OBJECTIVE: Age-related cataract (ARC) is the most common cause of visual impairment and blindness in older adults. However, the role of CUL4B in the ARC remains unclear. Therefore, we investigated CUL4B expression and its effects on apoptosis.
METHODS: CUL4B expression levels were detected by a quantitative real-time polymerase chain reaction from the anterior lens capsules of patients with ARC and HLE-B3 cells treated with different concentrations of H2O2. CUL4B expression was silenced by siRNA transfection to evaluate apoptosis. CUL4B and apoptotic proteins B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), caspase-3, cleaved caspase-3, Bax, Bak, and Bid were assessed using western blot analysis. Apoptosis was monitored using the TUNEL assay.
RESULTS: CUL4B expression was downregulated in the anterior lens capsules (P < 0.0001) and H2O2-treated HLE-B3 cells (P = 0.0405). CUL4B protein levels were significantly lower in 100 µmol/L (P = 0.0012) and 200 µmol/L (P = 0.0041) H2O2-treated HLE-B3 cells than in the untreated cells. CUL4B expression was significantly knocked down at the mRNA (P = 0.0043) and protein levels (P = 0.0002) in HLE-B3 cells. Bcl-2 (P = 0.0199), Mcl-1 (P = 0.0042), and caspase-3 (P = 0.0142) were significantly downregulated, whereas cleaved caspase-3 (P = 0.0089) and Bak (P = 0.009) were significantly upregulated in the knockdown group. The TUNEL assay showed a greater induction of apoptosis.
CONCLUSIONS: CUL4B downregulation promotes the apoptosis of lens epithelial cells. Our study may help in understanding the role of CUL4B in ARC pathogenesis.

The knowledge on responses of human lens epithelial cells (HLECs) to ionizing radiation exposure is important to understand mechanisms of radiation cataracts that are of concern in the field of radiation protection and radiation therapy. However, biological effects in HLECs following protracted exposure have not yet fully been explored. Here, we investigated the temporal kinetics of γ-H2AX foci as a marker for DNA double-strand breaks (DSBs) and cell survival in HLECs after exposure to photon beams at various dose rates (i.e., 150 kVp X-rays at 1.82, 0.1, and 0.033 Gy/min, and 137Cs γ-rays at 0.00461 Gy/min (27.7 cGy/h) and 0.00081 Gy/min (4.9 cGy/h)), compared to those in human lung fibroblasts (WI-38). In parallel, we quantified the recovery for DSBs and cell survival using a biophysical model. The study revealed that HLECs have a lower DSB repair rate than WI-38 cells. There is no significant impact of dose rate on cell survival in both cell lines in the dose-rate range of 0.033-1.82 Gy/min. In contrast, the experimental residual γ-H2AX foci showed inverse dose rate effects (IDREs) compared to the model prediction, highlighting the importance of the IDREs in evaluating radiation effects on the ocular lens.

Posterior Capsule Opacification (PCO), the most frequent complication of cataract surgery, is caused by the infiltration and proliferation of lens epithelial cells (LECs) at the interface between the intraocular lens (IOL) and posterior lens capsule (PLC). According to the \"no space, no cells, no PCO\" theory, high affinity (or adhesion force) between the IOL and PLC would decrease the IOL: PLC interface space, hinder LEC migration, and thus reduce PCO formation. To test this hypothesis, an in vitro hemisphere-shaped simulated PLC (sPLC) was made to mimic the human IOL: PLC physical interactions and to assess their influence on LEC responses. Three commercially available IOLs with different affinities/adhesion forces toward the sPLC, including Acrylic foldable IOL, Silicone IOL, and PMMA IOL, were used in this investigation. Using the system, the physical interactions between IOLs and sPLC were quantified by measuring the adhesion force and interface space using an adhesion force apparatus and Optical Coherence Tomography, respectively. Our data shows that high adhesion force and tight binding between IOL and sPLC contribute to a small interface space (or \"no space\"). By introducing LECs into the in vitro system, we found that, with small interface space, among all IOLs, acrylic foldable IOLs permitted the least extent of LEC infiltration, proliferation, and differentiation (or \"no cells\"). Further statistical analyses using clinical data revealed that weak LEC responses are associated with low clinical PCO incidence rates (or \"no PCO\"). The findings support that the in vitro system could simulate IOL: PLC interplays and predict IOLs\' PCO potential in support of the \"no space, no cells, no PCO\" hypothesis.

