Comparative study of FLACS vs conventional phacoemulsification for cataract patients with high myopia.

PURPOSE: To compare the short-term changes in cornea, retina and choroid of femtosecond laser-assisted cataract surgery (FLACS) with conventional phacoemulsification (CPS) in high myopia patients with cataract. SETTING: Affiliated Hospital of Nantong University, Jiangsu Province, China. DESIGN: Prospective single-center study. METHOD: Demographics, ocular clinical features, ultrasound power, absolute phacoemulsification time, and effective phacoemulsification time were recorded for each patient. Endothelial cell density (ECD), central corneal thickness (CCT), best corrected distance visual acuity (CDVA), intraocular pressure(IOP), center foveal thickness(CFT), subfoveal choroidal thickness (SFCT) and choroidal vascularity index (CVI) were evaluated preoperatively and at 1 week, 1 month, and 3months postoperatively. Intraoperative parameters and intraoperative /postoperative complications were recorded. RESULTS: Ninety-seven eyes (46 eyes and 51 eyes in the FLACS and CPS groups, respectively) were included and analyzed. Cumulative dissipated energy was lower in FLACS group compared with CPS group (P ＜0.05). The increase in CCT was significantly lower in the FLACS group compared with the CPS group at 1week and 1month (P ＜0.05). CDVA and IOP were similar in both groups at the final visit (P > 0.05). The ECD decreased was lower among CPS patients compare with FLACS patients. CFT, SFCT and CVI increase in both groups but were increase more in CPS group with high myopia patients. No serious complications occurred in either group. CONCLUSIONS: FLACS is a more safety and effective in cataract patients with high myopia. It has advantages in effectively reducing EPT and promoting faster recovery of the cornea, macular and choroidal thickness.

Comprehensive analysis of the miRNA-mRNA regulatory network involved in spontaneous recovery of an H2O2-induced zebrafish cataract model.

OBJECTIVE: To identify the hub miRNAs and mRNAs contributing to the spontaneous recovery of an H2O2-induced zebrafish cataract model. METHODS: Zebrafishes were divided into three groups, i.e., Group A, which included normal control fish (day 0), and Groups B and C, where fish were injected with 2.5% hydrogen peroxide into the anterior chamber and reared for 14 and 30 days, respectively. Fish eyes were examined by stereomicroscope photography and optical coherence tomography (OCT). RNA profiles of fish lenses were detected by RNA sequencing. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) were identified among three groups. The DEGs and DEmiRs, which changed in opposite positions between "B vs. A" and "C vs. B" were defined as ODGs (opposite positions changed DEGs) and ODmiRs (opposite positions changed DEmiRs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis were carried out by R language. The protein-protein interaction network (PPI) was constructed using STRING. Potential targets of miRNAs were obtained using miRanda. miRNA-mRNA networks were constructed by Cytoscape. RESULTS: The fish lens opacity formed on day 14 and recovered to transparent on day 30 after injection. Compared to group B, 1366 DEGs and 54 DEmiRs were identified in group C. "C vs. B" DEGs were enriched in gene clusters related to development and oxidative phosphorylation. Target genes of DEmiRs were enriched in clusters such as development and cysteine metabolism. Among three groups, 786 ODGs and 27 ODmiRs were identified, and 480 ODGs were predicted as targets of ODmiRs. Target ODGs were enriched in pathways related to methionine metabolism, ubiquitin, sensory system development, and structural constituents of the eye lens. In addition, we established an ODmiRs-ODGs regulation network. CONCLUSION: We identified several hub mRNAs and altered miRNAs in the formation and reversal of zebrafish cataracts. These hub miRNAs/mRNAs could be potential targets for the non-surgical treatment of ARC.

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.

Lens epithelium cell ferroptosis mediated by m6A-lncRNA and GPX4 expression in lens tissue of age-related cataract.

