Human Lens Epithelial Cell Line Research Articles

High ambient temperature may induce presbyopia via TRPV1 activation.

The prevalence of presbyopia and nuclear cataracts (NUC) is reported to be higher in tropical areas than that in other regions, suggesting a potential influence of high temperatures on lens health. Transient receptor potential vanilloid (TRPV) channels play a crucial role in detecting ambient temperatures across various species, with TRPV1 and TRPV4 expressed in lens epithelial cells. In this study, we investigated whether ambient temperatures affect TRPV1 and TRPV4 activity in the lens, potentially contributing to the development of presbyopia and NUC. We conducted experiments using cultured human lens epithelial cell lines under different temperature conditions. Our results revealed that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 pathways, downstream molecules of TRPV1, were activated, while Src family kinase, a downstream molecule of TRPV4, was inhibited at 37.5°C culture compared to 35.0°C. Confocal microscope images demonstrated higher expression of TRPV1 in 3D-structured cells under high-temperature culture conditions. Additionally, in organ culture lenses, higher elasticity was observed at elevated temperatures compared to that at lower temperatures. These results suggest that high ambient temperatures may induce lens sclerosis via TRPV1 activation, potentially contributing to the development of presbyopia and NUC.

Investigation of Mitochondrial Homeostasis Changes in Lens Epithelium of High-Myopic Cataract

Purpose High myopia is demonstrated as a pathogenic factor for nuclear cataract. The main mechanism of high-myopia cataracts (HMC) is oxidative damage, which causes mitochondrial homeostasis imbalance. This study aimed to explore the mitochondrial homeostasis alterations in lens epithelial cells (LECs) of HMC. Methods The lens epithelium tissues of 20 patients with HMC and 20 control subjects with age-related cataracts (ARC) were collected. The real-time quantitative PCR and western blot assays were performed for gene expressions. Immunofluorescence (IF) assays were performed for mitochondrial marker TOM20, DNA damage marker 15A3, and autophagosome marker LC3. Transmission electron microscopy (TEM) was used to observe the changes in mitochondria morphology. Mitochondrial ROS, and mitochondrial membrane potential were detected by MitoSOX fluorescence, and JC-1 MitoMP staining, respectively. Rat lenses cultured in vitro were pretreated with CCCP and H2O2 (10 and 400 µM) for 24 h. Results The copy number of mtDNA was decreased in HMC patients compared to the ARC patients. Increased mitochondrial-oriented oxidative stress response was detected in LECs of HMC compared to that of ARC. Altered expressions of mitochondrial homeostasis and mitophagy markers, including TFAM, PGC1α, MFN1, MFN2, Drp1, PINK1, Parkin and LC3, were found in HMC patients. Reciprocally, no significant differences in the expression of BNIP3 and FUNDC1 were found between HMC and ARC patients. Importantly, TEM revealed that the obvious mitochondrial fission and mitophagy phenomena occur in the LECs of HMC patients compared to the ARC patients. Moreover, CCCP aggreated the mitoROS production and depolarized mitochondrial membrane potential in the H2O2-treated human lens epithelial cells line (SRA01/04); Most important, rat lens organ culture experiments indicated a significant increase in H2O2-induced lens opacity following mitochondrial uncoupling CCCP treatment. Conclusion This study has identified for the first time the abnormal mitochondrial homeostasis in HMC, and provide a new perspective on the potential mechanisms of HMC, which occurs earlier and at a higher incidence rate than ARC.