Our study mainly analyzed the mechanism of C/EBP homologous protein (CHOP) and its interacting protein Nupr1 on endoplasmic reticulum stress (ERS) induced lens epithelial cells (LEC) apoptosis. Cell proliferation was detected by CCK-8. Apoptosis was detected by flow cytometry and TUNEL. Nupr1 expression was detected by RT-qPCR. The expressions of CHOP, Nupr1, apoptosis-related protein, and ERS-related protein were detected by Western blot. DCFH-DA probe was used to detect cell ROS. The SOD, GSH-PX, and MDA contents were detected by the kit. Co-IP was used to detect the interaction between CHOP and Nupr1. The morphology of the lens was detected by HE staining. The result shows that Tunicamycin (TU) can induce endoplasmic reticulum stress and apoptosis in LEC in a concentration-dependent manner. TU induction leads to the occurrence of CHOP nuclear translocation. Overexpression of CHOP can further enhance the inhibitory effect of TU on LEC proliferation and the promotion of apoptosis, while knockdown of CHOP has the opposite effect. CHOP and Nupr1 are interacting proteins, and knockdown of Nupr1 or addition of Nupr1 inhibitor ZZW-115 can reverse the effects of TU and overexpression of CHOP, respectively. It has been observed in animal experiments that treatment with oe-CHOP can further aggravate the pathological lesions of the rat lens, while ZZW-115 can reverse the effect of oe-CHOP to a certain extent and improve the lesions of the rat lens. Overall, CHOP interacts with Nupr1 to regulate apoptosis caused by ERS and mediate cataract progression in rats, and this study provides a new potential therapeutic target for the treatment of cataract.

Fibrotic cataracts, including anterior subcapsular cataract (ASC) as well as posterior capsule opacification (PCO), are a common vision-threatening cause worldwide. Still, little is known about the underlying mechanisms. Here, we demonstrate a miRNA-based pathway regulating the pathological fibrosis process of lens epithelium.
Gain- and loss-of-function approaches, as well as multiple fibrosis models of the lens, were applied to validate the crucial role of two miR-1225 family members in the TGF-β2 induced PCO model of human LECs and injury-induced ASC model in mice.
Both miR-1225-3p and miR-1225-5p prominently stimulate the migration and EMT process of lens epithelial cells (LECs) in vitro as well as lens fibrosis in vivo. Moreover, we demonstrated that the underlying mechanism for these effects of miR-1225-5p is via directly targeting Keap1 to regulate Keap1/Nrf2 signaling. In addition, evidence showed that Keap1/Nrf2 signaling is activated in the TGF-β2 induced PCO model of human LECs and injury-induced ASC model in mice, and inhibition of the Nrf2 pathway can significantly reverse the process of LECs EMT as well as lens fibrosis.
These results suggest that blockade of miR-1225-5p prevents lens fibrosis via targeting Keap1 thereby inhibiting Nrf2 activation. The \'miR-1225-Keap1-Nrf2\' signaling axis presumably holds therapeutic promise in the treatment of fibrotic cataracts.

