lens cells. The lens is a structure allocated in the eye that highly depends on Cx for the metabolic coupling between its cells, a condition necessary for its transparency. Therefore, we hypothesized that LA induces lens cells death by modulating hemichannel activity. In this work, we characterized the effect of LA on hemichannel activity and survival of HLE-B3 cells (a human lens epithelial cell line). We found that HLE-B3 cells expresses Cx43, Cx46, and Cx50 and can form functional hemichannels in their plasma membrane. The extracellular exposure to 10–50 μM of LA increases hemichannels activity (dye uptake) in a concentration-dependent manner, which was reduced by Cx-channel blockers, such as the Cx-mimetic peptide Gap27 and TATGap19, La3+, carbenoxolone (CBX) and the Akt kinase inhibitor. Additionally, LA increases intracellular calcium, which is attenuated in the presence of TATGap19, a specific Cx43-hemichannel inhibitor. Finally, the long exposure of HLE-B3 cells to LA 20 and 50 μM, reduced cell viability, which was prevented by CBX. Moreover, LA increased the proportion of apoptotic HLE-B3 cells, effect that was prevented by the Cx-mimetic peptide TAT-Gap19 but not by Akt inhibitor. Altogether, these findings strongly suggest a contribution of hemichannels opening in the cell death induced by LA in HLE-B3 cells. These cells can be an excellent tool to develop pharmacological studies in vitro.

Upregulation of miR-630 Induced by Oxidative Damage Resists Cell Migration Through Targeting ALCAM in Human Lens Epithelium Cells

ABSTRACTPurpose: To investigate the role of miRNAs in regulating oxidative damage during cataract formation.Methods: Microarray analysis and gene expression profiling assay were used to separately evaluate the miRNAs and mRNAs profiles in normal human lens epithelium cell line HLE-B3 treated by H2O2. The expression level of miR-630 was detected by RT-qPCR and the gene expression profiles were performed with gene ontology analysis using Bio Informatical database. The targets of miR-630 were predicted using miRecords and the results were used for screening targets of miR-630 combined with the GO analysis above. The mRNA levels of ALCAM, PCDH7, COL12A2, and EDIL3 in HLE-B3 cells after oxidative stimulation or miR-630 mimics transfection were measured by RT-qPCR, and the expression of ALCAM regulated by miR-630 was confirmed by Western blot and dual-luciferase reporter gene assay. The level of cell migration was measured by transwell assay and scratching test after transfection of miR-630 mimics and ALCAM siRNAs.Results: The microarray analysis demonstrated that miR-630 was significantly increased in HLE-B3 cells after oxidative stimulation. ALCAM, PCDH7, COL12A2, and EDIL3 were screened to be the possible targets of miR-630 by miRecords combined with GO analysis, but the results of RT-qPCR, Western blot and dual-luciferase reporter gene assay showed that only the expression of ALCAM was repressed by miR-630 transfection. Cell migration was inhibited through transfection of miR-630 mimics or ALCAM siRNAs and the upregulation of miR-630 partly reduced the cell migration increased by oxidative stimulation.Conclusion: miR-630 is one of the miRNAs increased by oxidative stimulation in human lens epithelium cells. Its upregulation may inhibit cell migration by targeting on ALCAM, which is important for HLECs to resist behavioral changes induced by oxidative damage and may delay the progression of cataract.

MicroRNA-15a modulates lens epithelial cells apoptosis and proliferation through targeting B-cell lymphoma-2 and E2F transcription factor 3 in age-related cataracts.