Arginase-1 promotes lens epithelial-to-mesenchymal transition in different models of anterior subcapsular cataract

BackgroundArginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in epithelial-to-mesenchymal transition (EMT)-associated cataracts were studied herein.MethodsC57BL/6 mice, a human lens epithelial cell line (HLEC-SRA01/04), and human lens capsule samples were used in this study. The right lens anterior capsule of the mouse eye was punctured through the central cornea with a 26-gauge hypodermic needle. Human lens epithelial cells (HLECs) were transfected with ARG1-targeted (siARG1) or negative control siRNA (siNC). For gene overexpression, HLECs were transfected with a plasmid bearing the ARG1 coding sequence or an empty vector. Medium containing 0.2% serum with or without transforming growth factor beta-2 (TGF-β2) was added for 6 or 24 h to detect mRNA or protein, respectively. The expression of related genes was measured by quantitative real-time polymerase chain reaction (RT–qPCR), western blotting, and immunohistochemical staining. Transwell assays and wound healing assays were used to determine cell migration. Cell proliferation, superoxide levels, nitric oxide (NO) levels, and arginase activity were estimated using Cell Counting Kit-8 assays, a superoxide assay kit, an NO assay kit, and an arginase activity kit.ResultsARG1, alpha-smooth muscle actin (α-SMA), fibronectin, and Ki67 expression increased after lens capsular injury, while zonula occludens-1 (ZO-1) expression decreased.Fibronectin and collagen type I alpha1 chain (collagen 1A1) expression increased, and cell migration increased significantly in ARG1-overexpressing HLECs compared with those transfected with an empty vector after TGF-β2 treatment. These effects were reversed by ARG1 knockdown.The arginase-related pathway plays an important role in EMT. mRNAs of enzymes of the arginase-related pathway were highly expressed after ARG1 overexpression. ARG1 knockdown suppressed these expression changes.Numidargistat (CB-1158) dihydrochloride (CB-1158), an ARG1 inhibitor, suppressed TGF-β2-induced anterior subcapsular cataract (ASC) by reducing the proliferation of lens epithelial cells (LECs) and decreasing fibronectin, α-SMA, collagen 1A1, and vimentin expression.Compared with that in nonanterior subcapsular cataract (non-ASC) patients, the expression of ARG1, collagen 1A1, vimentin, fibronectin, and Ki67 was markedly increased in ASC patients.ConclusionsARG1 can regulate EMT in EMT-associated cataracts. Based on the pathogenesis of ASC, these findings are expected to provide new therapeutic strategies for patients.Graphical abstract

Cellular Localization of FOXO3 Determines Its Role in Cataractogenesis

The transcription factor FOXO3 is a core regulator of cellular homeostasis, stress response, and longevity. The cellular localization of FOXO3 is closely related to its function. Here, we explored the role of FOXO3 in cataract formation. FOXO3 showed nuclear translocation in lens epithelial cells arranged in a single layer on lens capsule tissues from both human cataract and N-methyl-N-nitrosourea (MNU)-induced rat cataract, also in MNU-injured human lens epithelial cell lines (HLECs). FOXO3 knockdown inhibited the MNU-induced increase in expression of genes related to cell cycle arrest (GADD45α and CCNG2) and apoptosis (Bak and TP53). H2 is highly effective in reducing oxidative impairments in nuclear DNA and mitochondria. When we applied H2 to MNU-injured HLECs, FOXO3 underwent cleavage by MAPK1 and translocated into mitochondria, thereby increasing the transcription of oxidative phosphorylation-related genes (MTCO1, MTCO2, MTND1, and MTND6) in HLECs. Furthermore, H2 mediated the translocation of FOXO3 from the nucleus to the mitochondria within the lens epithelial cells of cataract capsule tissues of rats exposed to N-methyl-N-nitrosourea (MNU). This intervention ameliorated MNU-induced cataracts in the rat model. In conclusion, we demonstrated the correlation between the localization of FOXO3 and its function in cataract formation. We also determined that H2 protects HLECs from injury by leading FOXO3 mitochondrial translocation via MAPK1 activation. Mitochondrial FOXO3 can increase mtDNA transcription, stabilize mitochondrial function in HLECs.

Formulation and In-Vitro/Ex-Vivo Characterization of Pregelled Hybrid Alginate-Chitosan Microparticles for Ocular Delivery of Ketorolac Tromethamine.