BACKGROUND: In the present study, we explored the role of N6-methyladenosine (m6A) modification of long non-coding RNAs (lncRNAs) and its association with ferroptosis in lens epithelium cells (LECs) of age-related cataract (ARC). METHODS: Through m6A RNA immunoprecipitation sequencing (m6A-RIP-seq) and RNA sequencing (RNA-seq), we identified m6A mediated and differentially expressed lncRNAs (dme-lncRNAs) in ARC patients. Based on bioinformatics analysis, we selected critical dme-lncRNAs and pathways associated with ARC formation to reveal their potential molecular mechanisms. The downregulation of glutathione peroxidase 4 (GPX4), a key component of ferroptosis, was confirmed by real-time RT-PCR (RT-qPCR) and Western blotting in age-related cortical cataract (ARCC) samples. Transmission electron microscopy was used to assess the change in mitochondrial in LECs. RESULTS: The analysis revealed a total of 11,193 m6A peaks within lncRNAs, among which 7043 were enriched and 4150 were depleted. Among those, lncRNA ENST00000586817(upstream of the GPX4 gene) was not only significantly upregulated in the LECs of ARCC but also potentially augmented the expression of GPX4 through a cis mechanism. The expression of m6A-modified lncRNA (ENST00000586817) was correlated with that of GPX4 and was downregulated in ARC patients. The TEM results indicated significant mitochondrial changes in ARCC samples. GPX4 downregulation enhanced LEC ferroptosis and decreased viability via RSL3 in SRA01/04 cells. CONCLUSIONS: Our results provide insight into the potential function of m6A-modified lncRNAs. M6A-modified lncRNA ENST00000586817 might regulate the expression of GPX4 by a cis mechanism and be implicated in ferroptosis in ARCs.

Evaluation of Fundus Function in Mature Cataract Patients by Visual Electrophysiology.

Purpose: To explore the value of visual electrophysiology in evaluating the fundus function of mature cataract patients. Methods: 124 mature cataract patients (153 eyes) were examined before cataract surgery; the examinations included best corrected visual acuity (BCVA), pattern visual evoked potential (PVEP), full-field electroretinogram (ffERG), and multifocal electroretinogram (mfERG). According to the postoperative fundus conditions, the subjects were divided into two groups: the no fundus disease group and the fundus disease group. Approximately one month after the operation, BCVA was measured, and visual electrophysiology was performed on subjects who had a stable fundus condition and had not received treatment for fundus disease. Results: One month after cataract surgery, BCVA ≤ 0.3 logMAR was found in 60 eyes (96.8%) without fundus disease and 59 eyes (64.8%) with fundus disease. Compared with the group without fundus disease, the preoperative electrophysiological examination of the group with fundus disease showed that the amplitude of ffERG waves and the amplitude density of the P1 wave in the 2nd to 5th rings of mfERG were decreased (all P < 0.05). ffERG and mfERG can be used for differential diagnosis of fundus disease (all P < 0.05), while PVEP has no significant diagnostic value for fundus disease (all P > 0.05). In the group without fundus disease, the amplitude of the PVEP 15' P100 wave and the amplitude of dark-adapted (DA) 0.01 b-wave, DA 3.0 a-wave, and DA 10.0 a-wave were negatively correlated with postoperative logMAR BCVA (all P < 0.05). In the group with fundus disease, the amplitude of PVEP and ffERG and the amplitude density of mfERG were negatively correlated with postoperative logMAR BCVA (all P < 0.05). In the eyes of cortical cataracts, some parameters of PVEP, ffERG, and mfERG were significantly different before and after surgery. In the eyes of nuclear cataracts, some parameters of ffERG and mfERG were significantly different before and after surgery. In the eyes of posterior subcapsular cataracts, some parameters of PVEP and ffERG were significantly different before and after surgery. Conclusions: ffERG and mfERG can be used to detect fundus disease in mature cataract patients. The preoperative visual electrophysiological examination has high clinical value in predicting postoperative vision of mature cataract patients with fundus disease. Different types of cataracts have different effects on electrophysiological examination results. When interpreting the electrophysiological report, it is necessary to consider the existence of cataracts. This trial is registered with 2019-K068.

