Apoptosis Of Lens Epithelial Cells Research Articles

GJA8 missense mutation disrupts hemichannels and induces cell apoptosis in human lens epithelial cells

Autosomal dominant congenital cataract (ADCC), the most common hereditary disease, is a major cause of eye disease in children. Due to its high genetic and clinical heterogeneity, the identification of ADCC-associated gene mutations is essential for the development of molecular therapies. In this study, we examined a four-generation Chinese pedigree with ADCC and identified putative mutations in ADCC candidate genes via next-generation sequencing (NGS) followed by Sanger sequencing. A novel missense mutation in GJA8 (c.T217C) in ADCC patients causes a serine-to-proline substitution at residue 73 of connexin 50 (Cx50); no mutation was found in unaffected family members and unrelated healthy individuals. Functional analysis revealed that this missense mutation disrupts protein function in human lens epithelial cells (HLEpiCs), which fails to form calcium-sensitive hemichannels. Furthermore, mutant Cx50 leads to decreased ROS scavenging by inhibiting G6PD expression and thus induces cell apoptosis via aberrant activation of the unfolded protein response (UPR). In conclusion, we report a novel GJA8 heterozygous mutation in a Chinese family with a vital role in ADCC, broadening the genetic spectrum of this disease.

SP1-mediated lncRNA PVT1 modulates the proliferation and apoptosis of lens epithelial cells in diabetic cataract via miR-214-3p/MMP2 axis.

Emerging evidence illustrates the critical roles of long non‐coding RNAs (lncRNAs) in the diabetes. However, the deepgoing regulation of lncRNA PVT1 in the diabetic cataract (DC) is still unclear. Here, present research investigates the pathologic roles and underlying mechanism by which lncRNA PVT1 regulates the DC pathogenesis. Human lens epithelial (HLE) B‐3 cells were induced by the high glucose (HG) to simulate the DC microenvironment models. Results revealed that lncRNA PVT1 expression was up‐regulated in the HG‐induced HLE B‐3 cells as compared to the normal glucose group. Transcription factor SP1 could bind with the promoter region of PVT1 and activate its transcription. Functionally, PVT1 knock‐down could repress the proliferation and promote the apoptosis of HLE B‐3 cells. Mechanistically, PVT1 acted as the ‘miRNA sponge’ to target miR‐214‐3p/MMP2 axis. This finding revealed a novel insight of lncRNA PVT1 for the DC pathogenesis, providing an inspiration for the DC mechanism.

Protective Effects of Lanosterol Synthase Up-Regulation in UV-B-Induced Oxidative Stress.

UV-B radiation may be an important risk factor in cataract etiology. After exposure to UV-B radiation, cells show imbalances in the repair of DNA damage, which induce changes in the levels of certain proteins, including alpha-crystallin, which is the most abundant protein in the lens and crucial for the maintenance of lens transparency. Lanosterol synthase (LSS), an essential rate-limiting enzyme in cholesterol biosynthesis, might play significant roles in oxidative stress and in the maintenance of lens transparency. However, the roles of LSS in UV-B-induced apoptosis are not well understood. Therefore, we irradiated female Sprague-Dawley rats with ultraviolet radiation to establish an animal model for exploring the variations in LSS expression during the early stages of UV-B exposure. In addition, we cultured human lens epithelial (HLE) cells that overexpress LSS and exposed them to UV-B radiation to explore the function of increased LSS expression in UV-B-induced apoptosis. The data demonstrated that UV-B exposure induced oxidative stress and apoptosis in rat lens epithelial cells and that irradiance exposure increased the level of lenticular damage. Additionally, UV-B exposure decreased the alpha-crystallin content and increased the expressions of Bax and cleaved caspase-3 compared with the control levels. After exposure to UV-B, the apoptosis-related index of HLE cells overexpressing LSS was lower than that of the control cells. Furthermore, ROS overproduction might activate the sirtuin 1 (Sirt1) pathway, which induced protein expressions of sterol regulatory element-binding transcription factor 2 (SREBF2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and LSS. However, the specific mechanism of the Sirt1 pathway needed to be further studied. In summary, UV-B exposure induced oxidative injury and resulted in crystallin denaturation and apoptosis in lens epithelial cells, and LSS might play a protective role during the early stages of this process and could be an important target in the cataract prevention.