Age-related cataract remains a serious problem in the aged over the world. MicroRNAs are abnormally expressed in various diseases including age-related cataract. MicroRNA-15a (MicroRNA-15a) has been involved in various diseases and plays crucial roles in many cellular processes. However, the mechanism of microRNA-15a in the genesis of cataract remains barely known. We therefore aimed to investigate the role of microRNA-15a in the cataract. Herein, human lens epithelial B3 cells, HLE-B3 cells were treated with 200 μmol/l H2O2 for 24 h. H2O2 was utilized in our study to induce HLE-B3 cells injury. We observed that cell apoptosis was induced by the treatment of H2O2 and meanwhile, cell proliferation was repressed by 200 μmol/l H2O2. Then, it was found that microRNA-15a was significantly increased with the H2O2 exposure in vitro. Importantly, B-cell lymphoma-2 (BCL2) and E2F transcription factor 3 (E2F3) exert crucial roles in cell apoptosis and cell proliferation. We found that BCL2 and E2F3 were greatly reduced by 200 μmol/l H2O2 in human lens epithelial cells. In addition, microRNA-15a overexpression induced cell apoptosis and repressed cell proliferation through suppressing BCL2 and E2F3. Subsequently, BCL2 and E2F3 were predicted as a direct target of microRNA-15a. The direct correlation between microRNA-15a and BCL2/E2F3 was confirmed by dual luciferase reporter assay. In conclusion, we demonstrated that microRNA-15a triggered apoptosis and repressed the proliferation of HLE-B3 cells by modulating BCL2 and E2F3.

Inhibition of NLRP3 Protects Human Lens Epithelial Cells against Oxidative Stress-Induced Apoptosis by NF-κB Signaling

Backgrounds/Aims: To explore whether NLRP3 is involved in the development of cataract and to study the effect of NLRP3 on hydrogen peroxide (H₂O₂)-induced injury in human lens epithelial cells. Methods: Oxidative stress-induced apoptosis model was constructed by treating HLEB3 cells with 50 µM H₂O₂ at different times (6 h, 12 h) and was confirmed by flow cytometry and Western blot. HLEB3 were divided into NC, NC+H₂O₂, shNLRP3, and shNLRP3+H₂O₂ groups. Quantitative real-time polymerase chain reaction and Western blot were employed to detect mRNA and protein expressions, DCFH-DA to measure reactive oxygen species production, and Annexin V-FITC/PI staining to determine cell apoptosis. Results: NLRP3 expression significantly increased in H₂O₂-induced HLEB3 cells. shRNA interference of NLRP3 inflammasome protects HLEB3 cells against oxidative stress-induced apoptosis by decreasing the expression levels of caspase-3 and Bax and increasing Bcl-2 expression. shNLRP3 was able to effectively suppress H₂O₂-induced apoptosis via inhibition of NF-κB signaling. Conclusion: NLRP3 might be involved in the apoptosis of lens epithelial cells. The inhibition of NLRP3 obviously attenuated H₂O₂-induced oxidative stress injury of human lens epithelial cells via NF-κB signaling.

The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

BackgroundThe signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors.MethodsHuman lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-SrcY530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and Vimentin were examined at 48 h by RT-PCR and western blot. At 48 h and 72 h of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assays.ResultsActivity of c-Src kinase, which causes the expression of p-Src418, was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-SrcY530F, the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src418 increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src418 expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced.ConclusionsThe c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

Protective Effects of Trimetazidine in Retarding Selenite-Induced Lens Opacification

ABSTRACTPurpose: Cataracts are the leading cause of vision loss worldwide, and the over-production of reactive oxygen species (ROS) is the foremost underlying cause of cataracts. Reducing ROS levels can efficiently prevent lens opacification, as evidenced by many studies. Here, we inhibited ROS overproduction with trimetazidine (TMZ), which is an antioxidant, to explore the therapeutic effects of TMZ and the mechanism of lens opacification.Materials and methods: Sodium selenite-induced cataract formation resulted in a significant loss of lens transparency. This effect could be efficiently rescued by TMZ, which was further found to be an inhibitor of ROS production, as determined by assaying oxidative stress-related parameters (SOD activity, MDA, ·OH and H2O2 levels) during cataract formation. The experimental protocols involving animal research were approved by the Animal Care and Ethics Committee of Wenzhou Medical University and conducted according to the Association for Research in Vision and Ophthalmology under the guidelines of the Animal Welfare Act (SYXK 2015–0009).Results: Our study found that TMZ can retard the onset and progression of lens opacification in vivo in experiments using Sprague-Dawley (SD) suckling rats and can rescue the morphology of HLEB3 cells in vitro. The flow cytometry and DNA fragmentation assays showed that TMZ could prevent sodium selenite-induced apoptosis. The western blot analysing showed that the levels of apoptosis-associated Bcl-2 and Nrf2 were dramatically decreased following the sodium selenite treatment. In addition, the bisulfate DNA sequencing revealed that the demethylation of CpGs in the promoter region of Keap1 was stimulated, and that this demethylation could be inhibited by TMZ by rescuing the Nrf2 expression level.Conclusions: Our findings indicate that the antioxidant TMZ strongly reduces ROS production, which ultimately delays the progression of cataract formation, suggesting that treatment with TMZ represents a novel, promising antioxidant protection to retard cataract formation.