Comparison of three fundus inspection methods during phacoemulsification in diabetic white cataract.

AIM: To investigate whether Wild Field Imaging System (WFIS SW-8000), 25G endoilluminator, and intraoperative optical coherence tomography (iOCT) can perform real-time screening and diagnosing in patients with suspicious diabetic retinopathy (DR) during phacoemulsification, especially in cases of white cataract. METHODS: A cross-sectional study was carried out. A total of 204 dense diabetic cataractous eyes of 204 patients with suspected DR treated from April 2020 to March 2021 were included. Phacoemulsification combined with intraocular lens implantation was performed. Following the removal of the lens opacity, the 25G endo-illuminator, fundus photography, and iOCT were performed successively. Optical coherence tomography (OCT) and/or fundus fluorescein angiography (FFA) were used to verify the fundus findings postoperatively. Intraoperative and postoperative results were compared to verify the accuracy of intraoperative diagnosis in each group. RESULTS: Intraoperative and postoperative examinations revealed 58 and 62 eyes with DR, respectively (positive rate, 28.43% and 30.39%, respectively). During the phacoemulsification, WFIS SW-8000 detected 44 eyes with DR (the detection rate, 70.97%); 25G endo-illuminator found 56 eyes with DR (the detection rate, 90.32%); iOCT found 46 eyes with DR (the detection rate, 74.19%); and 58 eyes with DR were found by combining the three methods (the detection rate, 93.55%). There were statistically significant differences in the diagnostic sensitivity for DR among the methods (χ2=16.36, P=0.001). CONCLUSION: WFIS SW-8000, 25G endo-illuminator, iOCT, and especially their combination can be used to inspect the fundus and detect DR intraoperatively; they are helpful for the timely diagnosis and treatment of DR in patients with dense cataract.

Pro-Fibrotic Role of Interleukin-4 in Influencing Idiopathic Epiretinal Membrane in Cataract Patients: Analysis From Clinical-Experimental Approaches.

Purpose: To evaluate the role of interleukin-4 in influencing idiopathic epiretinal membrane (iERM) formation and early progression post cataract surgery (PCS) from clinical and experimental perspectives. Methods: We quantified levels of IL-4 in aqueous humor (AH) samples from 22 iERM patients and 31 control subjects collected before and 20 hours after cataract surgeries using ELISA. After a 3-month follow-up, the association between IL-4 levels and iERM progression measurements was identified. In addition, in vitro studies were conducted to investigate the effects of IL-4 on primary rat retinal Müller glia proliferation, migration, and glial-mesenchymal transition (GMT). Results: Concentrations of IL-4 were significantly higher in preoperative AH samples from iERM patients versus controls (P = 0.006). Postoperatively, although IL-4 levels were elevated in both groups compared to their respective preoperative levels, they were even more obviously so in the iERM group (P < 0.001). Multivariate linear regression analyses revealed that, postoperatively, IL-4 level elevation was positively associated with macular volume and thickness increase (both P < 0.05) in iERM patients. However, no correlations were observed between IL-4 level (changes) and clinical characters in the controls. In vitro studies demonstrated that IL-4 promoted Müller glia proliferation and migration and increased the expression of GMT-related markers in a manner independent of transforming growth factor-ß1 (TGF-ß1). Conclusions: IL-4 plays a crucial pro-fibrotic role in iERM formation and early progression 3 months PCS possibly by stimulating Müller glia proliferation, migration, and GMT in a TGF-ß1-independent manner. Translational Relevance: The current study suggests the potential of IL-4 as a novel therapeutic target for iERM.

ATP-P2X7R-mediated microglia senescence aggravates retinal ganglion cell injury in chronic ocular hypertension.