MiR-15a: a potential diagnostic biomarker and a candidate for non-operative therapeutic modality for age-related cataract

ABSTRACTIntroduction: In order to better understand the role of hsa-miR-15a in the pathogenesis of age-related cataracts, we hypothesised altered expression, and of target anti-apoptotic genes, BCL-2 and MCL-1, in lens epithelial cells amongst age-related cataract patients.Material and methods: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) quantified the expression of hsa-miR-15a and the target genes BCL-2 and MCL-1 in lens epithelial cells of 120 age-related cataract patients (40 patients with cortical cataracts, 40 patients with nuclear cataracts and 40 patients with posterior subcapsular cataracts) and 40 controls. Sixty specimens (15 normal and 45 cataracts) were stained immunohistochemically with BCL-2 and MCL-1 markers.Results: The expression of hsa-miR-15a was significantly increased (p = 0.003) in lens epithelial cells of cataract patients compared to the control group. BCL-2 and MCL-1 expression levels were significantly decreased in cataract patients (p < 0.001). A significant increase in hsa-miR-15a expression in the cortical subtype compared to the posterior subcapsular subtype (p = 0.003) and a significant decrease in BCL-2 and MCL-1 expressions in the cortical subtype compared to the nuclear and the posterior subcapsular subtype was detected.Conclusions: The increased expression of hsa-miR-15a in lens epithelial cells of cataract patients may repress the expression of BCL-2 and MCL-1. The expression of hsa-miR-15a and the subsequent apoptosis of lens epithelial cells are part of the pathogenesis of age-related cataracts.

MiR-23b-3p regulates apoptosis and autophagy via suppressing SIRT1 in lens epithelial cells.

Age-related cataract is one of the prior causes of blindness and the incidence rates of cataract are even rising. Oxidative stress plays an important role in the pathogenesis of cataracts. Under oxidative stress, lens epithelial cell (LEC cell) apoptosis is activated, which might lead to the opacity of the lens and accelerate the progression of cataract development. Meanwhile, autophagy is also active to face oxidative stress. miRNAs have been reported to involve cataract. However, the underlying mechanism is not clear. The present study aimed to investigate the regulatory effect of miR23b-3p on apoptosis and autophagy in LEC cells under oxidative stress. The expression levels of miR-23b-3p were examined in age-related cataract tissues and LEC cells treated with hydrogen peroxide, showing that miR23b-3p expression levels were upregulated. Knockdown of miR23b-3p expression in LEC cells brought about apoptosis significantly decreased while autophagy significantly increased during hydrogen peroxide. We predicted microRNA miRNA-23b-3p might participate in regulating silent information regulator 1 (SIRT1) by bioinformatics database of TargetScan. Luciferase reporter assays confirmed that miRNA-23b-p could suppress SIRT1 expression by binding its 3'UTR. In addition, overexpression or knockdown of miR-23b-3p could decrease or increase SIRT1 expression, which indicated that Mir-23b-3p could suppress SIRT1 expression. In addition, enhanced SIRT1 could attenuate the regulation of cell apoptosis and autophagy induced by overexpression of miR-23b-3p. Taken together, our findings revealed that miR-23b-3p regulated apoptosis and autophagy via suppressing SIRT1 in LEC cell under oxidative stress, which could provide new ideas for clinical treatment of cataract.

CASC15 Promotes Lens Epithelial Cell Apoptosis in Age-Related Cataracts by Regulating MiR-139-5p/caspase-3

Objective: The aim of this study was to reveal the role of CASC15 and miR-139-5p function in regulating agerelated cataracts (ARC). Methods: The expression of CASC15, miR-139-5p and caspase-3 were detected by qRT-PCR. Western blot was used to analyze the level of apoptosis-related proteins. Flow cytometry was used to measure the apoptosis of SAR01/04l cells treated with UV irradiation. Results: The expression of CASC15 and caspase-3 in the lens of ARC was higher than that in normal lens, but the expression of miR-139-5p had opposite trend. The level of caspase-3 and Bax were increased and Bcl-2 was decreased in ARC group. In addition, CASC15 negatively regulated miR-139-5p expression and promoted SAR01/04l cell apoptosis. CASC15 could combine with miR-139-5p, and miR-139-5p could combine with caspase-3 3’-UTR. Furthermore, pcDNA-caspase-3 could reverse the inhibitory effect of si-CASC15 and miR139-5p mimic on SRA01/04 cell apoptosis. Conclusion: CASC15 promoted lens epithelial cell apoptosis by regulating miR-139-5p/caspase-3. Keywords: ARC; CASC15; LECs apoptosis; miR-139-5p