Innovative hybrid chitosan-sodium alginate (Ch-Ag) microparticles (MPs) were fabricated using both the ionic gelation method as well as the pre-gelation technique. The hybrid Ch-Ag MPs were studied for size, zeta potential, morphology, mucoadhesion, in-vitro release, corneal permeation, and ocular irritation using lens and corneal epithelial cell lines. The average particle size ranged from 1322 nm to 396 nm. The zeta potential for the prepared formulations showed an increase with increasing Ch concentrations up to a value of >35 mV; the polydispersity index (PDI) of some optimized MPs was around 0.1. Compared to drug-free MPs, ketorolac-loaded Ch-Ag MPs demonstrated a drug proportion-dependent increase in their size. SEM, as well as TEM of KT-loaded MPs, confirmed that the formed particles were quasi-spherical to elliptical in shape. The KT release from the MPs demonstrated a prolonged release profile in comparison to the control KT solution. Further, mucoadhesion studies with porcine mucin revealed that the KT-loaded MPs had effective mucoadhesive properties, and polymeric particles were stable in the presence of mucin. Corneal permeation was studied on bovine eyes, and the results revealed that Ch-based MPs were capable of showing more sustained KT release across the cornea compared with that for the control drug solution. Conclusively, the cytotoxicity assay confirmed that the investigated MPs were non-irritant and could confer protection from direct drug irritation of KT on the ocular surface. The MTT cytotoxicity assay confirmed that KT-loaded MPs showed acceptable and reasonable tolerability with both human lens and corneal epithelial cell lines compared to the control samples.

The Long Noncoding RNA H19 Promotes Fibrotic Processes in Lens Epithelial Cells.

The purpose of this study was to investigate the role of lncRNA H19 in epithelial-mesenchymal transition (EMT) and its molecular mechanism in fibrotic cataracts. TGF-β2-induced EMT was induced in human lens epithelial cell line (HLECs) and rat lens explants to mimic posterior capsular opacification (PCO) in vitro and in vivo. Anterior subcapsular cataract (ASC) was induced in C57BL/6J mice. The long noncoding RNA (lncRNA) H19 (H19) expression was detected by RT-qPCR. Whole-mount staining of lens anterior capsule was used to detect α-SMA and vimentin. Lentiviruses carrying shRNA or H19 vector were transfected in HLECs to knockdown or overexpress H19. Cell migration and proliferation were characterized by EdU, Transwell, and scratch assay. EMT level was detected by Western blotting and immunofluorescence. The rAAV2 carrying mouse H19 shRNA was injected into ASC model mouse anterior chambers as a gene therapy to determine its therapeutic potential. PCO and ASC models were built successfully. We found H19 upregulation in PCO and ASC models in vivo and in vitro. Overexpression of H19 by lentivirus transfection increased cell migration, proliferation, and EMT. In addition, H19 knockdown by lentivirus suppressed cell migration, proliferation, and EMT levels in HLECs. Moreover, transfection of rAAV2 H19 shRNA alleviated fibrotic area in ASC mouse lens anterior capsules. Excessive H19 participates in lens fibrosis. Overexpression of H19 increases, whereas knockdown of H19 ameliorates HLECs migration, proliferation, and EMT. These results demonstrate H19 might be a potential target for fibrotic cataracts.

LncRNA TUG1 regulates Smac/DIABLO expression by competitively inhibiting miR-29b and modulates the apoptosis of lens epithelial cells in age-related cataracts.