BACKGROUND: Dysfunction of microglia during aging affects normal neuronal function and results in the occurrence of neurodegenerative diseases. Retinal microglial senescence attributes to retinal ganglion cell (RGC) death in glaucoma. This study aims to examine the role of ATP-P2X7R in the mediation of microglia senescence and glaucoma progression. METHODS: Forty-eight participants were enrolled, including 24 patients with primary open-angle glaucoma (POAG) and age-related cataract (ARC) and 24 patients with ARC only. We used ARC as the inclusion criteria because of the availability of aqueous humor (AH) before phacoemulsification. AH was collected and the adenosine triphosphate (ATP) concentration was measured by ATP Assay Kit. The chronic ocular hypertension (COH) mouse model was established by microbead occlusion. Microglia were ablated by feeding PLX5622 orally. Mouse bone marrow cells (BMCs) were prepared and infused into mice through the tail vein for the restoration of microglia function. Western blotting, qPCR and ELISA were performed to analyze protein and mRNA expression in the ocular tissue, respectively. Microglial phenotype and RGC survival were assessed by immunofluorescence. The mitochondrial membrane potential was measured using a JC-1 assay kit by flow cytometry. RESULTS: ATP concentrations in the AH were increased in older adults and patients with POAG. The expression of P2X7R was upregulated in the retinal tissues of mice with glaucoma, and functional enrichment analysis showed that P2X7R was closely related to cell aging. Through in vivo and in vitro approaches, we showed that pathological activation of ATP-P2X7R induced accelerated microglial senescence through impairing PTEN-induced kinase 1 (PINK1)-mediated mitophagy, which led to RGC damage. Additionally, we found that replacement of senescent microglia in COH model of old mice with BMCs from young mice reversed RGC damage. CONCLUSION: ATP-P2X7R induces microglia senescence by inhibiting PINK1-mediated mitophagy pathway. Specific inhibition of ATP-P2X7R may be a fundamental approach for targeted therapy of RGC injury in microglial aging-related glaucoma.

DCLRE1A Contributes to DNA Damage Repair and Apoptosis in Age-Related Cataracts by Regulating the lncRNA/miRNA/mRNA Axis.

PURPOSE: Age-related cataract (ARC) is associated with the deregulation of transcription and defects in DNA repair in lens epithelial cells (LECs). DCLRE1A acted in DNA interstrand cross-links pathway to improve DNA replication and transcription. The aim of this study was to examined the further regulatory effect on DCLRE1A in the lncRNA-miRNA-mRNA network using a cell model of DCLRE1A overexpression (OE-DCLRE1A) in LECs. METHODS: The expression level of DCLRE1A in ARC tissues and SRA01/04 cells after H2O2 treatment was measured as protein and mRNA by qRT-PCR and Western Blot(WB). CCK8, and TUNEL assays detected the change in cell viability and apoptosis, respectively. Furthermore, Immunofluorescence assays detect the expression of DNA damaged and repair marker proteins after OE-DCLRE1A. The global expression profiles of lncRNAs, miRNAs, and mRNAs were determined using high-throughput sequencing. KEGG and GO enrichment analysis disclose the possible function of differentially expressed (DE) lncRNA, miRNA, and mRNA. RESULTS: The protein and mRNA of DCLRE1A were decreased in the anterior capsule of ARC and SRA01/04 cells treated by H2O2. OE-DCLRE1A improved damaged-DNA repair and enhanced cell viability against apoptosis after H2O2 treatment. Furthermore, we demonstrated the DE-molecules between the OE-DCLRE1A and control groups including 595 DE-lncRNAs, 221 DE-miRNAs, and 4718 DE-mRNAs. Next, bioinformatics analysis not only found that the DE-mRNAs are mainly involved in DNA repair-related signaling pathways after OE-DCLRE1A, but also screened two lncRNA-miRNA-mRNA networks focusing on DNA damage activated by OE-DCLRE1A, which involved 2 lncRNAs, 2 miRNAs, and 53 mRNAs. CONCLUSION: We revealed that DCLRE1A activated the lncRNA/miRNA/DNA-repair network to take part in DNA repair processes, which not only represents a new regulatory mechanism employed by DCLRE1A but also uncovers the screening lncRNA may hold potential therapeutic values in ARC formation. However, these conclusions will need to be confirmed by future studies in vitro and in vivo models.