SUMOylation Evoked by Oxidative Stress Reduced Lens Epithelial Cell Antioxidant Functions by Increasing the Stability and Transcription of TP53INP1 in Age-Related Cataracts.

Oxidative stress plays an important role in the pathogenesis of cataracts. Small ubiquitin-like modifier (SUMO) proteins have great effects on cell stress response. Previous studies have shown that TP53INP1 can arrest cell growth and induce apoptosis by modulating p53 transcriptional activity and that both TP53INP1 and p53 are substrates of SUMOylation. However, no previous research has studied the effect of SUMOylation on the oxidative stress response in cataracts. This is the first study to investigate the effect of SUMOylation of TP53INP1 in oxidative stress-induced lens epithelial cell injury and age-related cataract formation. We found that the oxidative stress-induced endogenous SUMOylation of TP53INP1 promoted human lens epithelial cell (holed) apoptosis and regulated hLEC antioxidant effects by increasing the stability and transcription of TP53INP1 in age-related cataracts. SUMO-1, SUMOylation, and TP53INP1 were upregulated in lens tissues affected by age-related cataracts. A SUMO-1-specific protease, SENP1, acted as an oxidative stress-sensitive target gene in hLECs. This study identified for the first time that TP53INP1 can be SUMOylated in vivo, that the SUMOylation of TP53INP1 is induced by oxidative stress, and that SUMOylation/deSUMOylation can affect the stability and transcription of TP53INP1 in hLECs.

Effects of Krüppel-like factor 6 overexpression towards apoptosis of human lens epithelial cells with ultra violetradiation B treatment

Objective To investigate the effect of the overexpression of Kruppel-like factor 6 (KLF6) towards the apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B (UVB) radiation. Methods The eukaryotic expression plasmid pEGFP-C2-KLF6 which was successfully constructed were transfected into HLECs, followed by the detection of KLF6 level by using Western blot, and then companied by UVB stimulation.Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay.The morphology of the cells was observed by using hematoxylin-eosin staining method.The cell damage was examined by Live/Dead staining.The apoptotic markers bax and bcl-2 were detected by Western blot.Quantitative apoptotic levels were measured with the apoptosis detection kit; the expression level of reactive oxygen species (ROS) was analyzed by DCFH-DA probe. Results The cell viability of the 0.5 μg transfection group and the 1.0 μg transfection group was significantly lower than that of the blank vector control group (both at P<0.05). In high KLF6 expression group, the cells were sparse, long and narrow in size and shape, and the cytoplasm was concentrated.The cells in the normal control group were green living cells with stable morphology and even quantity.The number of red dead cells was increased significantly in the KLF6 high-expression group.After UVB irradiation, the apoptosis value, relative bax expression, bax/bcl-2 ratio and ROS expression of HLECs cells in the KLF6 high-expression group were all higher than those in the blank vector control group, with statistically significant differences between them (all at P<0.05). Conclusions Overexpression of KLF6 can exacerbate apoptosis of HLECs caused by UVB, by regulating the expression of apoptosis-related proteins and promoting the accumulation of ROS in the endoplasmic reticulum.Down-regulation of KLF6 expression by biological tools may play a protective role on LECs to a certain extent. Key words: Kruppel factor 6; Lens epithelial cells; Apoptosis; Ultra violetradiation B; Oxidative stress

The inhibitory effect of decorin on apoptosis of human lens epithelial cells under high glucose condition and its mechanism