As one of the early discovered long non-coding RNAs (lncRNA), taurine upregulation gene 1 (TUG1) has been widely expressed in a variety of tumors. Moreover, it promotes cell proliferation, differentiation, apoptosis, and migration. However, our understanding of its importance in the pathogenesis of cataracts remains limited. This study aimed to explore the mechanism by which lncRNA TUG1 mediates lens epithelial cell apoptosis in age-related cataracts (ARC) by regulating the microRNAs (miR-29b)/second mitochondria-derived activator of caspases axis, and to identify more non-surgical strategies for cataract treatment. The messenger RNA expression levels of TUG1, miR-29b, and Smac were detected using quantitative real-time polymerase chain reaction in vivo and in vitro. The expression of the Smac protein was analyzed by Western blotting and immunofluorescence. Flow cytometry and cell counting kit-8 assays were used to detect the cell apoptosis and proliferation rates, respectively. The targeted regulatory relationship between lncRNA TUG1, miR-29b, and Smac was verified by viral vector construction, co-transfection, nuclear and cytoplasmic separation, luciferase reporter assays, and RNA immunoprecipitation. TUG1 and Smac were expressed at high levels in ARC and HLE-B3 cells treated with 200 μmol/L H2O2, whereas miR-29b expression was decreased. In vitro cell experiments confirmed that down-regulation of TUG1 could inhibit the apoptosis of lens epithelial cells. Mechanistically, Smac expression was negatively regulated by miR-29b. TUG1 competitively inhibited miR-29b expression and caused greater release of Smac. In addition, miR-29b partially reversed the effects of TUG1 on human lens epithelial cell line cells. lncRNA TUG1 increases Smac expression and promotes apoptosis of lens epithelial cells in ARC by competitively inhibiting miR-29b. This mechanism is the cytological basis for ARC formation. Based on these results, the lncRNA TUG1/miR29b/Smac axis may be a new molecular pathway that regulates ARC development.

Hesperetin treatment attenuates glycation of lens proteins and advanced‑glycation end products generation.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia invivo and invitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using invitro in human lens epithelial cell lines and exvivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Response of a Human Lens Epithelial Cell Line to Hyperglycemic and Oxidative Stress: The Role of Aldose Reductase.

A common feature of different types of diabetes is the high blood glucose levels, which are known to induce a series of metabolic alterations, leading to damaging events in different tissues. Among these alterations, both increased polyol pathway flux and oxidative stress are considered to play relevant roles in the response of different cells. In this work, the effect on a human lens epithelial cell line of stress conditions, consisting of exposure to either high glucose levels or to the lipid peroxidation product 4-hydroxy-2-nonenal, is reported. The occurrence of osmotic imbalance, alterations of glutathione levels, and expression of inflammatory markers was monitored. A common feature of the two stress conditions was the expression of COX-2, which, only in the case of hyperglycemic stress, occurred through NF-κB activation. In our cell model, aldose reductase activity, which is confirmed as the only activity responsible for the osmotic imbalance occurring in hyperglycemic conditions, seemed to have no role in controlling the onset of the inflammatory phenomena. However, it played a relevant role in cellular detoxification against lipid peroxidation products. These results, in confirming the multifactorial nature of the inflammatory phenomena, highlight the dual role of aldose reductase as having both damaging but also protecting activity, depending on stress conditions.

Extracellular HSP90 promotes differentiation of lens epithelial cells to fiber cells by activating LRP1-YAP-PROX1 axis.

Capsular residual lens epithelial cells (CRLEC) undergo differentiation to fiber cells for lens regeneration or tansdifferentiation to myofibroblasts leading to posterior capsular opacification (PCO) after cataract surgery. The underlying regulatory mechanism remains unclear. Using human lens epithelial cell lines and the ex vivo cultured rat lens capsular bag model, we found that the lens epithelial cells secrete HSP90α extracellularly (eHSP90) through an autophagy-associated pathway. Administration of recombinant GST-HSP90α protein or its M-domain induces the elongation of rat CRLEC cells with concomitant upregulation of the crucial fiber cell transcriptional factor PROX1and its downstream targets, β- and γ-crystallins and structure proteins. This regulation is abolished by PROX1 siRNA. GST-HSP90α upregulates PROX1 by binding to LRP1 and activating LRP1-AKT mediated YAP degradation. The upregulation of GST-HSP90α on PROX1 expression and CRLEC cell elongation is inhibited by LRP1 and AKT inhibitors, but activated by YAP-1 inhibitor (VP). These data demonstrated that the capsular residue epithelial cells upregulate and secrete eHSP90α, which in turn drive the differentiation of lens epithelial cell to fiber cells. The recombinant HSP90α protein is a potential novel differentiation regulator during lens regeneration.