Hydroxycamptothecin regulates scar formation of the filtration channel under scleral flap by inhibiting the proliferation of scleral fibroblasts.

BACKGROUND: To investigate the inhibitory effect of a hyaluronic acid hydrogel loaded with hydroxycamptothecin (HCPT) on scar formation after filtration surgery in a rabbit model. METHODS: Scleral fibroblasts were isolated and extracted from rabbits' eyes. After treatment with different concentrations of HCPT, cytotoxicity was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and proliferation and extent of apoptosis were analysed using flow cytometry. Hydrogels loaded with different dosages of HCPT were prepared and placed under the scleral flap after the filtration surgery. One day, one week, and two weeks after surgery, follicular, conjunctival, corneal, and anterior chamber inflammation and iris and lens changes were observed. RESULTS: In vitro, compared with cells not treated with HCPT, cells treated with HCPT had decreased survival rate and proliferation, and the apoptosis level increased with increasing HCPT concentrations (p < 0.05). In vivo, the flattening time of filtering blebs in the three groups treated with different dosages of HCPT hydrogel was delayed. The degrees of oedema, inflammation, and bleeding were similar to those observed in the control group. The HCPT hydrogel effectively downregulated the expression of collagen 1 and 3 and tissue inhibitor of metalloproteinase 2 and upregulated the expression of matrix metalloproteinase 2 in a dose-dependent manner. CONCLUSIONS: HCPT significantly inhibited the growth of rabbits' scleral fibroblasts and effectively inhibited scar formation after filtering surgery by accelerating the degradation of extracellular matrix deposition.

Ubiquitination of Ku70 by Parkin promotes apoptosis of lens epithelial cells.

Oxidative damage-triggered apoptosis in lens epithelial cells is considered as a main risk factor in the pathogenesis of age-related cataracts. Ku70 is a key factor in the DNA repair process of double-strand breaks. In the present study, we aimed to investigate the role of Ku70 and its related E3 ubiquitin ligase in lens epithelial cell apoptosis. The levels of Ku70 in the anterior lens capsules of human cataracts and Emory mice were lower compared to controls. H2 O2 treatment resulted in decreased expression of Ku70 through accelerating Ku70 ubiquitination. Parkin, an E3 ubiquitin ligase, could interact with Ku70 and promote the ubiquitination and degradation of this protein. In addition, ubiquitinated Ku70 was regulated by ubiquitin-proteasome, autophagy-lysosome and mitophagy pathways. Ectopic expression of Ku70 protected SRA01/04 cells from H2 O2 -induced apoptosis, whereas silencing Ku70 exhibited the opposite trend. Co-transfected with Parkin non-ubiquitinatable Ku70 mutant could maintain its anti-apoptosis ability, whereas wild-type Ku70 failed. Moreover, Ku70 might facilitate mitochondrial fusion by increasing the expression of Mitofusin 1/2. The present study revealed that Parkin-mediated Ku70 ubiquitination facilitated H2 O2 -induced lens epithelial cell apoptosis through alleviating mitochondrial fusion, which could provide potential targets for age-related cataract treatment.

Age-related cataract: GSTP1 ubiquitination and degradation by Parkin inhibits its anti-apoptosis in lens epithelial cells.