Cx46 hemichannel modulation by nitric oxide: Role of the fourth transmembrane helix cysteine and its possible involvement in cataract formation

Under normal conditions, connexin (Cx) hemichannels have a low open probability, which can increase under pathological conditions. Since hemichannels are permeable to relatively large molecules, their exacerbated activity has been linked to cell damage. Cx46 is highly expressed in the lens and its mutations have been associated to cataract formation, but it is unknown whether Cx46 has a role in non-genetic cataract formation (i.e. aging and diabetes). Nitric oxide (NO) is a key element in non-genetic cataract formation and Cx46 hemichannels have been shown to be sensitive to NO. The molecular mechanisms of the effects of NO on Cx46 are unknown, but are likely to result from Cx46 S-nitrosation (also known as S-nitrosylation). In this work, we found that lens opacity was correlated with Cx46 S-nitrosation in an animal model of cataract. Consistent with this result, a NO donor increased Cx46 S-nitrosation and hemichannel opening in HLE-B3 cells (cell line derived from human lens epithelial cells). Mutagenesis studies point to the cysteine located in the fourth transmembrane helix (TM4; human C212, rat C218) as the NO sensor. Electrophysiological studies performed in Xenopus oocytes revealed that rat Cx46 hemichannels are sensitive to different NO donors, and that the presence of C218 is necessary to observe the NO donors’ effects. Unexpectedly, gap junctions formed by Cx46 were insensitive to NO or the reducing agent dithiothreitol. We propose that increased hemichannel opening and/or changes in their electrophysiological properties of human Cx46 due to S-nitrosation of the cysteine in TM4 could be an important factor in cataract formation.

Topical administration of a ROCK inhibitor prevents anterior subcapsular cataract induced by UV-B irradiation

The deposition of extracellular matrix (ECM)—which is mainly composed of type I collagen—in anterior subcapsular cataracts (ASCs) during epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs) decreases visual function. Transforming growth factor (TGF)-β is a key factor in the induction of EMT in LECs. Although Rho kinase (ROCK) plays an important role in EMT induced by TGF-β, it is unknown whether ROCK inhibition affects type I collagen expression in TGF-β-stimulated LECs and ASC formation. This was investigated in the present study both in vitro using human lens epithelium (HLE)-B3 cells and in vivo using mice with ultraviolet radiation (UVR)-B-induced cataracts. We found that TGF-β2 increased type I collagen mRNA expression in HLE-B3 cells; this was inhibited in a dose-dependent manner by treatment with the ROCK inhibitor Y-27632. UVR-B exposure caused ASC formation in mice. A histopathological examination revealed that LECs in the anterior subcapsular area were flattened and multi-layered, and had a spindle shape in cross section. Immunohistochemical analysis revealed the presence of α-smooth muscle actin and type I collagen around these flattened LECs; these opacities were reduced by topical instillation of Y-27632. These findings suggest that suppression of TGF-β signaling in LECs by topical application of a ROCK inhibitor can prevent the formation of ASCs.

MiR‑15a‑3p affects the proliferation, migration and apoptosis of lens epithelial cells.