Upregulation of histone H3 caused by CRYAA may contribute to the development of age-related cataract

Objective: Age-relate cataract (ARC) is a disease of the eyes with no effective drugs to prevent or treat patients. The aim of the present study is to determine whether histone H3, αA-crystallin (CRYAA), β-galactosidase (GLB1), and p53 are involved in the pathogenesis of ARC. Methods: A total of 99 anterior lens capsules (ALCs) of patients with ARC of various nuclear grades, ultraviolet models of ALCs, and two human lens epithelial cell lines (FHL-124 and SRA01/04) were used, and the expression of histone H3, CRYAA, GLB1, and p53 were detected by immunoblotting and reverse transcription and real time-quantitative polymerase chain reaction. The association between CRYAA with histone H3, GLB1, and p53 was assessed in FHL-124 and SRA01/04 cells following CRYAA overexpression. Results: Histone H3 and p53 in ALCs of patients with ARC were up-regulated in a grade-dependent manner, and the expression of CRYAA showed a positive association with histone H3, p53, and GLB1. In UV models of ALCs and human lens epithelial cell lines, the expression levels of histone H3, cell apoptosis factors (Bax/Bcl-2, cleaved caspase-3), and inflammation factors (interleukin-6, tumor necrosis factor-α) were all up-regulated. Furthermore, transfection of CRYAA in FHL-124 cells induced overexpression of histone H3. Conclusion: CRYAA-mediated upregulation of histone H3 may be involved in the pathogenesis of ARC. p53 may also have a role in ARC development, but not via the CRYAA-histone H3 axis. The results of the present study may assist in improving our understanding of the pathogenesis of ARC and in identifying potential targets for treatment.

Toll-like receptor 3 gene regulates cataract-related mechanisms via the Jagged-1/Notch signaling pathway

ABSTRACT Epithelial-melancholy transition (EMT) is the main cause of organ fibrosis and a common pathogenetic mechanism in most cataracts. This study aimed to explore the molecular mechanism of Toll-like receptor (TLR)-3 in the occurrence and development of post-cataract EMT and to provide new ideas for the prevention and treatment of posterior capsule opacification (PCO). In the presence or absence of TLR3, the human lens epithelial cell (LEC) line, SRA01/04, was treated with the transforming growth factor (TGF)-β2. Cell counting kit-8 (CCK-8) and Transwell assays were used to analyze the cell proliferation, migration, and invasion. The expression levels of proteins and RNAs were detected by western blotting and quantitative polymerase chain reaction (qPCR) experiments. Functional gain and loss studies showed that TLR3 regulates the proliferation, migration, and invasion of LECs and EMT induced by TGF-β2. Moreover, TLR3 regulates the expression of Jagged-1, Notch-1, and Notch-3 These findings indicate that TLR3 prevents the progression of lens fibrosis by targeting the Jagged-1/Notch signaling pathway to regulate the proliferation, migration, and invasion of LECs, and TGF-β2-induced EMT. Therefore, the TLR3-Jagged-1/Notch signaling axis may be a potential therapeutic target for the treatment of fibrotic cataracts.

Oxidative Stress-Induced TRPV2 Expression Increase Is Involved in Diabetic Cataracts and Apoptosis of Lens Epithelial Cells in a High-Glucose Environment.

Cataracts are a serious complication of diabetes. In long-term hyperglycemia, intracellular Ca2+ concentration ([Ca2+]i) and reactive oxygen species (ROS) are increased. The apoptosis of lens epithelial cells plays a key role in the development of cataract. We investigated a potential role for transient receptor potential vanilloid 2 (TRPV2) in the development of diabetic cataracts. Immunohistochemical and Western blotting analyses showed that TRPV2 expression levels were significantly increased in the lens epithelial cells of patients with diabetic cataracts as compared with senile cataract, as well as in both a human lens epithelial cell line (HLEpiC) and primary rat lens epithelial cells (RLEpiCs) cultured under high-glucose conditions. The [Ca2+]i increase evoked by a TRPV2 channel agonist was significantly enhanced in both HLEpiCs and RLEpiCs cultured in high-glucose media. This enhancement was blocked by the TRPV2 nonspecific inhibitor ruthenium red and by TRPV2-specific small interfering (si)RNA transfection. Culturing HLEpiCs or RLEpiCs for seven days in high glucose significantly increased apoptosis, which was inhibited by TRPV2-specific siRNA transfection. In addition, ROS inhibitor significantly suppressed the ROS-induced increase of TRPV2-mediated Ca2+ signal and apoptosis under high-glucose conditions. These findings suggest a mechanism underlying high-glucose–induced apoptosis of lens epithelial cells, and offer a potential target for developing new therapeutic options for diabetes-related cataracts.