Objective To investigate the effects of decorin (DCN) on apoptosis and oxidative stress in human lens epithelial cells (LECs) under high glucose condition. Methods HLE-B3 cells were cultured in vitro and the effect of DCN with different concentrations on HLE-B3 viability was determined by using cell counting kit-8 (CCK-8). The cultured cells were divided into normal control group, DCN group, high glucose group and DCN + high glucose group.Flow cytometry was used to detect the apoptosis rate and the expression of reactive oxygen species (ROS) in the cells.Microplate spectrophotometer was used to measure total superoxide dismutase (SOD) enzyme activity and the radio of glutathione (GSH)/glutathione disulfide (GSSG). Western blot was used to detect the expressions of bax and bcl-2 proteins. Results HLE-B3 cells were spindle shaped, with centered and clearly visible nuclei and neatly cell arrangment.According to CCK-8 method, survival rates of HLE-B3 in all groups were more than 90%.Different concentrations of DCN showed no significant effect on HLE-B3 survival rate (all at P>0.05). After 48 hours of cell culture, the apoptosis rate of high glucose group was significantly higher than that of normal control group, and the apoptosis rate of DCN+ high glucose group was significantly lower than that of high glucose group (both at P=0.000). The mean fluorescence intensity of intracellular ROS in the high-glucose group was significantly higher than that in the normal control group, and the mean fluorescence intensity of ROS in the DCN group was significantly higher than that in the high glucose group (both at P=0.000). The total SOD activity in the high glucose group was significantly lower than that in the normal control group and DCN group (P=0.007, 0.004). The GSH/GSSG ratio of the high-glucose group was significantly lower than that of the normal control group and DCN group (both at P=0.000). Conclusions DCN can inhibit the apoptosis and oxidative stress of HLE-B3 under high glucose, which provides the basis for the treatment of diabetic cataract. Key words: Decorin; Human lens epithelial cell; High glucose condition; Oxidative stress; Apoptosis; Inhibitory effect; Mechanism

Sanguinarine induces apoptosis of human lens epithelial cells by increasing reactive oxygen species via the MAPK signaling pathway.

Posterior capsular opacification (PCO) remains a major complication of cataract surgery and is the most common reason for loss of vision. PCO is primarily associated with uncontrolled proliferation of residual human lens epithelial cells (HLEs). Sanguinarine is a type of benzophenanthridine alkaloid extracted from the herbaceous plant Sanguinaria canadensis, which is widely used for its anti-microbial, anti-inflammatory, anti-oxidative and anti-proliferative properties. However, studies examining the effect of sanguinarine on HLEs and the underlying mechanism are scarce. The present study aimed to investigate the effects of sanguinarine on HLEs. An MTT assay was used to determine the effect of sanguinarine on cell viability. Flow cytometry was used to evaluate cell apoptosis, and the mitochondrial membrane potential and reactive oxygen species (ROS) levels. A caspase 3/7 activity assay was also used to evaluate cell apoptosis, while western blotting was performed to determine protein levels. The results demonstrated that sanguinarine exerted an anti-proliferative effect by inducing ROS, and caused cell apoptosis via mitochondrial and caspase-dependent pathways. Treatment with sanguinarine led to the loss of mitochondrial membrane potential. Sanguinarine also significantly increased the phosphorylation levels of c-Jun N-terminal kinase and p38, which indicated the involvement of the mitogen-activated protein kinase signaling pathway. These results suggested that sanguinarine may have a noteworthy pro-apoptotic effect on HLEs, and may be used as a potential drug for PCO or even cataract prevention.

MiR-221 promotes lens epithelial cells apoptosis through interacting with SIRT1 and E2F3