The present study investigated the effect of microRNA (miR)-15a-3p on the proliferation, migration and apoptosis of lens epithelial cells and its potential mechanism, in order to further elucidate the pathogenesis of age-related cataracts (ARCs). The HLE-B3 human lens epithelial cell line was transfected with miR-15a-3p mimic. Expression of the miR-15a-3p mimic was measured by fluorescence-based reverse transcription-quantitative polymerase chain reaction analysis. Cell proliferation, apoptosis, invasion and migration were investigated using MTT and plate clone formation assays, terminal deoxynucleotidyl transferase dUTP nick end labeling and flow cytometry, and a wound healing assay and Transwell assay, respectively. The protein expression levels of B-cell lymphoma 2 (BCL2) and myeloid cell leukemia sequence 1 (MCL1) were also compared between transfected and wild-type HLE-B3 cells by western blot analysis. The results showed that transfection with the miR-15a-3p mimic significantly suppressed the proliferation of HLE-B3 cells, induced cell apoptosis and increased the proportion of early apoptotic cells. The migration of HLE-B3 cells was significantly inhibited following transfection with miR-15a-3p mimic (P<0.01), whereas cell invasion was unaffected (P>0.05). In addition, reduced protein levels of BCL2 and MCL1 were observed in the miR-15a-3p mimic-transfected HLE-B3 cells (P<0.01). In conclusion, miR-15a-3p may suppress cell proliferation and migration, and induce cell apoptosis in lens epithelial cells through inhibiting the expression of BCL2 and MCL1, which contributes to the onset of ARCs.

Epithelial-Mesenchymal Transdifferentiation in Pediatric Lens Epithelial Cells.

Posterior capsule opacification (PCO) is a complication after cataract surgery, particularly in children. Epithelial-mesenchymal transition (EMT) of lens epithelial cells, mediated by transforming growth factor beta (TGFβ), contributes to PCO. However, its pathogenesis in children is poorly understood. We correlated cell growth in culture with patient characteristics, studied gene expression of pediatric lens epithelial cells (pLEC), and examined the effects of TGFβ-2 on these cells in vitro. Clinical characteristics of children with cataracts correlated with growth behavior of pLEC in vitro. mRNA expression of epithelial (αB-crystallin, connexin-43) and mesenchymal (αV-integrin, α-smooth muscle actin, collagen-Iα2, fibronectin-1) markers was quantified in pLEC and in cell line HLE-B3 in the presence and absence of TGFβ-2. Fifty-four anterior lens capsules from 40 children aged 1 to 180 months were obtained. Cell outgrowth occurred in 44% of the capsules from patients ≤ 12 months and in 33% of capsules from children aged 13 to 60 months, but in only 6% of capsules from children over 60 months. TGFβ-2 significantly upregulated expression of αB-crystallin (HLE-B3), αV-integrin (HLE-B3), collagen-Iα2, and fibronectin-1 (in pLEC and HLE-B3 cells). Patient characteristics correlated with growth behavior of pLEC in vitro, paralleling a higher clinical incidence of PCO in younger children. Gene expression profiles of pLEC and HLE-B3 suggest that upregulation of αV-integrin, collagen-Iα2, and fibronectin-1 are involved in EMT.

The effect of L-carnitine on the apoptosis of human lens epithelial cells through endoplasmic reticulum stress