PURPOSE: Oxidative stress-induced apoptosis of lens epithelial cells (LECs) contributes to the pathogenesis of age-related cataract (ARC). The purpose of this research is to underlie the potential mechanism of E3 ligase Parkin and its oxidative stress-associated substrate in cataractogenesis. METHODS: The central anterior capsules were obtained from patients with ARC, Emory mice, and corresponding controls. SRA01/04 cells were exposed to H2O2 combined with cycloheximide (a translational inhibitor), MG-132 (a proteasome inhibitor), chloroquine (an autophagy inhibitor), Mdivi-1 (a mitochondrial division inhibitor), respectively. Co-immunoprecipitation was employed to detect protein-protein interactions and ubiquitin-tagged protein products. Levels of proteins and mRNA were evaluated by western blotting and quantitative RT-PCR assays. RESULTS: Glutathione-S-transferase P1 (GSTP1) was identified as a novel Parkin substrate. Compared with corresponding controls, GSTP1 was significantly decreased in the anterior lens capsules obtained from human cataracts and Emory mice. Similarly, GSTP1 was declined in H2O2-stimulated SRA01/04 cells. Ectopic expression of GSTP1 mitigated H2O2-induced apoptosis, whereas silencing GSTP1 aggregated apoptosis. In addition, H2O2 stimulation and Parkin overexpression could promote the degradation of GSTP1 through the ubiquitin-proteasome system, autophagy-lysosome pathway, and mitophagy. After co-transfection with Parkin, the non-ubiquitinatable GSTP1 mutant maintained its anti-apoptotic function, while wildtype GSTP1 failed. Mechanistically, GSTP1 might promote mitochondrial fusion through upregulating Mitofusins 1/2 (MFN1/2). CONCLUSION: Oxidative stress induces LECs apoptosis via Parkin-regulated degradation of GSTP1, which may provide potential targets for ARC therapy.

Anterior vitreous detachment and retrolental material during cataract surgery: incidence and risk factors, with pathological evidence.

PURPOSE: To determine the incidence of anterior vitreous detachment (AVD) and retrolental material occurrence in cataract surgery, determine the influence of surgical factors on it, and confirm the source of the material. SETTING: Affiliated Hospital of Nantong University, Jiangsu Province, China. DESIGN: Prospective single-center study. METHODS: Age, sex, ocular complication, nuclear sclerosis grade, ultrasonic time, mean longitudinal power, cumulative dissipated energy (CDE), total aspiration time, and estimated fluid usage were recorded for each patient. Retrolental anatomy was observed before and during surgery using real-time optical coherence tomography integrated into a microscope. The eyes with AVD were carefully observed and recorded during illumination with an optical fiber. Retrolental material was stained using immunohistochemistry. RESULTS: 205 eyes from 205 patients were included in this study. Spontaneous AVD was found in 5 cases. Intraoperatively, AVD was identified in 115 eyes (56.1%). Retrolental material presence was observed in 75 eyes (36.6%). A logistic regression model showed that CDE and aspiration time had a statistically significant effect on AVD ( P < .05, 95% CI, 1.011-1.558; P < .05, 95% CI, 1.026-1.051), and CDE was positively correlated with retrolental material occurrence ( P < .05, 95% CI, 1.052-1.534). Samples from 5 cases expressed large amounts of αA- and ßA-crystallins. CONCLUSIONS: Spontaneous AVD is rare in phakic eyes. There was a marked increase in AVD during surgery, with retrolental material occurring in more than a third of cases. Higher CDE and longer total aspiration time were risk factors for AVD. Immunohistochemistry revealed that most of the retrolental materials were lens fragments.

Concentrations of glucose metabolites in the aqueous humour of diabetic cataract eyes.