MicroRNAs (miRNAs) have been regarded as potential modulators in varying ocular diseases, including age-related cataract (ARC). However, the roles of miR-221 in ARC progression and its underlying mechanism remain poorly understood. In this study, human lens epithelial cell line (SRA01/04) was used to investigate the potential function of miR-221 in vitro. The expressions of miR-221, sirtuin-1 (SIRT1) and E2F transcription factor 3 (E2F3) were measured in ARC tissues by quantitative real-time polymerase chain reaction and western blotting, respectively. To investigate the effect of miR-221, SIRT1 and E2F3 on cell apoptosis, SRA01/04 cells were transfected with miR-221 inhibitor, negative control inhibitor, pcDNA3.1-SIRT1 overexpression vector, pcDNA3.1-E2F3 overexpression vector or pcDNA3.1 empty vector. After the transfection, cell viability was detected in SRA01/04 cells at 0, 24, 48 or 72 h by cell counting kit-8 assay. Cell apoptosis was evaluated in transfected SRA01/04 cells by flow cytometry and western blotting at 72 h. The interaction between miR-221 and SIRT1 or E2F3 was probed by luciferase activity and RNA immunoprecipitation assays. Results showed that high expression of miR-221 was exhibited in ARC tissues compared with that in normal samples and associated with Lens Opacities Classification System III grades. Knockdown of miR-221 promoted cell viability and inhibited apoptosis in SRA01/04 cells. Moreover, both of SIRT1 and E2F3 levels were directly targeted by miR-221 and down-regulated in ARC tissues. Besides, overexpression of SIRT1 or E2F3 increased cell viability and suppressed apoptosis in SRA01/04 cells, which was reversed by addition of miR-221. We concluded that miR-221 promoted lens epithelial cells apoptosis through regulating SIRT1 and E2F3, providing a novel biomarker for treatment of ARC.

TRIM69 inhibits cataractogenesis by negatively regulating p53

Ultraviolet B (UVB) irradiation can induce reactive oxygen species (ROS) production and apoptosis in human lens epithelial cells (HLECs), thus leading to the formation of cataracts. We studied the role of tripartite motif 69 (TRIM69) in cataract formation. The expression of TRIM69 protein was down-regulated in both human cataract capsule tissues and HLECs treated with UVB, whereas the expression of p53 protein exhibited an opposite trend. Ectopic expression of TRIM69 in HLECs significantly suppressed UVB-induced apoptosis and ROS production, whereas knockdown of TRIM69 promoted apoptosis and ROS production. TRIM69 can interact with p53 and induce its ubiquitination. The effects of TRIM69 overexpression in UVB-induced cell apoptosis and ROS production was clearly weakened by p53 overexpression, thus suggesting a role for p53 in TRIM69 functions. Furthermore, inhibition of ROS mitigated the effects of UVB irradiation on ROS production, cell apoptosis, forkhead box protein 3a (Foxo3a) phosphorylation, and TRIM69 expression. Additionally, Foxo3a overexpression significantly enhanced TRIM69 promoter activity, whereas Foxo3a knockdown had the opposite effect. In conclusion, we provide the first demonstration that Foxo3a is a potential transcription factor for TRIM69, and TRIM69 induces p53 ubiquitination. These results suggest that the Foxo3a/TRIM69/p53 regulatory network may be involved in cataract formation.

Orai3 exacerbates apoptosis of lens epithelial cells by disrupting calcium homeostasis in diabetic cataract

Orai3 exacerbates apoptosis of lens epithelial cells by disrupting calcium homeostasis in diabetic cataract

A novel mutation of PANK4 causes autosomal dominant congenital posterior cataract.

Though many mutations have been identified to be associated with the occurrence of congenital cataract, pathogenic loci in some affected families are still unknown. Clinical data and genomic DNA were collected from a four-generation Chinese family. Candidate mutations were independently verified for cosegregation in the whole pedigree. Linkage analysis showed that the disease-causing mutation was located between 1p36.21 and 1p36.33. Analysis of the whole-exome sequencing data combined with linkage analysis identified a novel pathogenic variant (g.2451906C>T) at intron 4 of Pantothenate kinase 4 (PANK4 protein, PANK4 gene) in 1p36.32|606162. This variant showed complete cosegregation with the phenotype in the pedigree. The mutation was not detected in 106 normal controls nor in 40 sporadic congenital cataract patients. The mutation was demonstrated to significantly reduce the expression of the PANK4 protein level in the blood of cataract patients than that in normal individuals by ELISA. Pank4-/- mice showed a cataract phenotype with increased numbers of apoptotic lens epithelial cells, fiber cell aggregation, and significant mRNA variation of crystallin family members. Thus, the association of a new entity of an autosomal dominant cataract with mutations in PANK4, which influences cell proliferation, apoptosis of lens epithelial cells, crystallin abnormalities, and fiber cell derangement, subsequently induces cataract.