Objective: To investigate the effect of L-carnitine on the apoptosis of human lens epithelial cells through endoplasmic reticulum (ER) stress pathway. Methods: HLE-B3 cell lines were used to set up an oxidative stress model with H(2)O(2) treatment for 12 h, and lead to ER stress. Cells were divided into four groups: H(2)O(2) group, L-carnitine (100 μmol/L) with H(2)O(2) group, phosphate buffered saline (PBS) group and L-carnitine (100 μmol/L) group. Cell counting kit-8 was used to detect the cell viability under the treatment of different concentrations (200, 400, 600 and 800 μmol/L) of H(2)O(2). The apoptosis ratio of HLE-B3 treated by 400 μmol/L H(2)O(2) was tested by flow cytometry. When HLE-B3 was treated by 400 μmol/L H(2)O(2), the expression levels of cysteinyl aspartate specific proteinase 3 (caspase-3) gene and glucose-regulated protein 78 (GRP78) gene were measured by RT-PCR, and the expression levels of caspase-3 protein and GRP78 protein were assayed by Western blotting. Data from groups was analyzed by the one-way analysis of variance, and the LSD-t test was used for the comparison of groups. Results: Cell counting kit-8 assay showed that when H(2)O(2) concentration was 200, 400, 600 and 800 μmol/L, there was significant difference in the H(2)O(2) group(77.6%±0.8%,58.1%±3.1%,39.2%±1.5%,28.1%±2.2%), L-carnitine with H(2)O(2) group(83.3%±4.2%,74.5%±3.1%,46.4%±1.7%,32.4%±1.2%), PBS group(97.6%±2.1%,98.3%±0.2%,96.3%±2.2%,98.5%±1.1%) and L-carnitine group(98.5%±1.3%, 96.1%±2.1%, 98.1%±0.2%, 97.3%±1.4%) (all P<0.05). There was no significant difference between groups (PBS group compared to L-carnitine group, all P>0.05). When the concentration of H(2)O(2) was 400 μmol/L, the survival rate of the L-carnitine with H(2)O(2) group was higher than the H(2)O(2) group. The difference was statistically significant (t=18.14, P=0.020). With increasing of the H(2)O(2) concentration, cell necrosis was increased. The cell survival rate had no significant difference between the L-carnitine with H(2)O(2) group and H(2)O(2) group (both P>0.05). Flow cytometry results of the H(2)O(2) group, L-carnitine with H(2)O(2) group, PBS group and L-carnitine group were 31.4%±4.5%, 16.5%±2.8%, 2.1%±0.2% and 1.9%±1.8%, respectively (F=126.784, P=0.024) . The rate of apoptosis in the L-carnitine with H(2)O(2) group was lower than that in the H(2)O(2) group (t=24.67, P=0.013). There was no significant difference between the PBS group and L-carnitine group (P>0.05). The results of RT-PCR showed that the expression of caspase-3 mRNA in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.424±0.041 vs. 0.752±0.203), and the expression of GRP78 mRNA in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.521±0.223 vs. 0.821±0.103). The difference was statistically significant (caspase-3: t=27.92, P=0.018;GRP78: t=16.31, P=0.019). Western blotting showed that the protein expression of caspase-3 in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.712±0.212 vs. 1.126±0.251), and the GRP78 protein expression in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.512±0.012 vs. 0.735±0.051). The difference was statistically significant (caspase-3: t=15.43, P=0.010;GRP78: t=20.62, P=0.018). Conclusion: L-carnitine can reduce the apoptosis rate of HLE-B3 during oxidative stress through ER stress pathway. (Chin J Ophthalmol, 2018, 54: 363-368).

The influence of EphA2 overexpression on proliferation and apoptosis of human lens epithelial cells exposed to high-concentration dexamethasone in vitro