PURPOSE: Glucose metabolism underpins diabetic cataracts (DCs), but the relationship between the two remains unclear. Here, we tested the aqueous humour (AH) of patients with DCs to elucidate glucose metabolite levels. METHODS: In this study, aqueous humour (AH) samples were collected preoperatively from DC eyes (n = 37) and age-related cataract eyes (n = 37) from 74 patients (74 eyes) undergoing uncomplicated cataract surgery. The content of glucose, pyruvate, L-lactate were detected by biochemical methods and advanced glycation end-products (AGEs) was detemined using enzyme-linked immunosorbent assay (ELISA) technique. Furthermore, the ratios of glucose/pyruvate and L-lactate/pyruvate in the AH were calculated. In addition, we calculated the correlation between glucose levels and AGEs in the AH. RESULTS: The concentrations of glucose, pyruvate and AGEs in the DC group were higher than those in the control group. Significantly lower levels of L-lactate in the AH were found in the DC group. We calculated the glucose/pyruvate ratio and the L-lactate/pyruvate ratio in the AH, which showed that glucose metabolism was changed in the AH from DC patients. Interestingly, we observed that AGEs in the AH were significantly correlated with increased anterior chamber glucose permeability. A stronger correlation was found in the subgroups of male patients, younger patients, and patients with poor glycaemic control status. CONCLUSIONS: Comparison of the levels of glucose metabolism-related products in the AH in the DC group highlight a potential pathological mechanism for DC from a glucose metabolism perspective. The findings indicated an alteration in the metabolic pathways of energy metabolism and amino acids in the AH of DC patients.

Upadacitinib inhibits corneal inflammation and neovascularization by suppressing M1 macrophage infiltration in the corneal alkali burn model.

Alkali burn-induced corneal inflammation and subsequent corneal neovascularization (CNV) are major causes of corneal opacity and vision loss. M1 macrophages play a central role in inflammation and CNV. Therefore, modulation of M1 macrophage polarization is a promising strategy for corneal alkali burns. Here, we illustrate the effect and underlying mechanisms of upadacitinib on corneal inflammation and CNV induced by alkali burns in mice. The corneas of BALB/c mice were administered with 1 M NaOH for 30 s and randomly assigned to the vehicle group and the upadacitinib-treated group. Corneal opacity and corneal epithelial defects were assessed clinically. Quantitative real-time PCR (qRT-PCR), immunohistochemistry, and western blot analysis were performed to detect M1 macrophage polarization and CD31+ corneal blood vessels. The results showed that upadacitinib notably decreased corneal opacity, and promoted corneal wound healing. On day 7 and 14 after alkali burns, upadacitinib significantly suppressed CNV. Corneal alkali injury caused M1 macrophage recruitment in the cornea. In contrast to the vehicle, upadacitinib suppressed M1 macrophage infiltration and decreased the mRNA expression levels of inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, and vascular endothelial growth factor A (VEGF-A) in alkali-injured corneas. Moreover, upadacitinib dose-dependently inhibited M1 macrophage polarization by suppressing interferon (IFN)-γ-/lipopolysaccharide-stimulated STAT1 activation in vitro. Our findings reveal that upadacitinib can efficiently alleviate alkali-induced corneal inflammation and neovascularization by inhibiting M1 macrophage infiltration. These data demonstrate that upadacitinib is an effective drug for the treatment of corneal alkali burns.

miR-125a-3p regulates apoptosis by suppressing TMBIM4 in lens epithelial cells.