Oxyresveratrol protects human lens epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis by activation of Akt/HO-1 pathway

Oxidative stress induced by hydrogen peroxide (H2O2) triggers human lens epithelial cell (HLEC) apoptosis and initiates cataract formation. Oxyresveratrol (Oxy) was reported to possess antioxidant and free radical scavenging activities. Herein, we investigated the effects of Oxy on H2O2-induced oxidative stress and apoptosis in HLECs and the associated mechanisms. Cell viability was detected by MTT assay. The oxidative damage was assessed by measuring the activities of superoxide dismutases-1 (SOD-1), catalase (CAT), glutathione reductase (GSH), and malondialdehyde (MDA). Apoptosis was analyzed by flow cytometry analysis. The changed expressions of heme oxygenase-1 (HO-1) and protein kinase B (Akt) pathways were evaluated by qRT-PCR and western blot. We found that exposure to H2O2 dose-dependently reduced cell viability, and induced oxidative stress and apoptosis in HLECs, which were reversed by pretreatment with Oxy. Oxy increased p-Akt and HO-1 expressions in H2O2-stimulated HLECs. Akt and HO-1 expressions form a regulatory axis and Oxy activated the Akt/HO-1 pathway in H2O2-stimulated HLECs. Inhibition of the Akt/HO-1 pathway by LY294002 or ZnPP attenuated the effects of Oxy on oxidative stress and apoptosis in H2O2-stimulated HLECs. In conclusion, Oxy protected H2O2-induced oxidative stress and apoptosis through activating the Akt/HO-1 pathway, suggesting the protective effect of Oxy against H2O2-induced cataract.

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.

MiR-326 antagomir delays the progression of age-related cataract by upregulating FGF1-mediated expression of betaB2-crystallin

Age-related cataract, the most common cause of blindness worldwide, has been found closely associated with β-crystallin B2 (βB2 or CRYBB2). MicroRNAs (miRNAs) are the primary epigenetic regulators important for various biological processes. However, the role of miRNAs in the progression of lens cataract remains to be elucidated. In this study, we found a novel signal cascade miR-326–fibroblast growth factor 1 (FGF1)–βB2 modulating the progression of lens cataract. In brief, miR-326 exacerbated but its antagomirs attenuated H2O2-induced apoptosis of HLEC-B3 human lens epithelial cells. Dual-luciferase reporter assay and Western blot showed that miR-326 inhibited FGF1 expression by directly targeting its mRNA 3′-UTR. Consistent with this result, miR-326 antagomir enhanced FGF1 protein level. In addition to FGF1, miR-326 antagomir also enhanced βB2 expression and this enhancement was abolished by transfection of HLEC-B3 cells with FGF1 shRNA. These data demonstrated that miR-326 antagomir increased βB2 expression via upregulating FGF1, which was further confirmed by the studies in a rat model of selenite-induced cataract. This work suggests that miR-326 antagomir might be a promising candidate to prevent progression of age-related cataract.

MicroRNA-124 Prevents H2O2-Induced Apoptosis and Oxidative Stress in Human Lens Epithelial Cells via Inhibition of the NF-κB Signaling Pathway

Aim: To investigate the regulation of microRNA-124 ­(miRNA-124) on NF-κB pathway from H₂O₂-induced apoptosis and oxidative stress in human lens epithelial cells (hLEC). Methods: The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to detect hLEC ­viability. HLECs were divided into Blank, H₂O₂, mimics (miRNA-124 mimics) + H₂O₂, NC+ H₂O₂, pyrrolidine dithiocarbamate (PDTC; NF-κB signaling pathway inhibitor) + H₂O₂, and inhibitors (miRNA-124 inhibitors) + PDTC + H₂O₂ groups. Quantitative real-time polymerase chain reaction and Western blot were employed to detect mRNA and protein expressions, Dichloro-dihydro-fluorescein diacetate to measure reactive oxygen species (ROS) production, and AnnexinV-FITC/PI staining to determine cell apoptosis. The mitochondrial membrane potential (MMP) was detected by fluorescence probe JC-1. Results: The H₂O₂-induced hLEC showed reductions in cell viability with decreased miRNA-124 but increased p-p65 in a dose-/time-dependent manner. Furthermore, ROS production, malondialdehyde content, Bax and Caspase-3 expressions, and cell apoptosis were elevated in H₂O_2-induced hLEC, whereas the activities of superoxide dismutase and glutathione peroxidase, Bcl-2 expression, MMP, as well as the mitochondrial energy metabolism genes were reduced. Additionally, miRNA-124 mimics and PDTC both decreased the p-p65 and reversed the cytotoxicity in H₂O₂-induced hLEC. Conclusion: MiRNA-124 prevents H₂O₂-induced oxidative stress and apoptosis in hLEC through suppressing the activation of the NF-κB pathway.