Objective: To construct lentiviral-mediated EphA2 overexpression vectors, transfect them into human lens epithelial cells (HLE-B3) in vitro, and investigate the effect of EphA2 gene overexpression on the proliferation and apoptosis of HLE-B3 exposed to high-concentration dexamethasone. Methods: Experimental Study. The pCDH-CMV- MCS-EF1-RFP plasmid was set up by the digestion of NOTⅠand XbaⅠ double restriction enzyme and ligation of CE ligase, and then the plasmid was transformed into DH10B cells. Seven clons were picked for enzymatic digestion and the clons with correct results were chosen for sequencing. The 293 T/17 cells were co-transfected with the pCDH-CMV-MCS-EF1-RFP-EphA2 and the packaging mixture by Lipofectamine 2000. At different multiplicities of infection (MOI=20, 50, 100, and 200) after 72-hour infection, we observed the expression of RFP and morphological changes of HLE-B3 by an inverted fluorescence microscope, and calculated the transfection efficiency through the flow cytometry. EphA2 protein expression was detected by Western blot. The following experiments were divided into four groups: normal control group (group A), EphA2 overexpression vector transfection group (group B), HLE-B3 cells exposed to dexamethasone group (group C) and EphA2 overexpression vector transfection HLE-B3 cells exposed to dexamethasone group (group D). Statistical analysis method was single factor or two factors variance analysis. Cell survival rate was detected by the Cell Counting Kit-8 assay. Cell apoptosis index was detected by Tunel. Results: Restriction enzyme digestion and sequencing indicated that EphA2 cDNA fragment was successfully inserted in the vector. The infection efficiency was up to 38.6%±3.9%, 49.2%±4.2%, 79.5%±5.5% and 80.2%±6.0% when the MOI was 20, 50, 100 and 200, respectively. There was statistically significant difference (F=2 600.8, P=0.001) among the four groups and between any two groups except between the MOI=100 group and MOI=200 group (P=2.507) . The relative quantity of EphA2 protein of the normal control group, empty vector transfection group and EphA2 gene overexpression vector transfection group was (0.561 2±0.031 7) , (0.559 7±0.012 8) and (3.032 0±0.041 9) , respectively. There was statistically significant difference (F=2 646.0, P=0.001) among the three groups and between any two groups except between the normal control group and empty vector transfection group (P=0.868) . The survival rate of groups A, B, C and D was 98.18%±1.85%, 122.01%±3.89%, 52.32%±1.99% and 76.18%±3.74%, respectively. There was statistically significant difference among the four groups (F=497.6, P=0.001) . The survival rate of group B was greater than group A (P=0.001) . The survival rate of group D was greater than group C (P=0.001) . Tunel results showed that the apoptosis index of groups A, B, C and D was 5.4%±1.5%, 5.0%±1.3%, 23.0%±3.9% and 14.4%±2.7%, respectively. There was statistically significant difference among the four groups (F=397.6, P=0.001) . The apoptosis index of group B was lower than group A, but there was no statistically significant difference between them (P=0.415) ; the apoptosis index of group D was lower than group C (P=0.018). Conclusions: The lentiviral vector carrying human EphA2 gene has been successfully constructed and efficiently expressed in HLE-B3 cells. EphA2 gene overexpression could increase the HLE-B3 cell survival rate and protect HLE-B3 cells from high-concentration dexamethasone-induced reduction of the cell survival rate. EphA2 gene overexpression could protect HLE-B3 cells from high-concentration dexamethasone-induced apoptosis, but it has no remarkable effect on apoptosis of HLE-B3 cells under physiological conditions. (Chin J Ophthalmol, 2018, 54: 125-132).

Effects of SMP-30 overexpression on apoptosis of human lens epithelial cells induced by ultraviolet B irradiation