UNASSIGNED: Cataract is the most common cause of blindness worldwide, a visual disorder caused by a clouded lens that seriously affects People\'s Daily lives. Age-related cataract (ARC) is the most common type of cataract due to long-term combined effects of many factors, and its pathogenesis is varied. At present, the surgery is the main treatment for cataracts, but it is still limited to the prevention, treatment of early cataracts and the postoperative complications care. While, its drug treatments are still in the stage of exploration and research. Traditional Chinese Medicine (TCM), a unique resource in China, is conceived under the guidance of traditional Chinese medicine theory and has little toxicity and side effects, but it has made great progress in the treatment and prevention of ARC.
OBJECTIVE: This review presents an overview of the pathogenesis of ARC in both traditional and modern medicines and summarizes the history and therapeutic effect of TCM on ARC including their formula, crude drugs and active components, and also the other auxiliary methods.
METHODS: A number of recognized databases like SciFinder, PubMed, Science Direct, Google Scholar, and China National Knowledge Infrastructure (CNKI) were extensively explored by using keywords and phrases such as \"cataract\", \"age-related cataract\", \"traditional medicine\", \"ethnopharmacology\", \"herbs\", \"medicinal plants\", or other relevant terms, and the plants/phytoconstituents that are evaluated in the models of age-related cataract. As well as the current TCM adjuvant therapy used in the clinical treatment were summarized.
RESULTS: TCM revealed to plays an active role in treating age-related cataract, via multi-pathway and multi-target, and can treat or delay ARC by inhibiting abnormal glucose metabolism, antioxidant damage, inhibiting LEC apoptosis, and so on, which is in concordance with the good effects of the global use of TCM in clinical application. Concerning the early prevention and treatment of cataract and postoperative complications, TCM and auxiliary methods remain to achieve better clinical effects.
CONCLUSIONS: ARC belongs to the category of \"Yuan Yi Nei Zhang\" in TCM theory, showing that there are many causes of ARC including aging, and kidney-yang, spleen, sperm and blood deficiencies. At the same time, the viscera gradually decline, as well as yin or yang progressively become weak, especially in the elder people. So, TCM could be mainly based on liver, kidney, and spleen syndrome differentiation, personalizing diagnosis and treatment, following multiple targets, regulating fundamentally yin and yang, and thus justifying the advantages of Chinese medicine in the prevention and treatment of ARC.

To identify the inactive genes in cataract lenses and explore their function in lens epithelial cells (LECs).
Lens epithelium samples obtained from both age-related cataract (ARC) patients and normal donors were subjected to two forms of histone H3 immunoprecipitation: H3K9ac and H3K27me3 chromatin immunoprecipitation (ChIP), followed by ChIP-seq. The intersection set of \"active genes in normal controls\" and \"repressed genes in cataract lenses\" was identified. To validate the role of a specific gene, ETV1, within this set, quantitative polymerase chain reaction (qPCR), western blot, and immunofluorescence were performed using clinical lens epithelium samples. Small interference RNA (siRNA) was utilized to reduce the mRNA level of ETV1 in cultured LECs. Following this, transwell assay and western blot was performed to examine the migration ability of the cells. Furthermore, RNA-seq analysis was conducted on both cell samples with ETV1 knockdown and control cells. Additionally, the expression level of ETV1 in LECs was examined using qPCR under H2O2 treatment.
Six genes were identified in the intersection set of \"active genes in normal controls\" and \"repressed genes in ARC lenses\". Among these genes, ETV1 showed the most significant fold-change decrease in the cataract samples compared to the control samples. After ETV1 knockdown by siRNA in cultured LECs, reduced cell migration was observed, along with a decrease in the expression of β-Catenin and Vimentin, two specific genes associated with cell migration. In addition, under the oxidative stress induced by H2O2 treatment, the expression level of ETV1 in LECs significantly decreased.
Based on the findings of this study, it can be concluded that ETV1 is significantly reduced in human ARC lenses. The repression of ETV1 in ARC lenses appears to contribute to the disrupted differentiation of lens epithelium, which is likely caused by the inhibition of both cell differentiation and migration processes.