Effects of UV on apoptotic factors in lens epithelial cells of an animal model.

Effect of ultraviolet (UV) on the apoptotic factors p53, Bax and Bcl-2 in lens epithelial cells (LECs) was explored. Sixty SD rats were randomly divided into control group (n=12) and model group (n=48). Rats in model group were used to construct UV radiation eyeball models. Twelve rats in model group and 3 rats in control group were sacrificed at day 1, 3, 5 and 7, and the expression of p53, Bax and Bcl-2 in LECs was detected by RT-qPCR. There was no significant difference in p53 expression between model and control group on day 1 (P>0.05), while expression level of p53 was significantly higher in model than in control group at day 3, 5 and 7 (P<0.05), and the highest expression level was found on day 7 (P<0.05). Relative expression level of Bax in model group was significantly higher than that in control group (P<0.05), and the highest expression level was found on day 7 (P<0.05). On day 1, expression level of Bcl-2 in model group was significantly lower than that in control group (P<0.05), but it began to rise on day 3 but did not differ significantly from the control group (P>0.05), and was significantly higher in model than in control group on day 5 and 7 (P<0.05), and the highest expression level was observed on day 7 (P<0.05). Linear correlation analysis showed that there was a positive correlation between p53, Bax and Bcl-2 expression and UV exposure time in LEC (r=0.82, 0.90, 0.95, P<0.05). Expression levels of p53, Bax, and Bcl-2 in LEC of rats are positively correlated with the time after UV irradiation, suggesting that UV could induce apoptosis of LEC by promoting p53, Bax and Bcl-2 expression.

Ketamine delays progression of oxidative and damaged cataract through regulating HMGB-1/NF-κB in lens epithelial cells

Objective: Lens epithelial cell (LEC) membrane damage is one of the pathogenesis of cataract. High mobility group box-1 (HMGB-1) and nuclear factor-κB (NF-κB) play vital roles in a variety of diseases, such as inflammation. Ketamine has numerous pharmacological effects that can inhibit inflammation. However, its role in cataract rats LECs has not yet been elucidated.Materials and methods: LECs were isolated from SD rats and cultured in vitro. The cells were randomly divided into three groups, including the control group, cataract model group induced by H2O2, and ketamine group treated by 10 mM ketamine under H2O2 environment. LECs proliferation was assessed by MTT assay. LECs apoptosis was evaluated by Caspase-3 activity detection. NF-κB mRNA and protein expressions were tested by real-time PCR and Western blot. HMGB-1 expressions in cells and supernatant were detected by real-time PCR and ELISA. TNF-α and IL-1β secretions were detected by ELISA.Results: In H2O2 model group, the LECs proliferation was significantly inhibited, the caspase-3 activity significantly increased, HMGB-1 mRNA and secretion significantly enhanced, NF-κB mRNA and protein levels significantly elevated, compared to the Control group (p < .05). While the TNF-α and IL-1β secretions significantly up-regulated in H2O2 model group compared to the Control group (p < .05). Ketamine significantly promoted the LECs proliferation, significantly reduced the caspase-3 activity, and significantly declined the HMGB expression compared to H2O2 model group (p < .05). The NF-κB mRNA and protein levels were significantly decreased, TNF-α and IL-1β secretions were significantly decreased in the Ketamine group compared with the model group (p < .05).Conclusions: Ketamine delays the progression of oxidative and damaged cataract by regulating HMGB-1/NF-κB expression, inhibiting TNF-α, IL-1β, and apoptosis, and promoting cell proliferation.