Objective: This study was to observe the effect of SMP-30 on ultraviolet B (UVB)-induced apoptosis of human lens epithelial cells(HLE-B3) in vitro. Methods: Experimental study. The SMP-30 cDNA was amplified by PCR and inserted into the pRFP-N1 expressing vector which had been double digested by XhoI/HindIII. HLE-B3 cells were cultured and divided into three groups: normal group, pRFP-N1 vector plasmid group and pRFP-N1-SMP-30 plasmid group (SMP-30). Then cells were exposed to UVB and the survival rate of cells was detected by MTT assay. The effects of SMP-30 on UVB-induced HLE-B3 apoptosis were measured by the Cell Death Detection ELISA kit. Meanwhile, the influence of SMP-30 on UVB-induced apoptosis-relative protein expression in HLE-B3 cells was tested by Western blots. Moreover, 2', 7'-Dichlorofluorescin diacetate staining was performed to monitor the protective effects of SMP-30 on UVB-induced HLE-B3 reactive oxygen species(ROS). One-way analysis of variance combined with Dunnett's statistical method were performed to analyze the data. Results: The full length of PSF cDNA fragment was correctly inserted into the pRFP-N1 vector, which was confirmed by DNA sequencing. The SMP-30 fragment was inserted to the plasmid pRFP-N1 correctly, which was also confirmed by DNA sequencing. The PRFP-N1-SMP-30 plasmid was transfected into HLE-B3 successfully. SMP-30 expression was up-regulated in the transfection group, compared with the control group. Data showed that the survival rate of HLE-B3 after the pRFP-N1-SMP-30 plasmid transfection was 0.90±0.14, while the apoptosis rate was 0.43±0.06 and the ROS production was 0.52±0.02, showing significant difference in comparison with the vector plasmid group and the normal group(t=5.830, 9.934, 12.19, P<0.05). In the meantime, SMP-30 overexpression down-regulated the levels of Bax and cleav-caspase-3, but up-regulated the Bcl-2 and Pro-caspase-3 expression levels under UVB irradiation. Conclusion: SMP-30 overexpression plays a protective role in UVB-induced apoptosis via regulating the expression of apoptosis-related proteins and inhibiting the production of ROS in HLE-B3 cells. (Chin J Ophthalmol, 2017, 53: 835-841).

Activation of a Ca2+-dependent cation conductance with properties of TRPM2 by reactive oxygen species in lens epithelial cells

Ion channels are crucial for maintenance of ion homeostasis and transparency of the lens. The lens epithelium is the metabolically and electrophysiologically active cell type providing nutrients, ions and water to the lens fiber cells. Ca2+-dependent non-selective ion channels seem to play an important role for ion homeostasis. The aim of the study was to identify and characterize Ca2+- and reactive oxygen species (ROS)-dependent non-selective cation channels in human lens epithelial cells. RT-PCR revealed gene expression of the Ca2+-activated non-selective cation channels TRPC3, TRPM2, TRPM4 and Ano6 in both primary lens epithelial cells and the cell line HLE-B3, whereas TRPM5 mRNA was only found in HLE-B3 cells. Using whole-cell patch-clamp technique, ionomycin evoked non-selective cation currents with linear current-voltage relationship in both cell types. The current was decreased by flufenamic acid (FFA), 2-APB, 9-phenanthrol and miconazole, but insensitive to DIDS, ruthenium red, and intracellularly applied spermine. H2O2 evoked a comparable current, abolished by FFA. TRPM2 protein expression in HLE-B3 cells was confirmed by means of immunocytochemistry and western blot. In summary, we conclude that lens epithelial cells functionally express Ca2+- and H2O2-activated non-selective cation channels with properties of TRPM2.

Dickkopf-1 inhibits Wnt3a-induced migration and epithelial-mesenchymal transition of human lens epithelial cells

Posterior capsular opacification (PCO) is a major post-operative complication of cataract surgery. Epithelial-mesenchymal transition (EMT) contributes to PCO. We previously indicated that Wnt3a induces the EMT of human lens epithelial cells (LECs) and plays an important role in the development of PCO. The present study aimed to test the potential effect of Dickkopf-1 (Dkk1) on Wnt3a-induced cell migration and the EMT of LECs and to explore possible cellular mechanisms. The secretion of Dkk1 was reduced in the rabbit PCO model, and Dkk1 injected into the eyes post-surgical manipulation prevented PCO formation. Cultured HLE-B3 cells were then transfected with Wnt3a in the presence or absence of Dkk1. Dkk1 treatment restored the epithelial phenotype and reversed the expression of EMT-associated proteins induced by Wnt3a. Dkk1 suppressed LEC migration and the expression of matrix metalloproteinase-1 (MMP-1), and the activity of MMP-2 and MMP-9. Dkk1 inhibited the nuclear accumulation of β-catenin, which is the key regulator of the canonical Wnt signaling. Our results indicate that Dkk1 inhibits Wnt3a-induced migration and the EMT of human LECs.The results contribute to the prevention of PCO formation and development.