Astragaloside IV alleviates oxidative stress-related damage via inhibiting NLRP3 inflammasome in a MAPK signaling dependent pathway in human lens epithelial cells.

Oxidative stress alters cellular microenvironment, facilitating cell apoptosis and inflammatory response, and oxidation of lens constituents may ultimately result in cataracts. Astragaloside IV (AS-IV) exhibits a variety of pharmacological activities, such as antioxidative and anti-inflammatory activity via regulating various signaling pathways. However, the effect of AS-IV on lens epithelial cells and its potential therapeutic role in cataracts remained to be investigated. In this study, AS-IV prevented H2 O2 -induced injury and inflammatory response in human lens epithelial cell line HLE-B3 through inhibiting NLRP3 inflammasome in a mitogen-activated protein kinase-dependent pathway, providing a potential novel therapeutic strategy for cataracts.

GSH Levels Serve As a Biological Redox Switch Regulating Sulforaphane-Induced Cell Fate in Human Lens Cells.

PurposeSulforaphane (SFN) is a therapeutic phytochemical agent for many health conditions. SFN-induced cytotoxicity is shown to have promise in preventing posterior capsule opacification (PCO). In the current study, we aimed to elucidate key processes and mechanisms linking SFN treatment to lens cell death.MethodsThe human lens epithelial cell line FHL124 and central anterior epithelium were used as experimental models. Cell death was assessed by microscopic observation and cell damage/viability assays. Gene or protein levels were assessed by TaqMan RT-PCR or immunoblotting. Mitochondrial networks and DNA damage were assessed by immunofluorescence. Mitochondrial membrane potential, activating transcription factor 6 (ATF6) activity, ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and glutathione reductase (GR) activity were measured using different light reporter assays. SFN metabolites were analyzed by LC-MS/MS.ResultsTreatment with N-acetylcysteine (NAC), a reactive oxygen species scavenger, prevented SFN-induced cell death in both models. NAC also significantly protected FHL124 cells from SFN-induced mitochondrial dysfunctions, endoplasmic reticulum stress (ERS), DNA damage and autophagy. SFN significantly depleted GSH, the major antioxidant in the eye, and reduced GR activity, despite doubling its protein levels. The most abundant SFN conjugate detected in lens cells following SFN application was SFN–GSH. The addition of GSH protected lens cells from all SFN-induced cellular events.ConclusionsSFN depletes GSH levels in lens cells through conjugation and inhibition of GR activity. This leads to increased reactive oxygen species and oxidative stress that trigger mitochondrial dysfunction, ERS, autophagy, and DNA damage, leading to cell death. In summary, the work presented provides a mechanistic understanding to support the therapeutic application of SFN for PCO and other disorders.

MiRNA-26b suppresses the TGF-β2-induced progression of HLE-B3 cells via the PI3K/Akt pathway