Posterior capsule opacification (PCO) is the most common postoperative complication of cataract surgery. Transforming growth factor-β (TGF-β) is related to epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) that is proven to induce PCO formation in clinical and experimental studies. In this study, CRISPR sequences targeting exon of TGF-βRII were knocked out with lentiviral transfection in LECs. Rabbits\' PCO model was established and recombinant adeno-associated virus (AAV) for transferring the gRNA of TGF βRII were intravitreally injected. SgRNA inhibited TGF-βRII expression and human LECs proliferation. In TGF-βRII knockout group, LECs motility and migration were suppressed, N-cadherin and vimentin expressions were significantly decreased, whereas E-cadherin was increased. The animal model showed that TGF-βRII knockout in vivo was effective in suppressing PCO. The current study suggested that the CRISPR/Cas9 endonuclease system could suppress TGF-βRII secretion, which participates in the EMT procedure of LECs in vitro and PCO in vivo. These findings might provide a new gene-editing approach and insight into a novel therapeutic strategy for PCO.

To observe the effects of oxidative stress on vascular endothelial growth factor (VEGF) and connections of lens epithelial cells.
Human lens epithelium of patients with age-related cataract (ARC), both SRA01/04 cells and whole mice lens stimulated by H2O2 were employed. VEGF in human aqueous humor of ARC-patients and the supernatant of SRA01/04 cells was determined by ELISA. The expressions of VEFG in human lens epithelium were detected by immunofluorescence staining. Multiple linear regression analysis and spearman rank-order correlation were used to determine the associations between VEGF and parameters of ARC individuals. In H2O2-induced SRA01/04 cells, Catalase (CAT), PP1 (inhibitor of c-Src kinase) and Avastin (VEGF antibody) were used to inhibit the effects of H2O2, activation of c-Src kinase and VEGF, which were detected by Western blot. The alterations of ZO-1 and N-cadherin were tested by immunofluorescence staining and Western blot. In H2O2-induced whole lens, the changes of opacification area in different treatment of inhibitors were observed.
The secretion of VEGF in aqueous humor and expression of VEGF in the lens epithelium of ARC patients increased significantly with age. In H2O2-induced SRA01/04 cells, the VEGF in the supernatant was increased with the culture duration and the dose of H2O2. The expressions of p-Src418 and VEGF were also up-regulated, whereas the expressions of ZO-1 and N-cadherin were down-regulated. CAT effectively prevented these changes induced by H2O2, while PP1 inhibited not only p-Src418 but also up-regulation of VEGF, Avastin partially inhibited VEGF up-regulation. Both PP1 and Avastin prevented down-regulation of ZO-1 and N-cadherin, respectively, but Avastin combined with PP1 had no significant synergistic effects. In H2O2-induced cataract, CAT prevented development of opacification area effectively, and PP1 and Avastin did partially.
Oxidative stress disrupts connections of lens epithelial cells by activating c-Src/VEGF, inhibiting which may prevent cataract.

Accumulated evidence has shown that microRNAs (miRNAs) are closely related to the pathogenesis and progression of senile cataracts. Here we investigate the effect of miR-29a-3p in cataractogenesis and determined the potential molecular mechanism involved.
In this study, we constructed a selenite cataract model in rats and obtained the miRNAs related to cataracts by whole transcriptome sequencing. To investigate the effect and mechanism of miR-29a-3p on cataracts, we performed several in vivo and in vitro experiments, including CCK8 assay, flow cytometry, luciferase reporter assay, Edu assay, and western blot analysis.
Sequencing data showed downregulation of miR-29a-3p in rats with selenite cataracts. Down-regulation of miR-29a-3p could promote lens epithelial cells (SRA01/04) proliferation and inhibit cell apoptosis, and miR-29a-3p silence could inhibit the development of cataracts. Additionally, CAND1 was a direct target gene for miR-29a-3p.
These data demonstrate that miR-29a-3p inhibits apoptosis of lens epithelial cells by regulating CAND1, which may be a potential target for senile cataracts.