To study the effect of miR-26b on lens epithelial cells induced by transforming growth factor beta (TGF-β) 2 and the underlying signaling pathways. Human lens epithelial cell line B-3 (HLE-B3) was incubated with TGF-β2 (5 ng/mL) and then transfected with miR-26b mimics. The expression of miR-26b was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), while 5'-bromodeoxyuridine (BrdU) and wound-healing assays were used to measure the growth and migration of HLE-B3 cells, respectively. The expression of epithelial-mesenchymal transition (EMT) markers and the activity of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway were measured by Western blotting assay and immunofluorescence staining. Electron microscopy was also used to observe cellular morphology. The expression levels of miR-26b were significantly reduced in human posterior capsular opacification-attached lens tissue and TGF-β2-stimulated HLE-B3 cells. In the presence of TGF-β2, the growth, migration, and EMT of HLE-B3 cells were distinctly enhanced; these effects were attenuated by the administration of miR-26b mimics. Furthermore, the overexpression of miR-26b significantly reduced upregulation of the PI3K/Akt pathway when stimulated by TGF-β2 in HLE-B3 cells. Moreover, the addition of an activator (740 Y-P) led to the upregulation of the PI3K/Akt pathway and abolished the protective effect of miR-26b on the HLE-B3 cells that was mediated by TGF-β2. The miR-26b suppresses TGF-β2-induced growth, migration, and EMT in HLE-B3 cells by regulating the PI3K/Akt signaling pathway.

Aqueous extract prepared from steamed red amaranth (Amaranthus gangeticus L.) leaves protected human lens cells against high glucose induced glycative and oxidative stress.

HLE-B3 cell line, a human lens epithelial cell line, was used to examine the anti-glycative and anti-oxidative protection of aqueous extract prepared from steamed red amaranth leaves against high glucose induced injury. Phytochemical profile of this aqueous extract was analyzed. HLE-B3 cells were pretreated by this aqueous extract at 0.25%, 0.5%, or 1%, and followed by high glucose treatment. Results showed that the content of phenolic acids, flavonoids, anthocyanins, carotenoids, and triterpenoids in this aqueous extract was in the range of 1,107-2,861mg/100g dry weight. High glucose decreased cells viability and suppressed Bcl-2 mRNA expression. This aqueous extract pretreatments raised 11-42% cell survival and upregulated 20-47% Bcl-2 mRNA expression. High glucose reduced Na+ -K+ ATPase activity and mitochondrial membrane potential (MMP). This aqueous extract raised 27-40% Na+ -K+ ATPase activity, and 18-51% MMP. High glucose stimulated the generation of total advanced glycative endproducts (AGEs), methylglyoxal, and reactive oxygen species (ROS). This aqueous extract pretreatments lowered total AGEs, methylglyoxal, and ROS levels in the range of 0.38-1.17 folds, 1.7-4.9nmol/mg protein, and 0.35-1.06 relative fluorescence unit/mg protein. High glucose upregulated mRNA expression of aldose reductase, nuclear factor kappa B, and p38. This aqueous extract pretreatments decreased mRNA expression of these factors in the range of 75-159%, 57-151%, and 54-166%. High glucose downregulated mRNA expression of nuclear factor E2-related factor 2 (Nrf2). This aqueous extract pretreatments increased 12-38% Nrf2 mRNA expression. These results suggested that this aqueous extract might be a potent nutritional supplement to prevent diabetic retinopathy.

Curcumin induces human lens epithelial cell apoptosis and cell cycle arrest by inhibiting Wnt/β-catenin signaling pathway

To investigate the effect of curcumin on cell cycle and apoptosis of human lens epithelial cells and the possible molecular mechanism. Cultured human lens epithelial cell line HLEC-SRA01/04 was treated with 20, 40 and 60 μmol/L curcumin for 24 or 48 h. The cell proliferation inhibition rate was determined using MTT assay, and the changes in cell cycle, mitochondrial membrane potential and apoptosis rate were analyzed with flow cytometry. Western blotting was used to detect the expression levels of caspase-9, caspase-3, Bcl-2, Bax, cyclin B1, CDK1, β-catenin, c-myc, and cyclin D1 in the cells. Curcumin concentration- and time-dependently inhibited the proliferation of in HLEC-SRA01/04 cells as compared with the control cells (P < .05). Flow cytometric analysis showed that curcumin significantly increased apoptosis rate and cell percentage in G2/M phase and lowered mitochondrial membrane potential of HLEC-SRA01/04 cells in a concentrationdependent manner (P < 0.05). The results of Western blotting showed that curcumin also concentration-dependently increased the cellular expressions of caspase-3, caspase-9 and Bax and lowered the expressions of Bcl-2, cyclin B1, CDK1 and β-catenin along with the downstream proteins cyclin D1 and c-myc in the Wnt/β-catenin signaling pathway (P < 0.05). Curcumin inhibits the proliferation of HLEC-SRA01/04 cells possibly by inhibiting the Wnt/β-catenin signaling pathway and causing cell cycle arrest to induce cell apoptosis.

MicroRNA-124 facilitates lens epithelial cellapoptosis by inhibiting SPRY2 and MMP-2.

Age-related cataract (ARC) is the primary cause of blindness worldwide. Abnormal expression of microRNAs (miRNAs/miRs) has been reported to be associated with multiple diseases, including ARC. However, the potential role of miR-124 in ARC remains unclear. The present study used the human lens epithelial cell line, SRA01/04, to investigate the potential role of miR-124 in ARC. Reverse transcription-quantitative PCR analysis was performed to detect the expression levels of miR-124, protein sprouty homolog 2 (SPRY2) and matrix metalloproteinase-2 (MMP-2) in ARC tissues, while western blotting was performed to detect the protein levels of SPRY2 and MMP-2. Cell viability and apoptosis of SRA01/04 cells were assessed via Cell Counting Kit-8 and TUNEL assays, respectively. The interaction between miR-124 and SPRY2 or MMP-2 was confirmed via the dual-luciferase reporter and RNA immunoprecipitation assays. The results of the present study demonstrated that miR-124 expression was significantly upregulated in ARC tissues, and knockdown of miR-124 increased SRA01/04 cell viability and suppressed apoptosis. In addition, SPRY2 and MMP-2 expression was decreased in ARC tissues, and were demonstrated to directly bind to miR-124. Overexpression of SPRY2 or MMP-2 increased SRA01/04 cell viability and repressed apoptosis, the effects of which were reversed following overexpression of miR-124. Taken together, these results suggested that miR-124 facilitates lens epithelial cell apoptosis by modulating SPRY2 or MMP-2 expression, providing a novel treatment approach for ARC.

Fluorofenidone inhibits epithelial-mesenchymal transition in human lens epithelial cell line FHL 124: a promising therapeutic strategy against posterior capsular opacification.

The present study aimed to investigate the inhibitory effect of fluorofenidone against transforming growth factor β2-induced proliferation and epithelial-mesenchymal transition in human lens epithelial cell line FHL 124 and its potential mechanism. We evaluated the effect of fluorofenidone on proliferation and epithelial-mesenchymal transition of human lens epithelial cell line FHL 124 in vitro. After treatment with 0, 0.1, 0.2, 0.4, 0.6, and 1.0 mg/mL fluorofenidone, cell proliferation was measured via MTT assay. Cell viability was evaluated by lactate dehydrogenase activity from damaged cells. FHL 124 cells were treated with different transforming growth factor β2 concentrations (0-10 ng/mL) for 24 h and the expression of CTGF, α-SMA, COL-I, E-cadherin, and Fn were detected via quantitative polymerase chain reaction and Western blot analysis. After treatment with 0, 0.2, and 0.4 mg/ml fluorofenidone, the expressions of transforming growth factor β2 and SMADs were detected with real-time polymerase chain reaction and Western blot analysis. Expressions of CTGF, α-SMA, COL-I, and Fn were analyzed by immunocytochemistry assay. The viability of FHL 124 cells was not inhibited when the fluorofenidone concentration was ≤0.4 mg/mL after the 24h treatment. Cytotoxicity was not detected via lactate dehydrogenase assay after the 24h and 36h treatment with 0.2 and 0.4 mg/mL fluorofenidone. Transforming growth factor β2 increased mRNA and protein expression of CTGF, α-SMA, COL-I, and Fn. However, fluorofenidone significantly suppressed expression of SMADs, CTGF, α-SMA, COL-I, and Fn in the absence or presence of transforming growth factor β2 stimulation. Fluorofenidone significantly inhibited expression of SMADs, CTGF, α-SMA, COL-I, and Fn in FHL 124 cells. Due to noncompliance in infants, fluorofenidone may become a novel therapeutic drug against posterior capsular opacification in infants.