PURPOSE: To explore the regulatory effect of miR-125a-3p on lens epithelial cells (LECs) under ultraviolet radiation B (UVB) irradiation. METHODS: The expression of miR-125a-3p in age-related cataract (ARC) specimens and cell models was detected by qRT-PCR. UVB was utilized to establish DNA damage model of LECs. Cell count kit-8 was applied in detecting cell viability. Cell apoptosis ratio was analyzed by flow cytometry. Dual luciferase reports were applied to analyze the mechanism between miRNA and target genes. Nanoparticle tracking analysis, and Western blot were used to identify whether the exosomes were typical exosomes. RESULTS: miR-125a-3p was upregulated in ARC tissues and LECs treated with UVB. Knockdown of miR-125a-3p in LECs significantly decreased apoptosis and increased viability of UVB-irradiated LECs. We predicted that miR-125a-3p could regulate transmembrane Bax inhibitor motif containing 4 (TMBIM4) by the bioinformatics databases TargetScan, miRBase, and miRWalk. Luciferase reporter assays demonstrated that miR-125a-3p may suppress TMBIM4 protein translation by binding to 3'UTR of TMBIM4 mRNA. Overexpression of miR-125a-3p decreased TMBIM4, which suggested that miR-125a-3p could inhibit TMBIM4. Moreover, knockdown of TMBIM4 decreased cell viability and enhanced cell apoptosis during UVB irradiation. In addition, the exosome secretion of LECs irradiated by UVB was enhanced, and the expression of miR-125a-3p was high. Cell viability was significantly decreased, and cell apoptosis was increased during UVB-exos treatment. CONCLUSION: This study indicated that miR-125a-3p regulated apoptosis by suppressing TMBIM4 in LECs under oxidative damage, providing a new idea for clinical therapeutic target of cataract.

Exosomal microRNA-222-3p increases UVB sensitivity of lens epithelium cells by suppressing MGMT.

BACKGROUND: Age-related cataract (ARC) is a leading cause of blindness worldwide with multiple pathogenic factors. Oxidative damage of lens epithelium cells (LECs) is one of the well-accepted pathogenesis of ARC which can be regulated by DNA repair genes (DRGs). The present research aimed to clarify the regulatory mechanism of exosomal microRNAs (miRNAs) on DRGs in LECs. METHODS: The LECs oxidative damage model was established by UVB-irradiation on SRA01/04 (human lens epithelium cell line). Exosomes from UVB-irradiated cells (UVB-exo) and exosomes from normal control cells (NC-exo) were collected from the culture medium. To explore the functions of LECs exosomes, SRA01/04 were incubated with UVB-exo/NC-exo. Then, we detected SRA01/04 proliferation, viability and apoptosis respectively using 5'-ethynyl-2'-deoxyuridine (EdU), cell-counting kit-8 (CCK-8) and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. Next, the miRNA expression profiles of UVB-exo and NC-exo were identified by miRNA microarrays. RNA expression in exosomes, cells, and clinical samples was verified by qRT-PCR. The location and expression of MGMT and CD63 proteins were detected by immunofluorescence and western blot. The 3'UTR regulation of miR-222-3p to MGMT was verified by luciferase analyses. RESULTS: MGMT down-regulated while miR-222-3p up-regulated in LECs sub-central anterior capsule from ARC lenses. MGMT and miR-222-3p expressions in central and peripheral LECs from anterior lens capsules were differential. UVB-exo can transport the up-regulated miR-222-3p from oxidative-damaged LECs to normal LECs, which could suppress MGMT expression and increase UVB sensitivity of LECs. CONCLUSIONS: Findings on exosomal miRNA functions provided novel insights into pathogenesis of ARC. Exosomal miR-222-3p can be a potential target for prevention and cure of ARC.

Identification of the circRNA-miRNA-mRNA Regulatory Network in Pterygium-Associated Conjunctival Epithelium.

To investigate the regulatory mechanism of pterygium formation, we detected differentially expressed messenger RNAs (DE-mRNAs) and differentially expressed circular RNAs (DE-circRNAs) in pterygium-associated conjunctival epithelium (PCE) and normal conjunctival epithelium (NCE). Genome-wide mRNA and circRNA expression profiles of PCE and NCE were determined using high-throughput sequencing. Bioinformatics analyses, including Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) analysis, were conducted. The microRNAs (miRNAs) interacting with the hub DE-mRNAs and DE-circRNAs were predicted and verified using real-time quantitative PCR (RT-qPCR). The data showed that there were 536 DE-mRNAs (280 upregulated and 256 downregulated mRNAs) and 78 DE-circRNAs (20 upregulated and 58 downregulated circRNAs) in PCE. KEGG enrichment analysis indicated that the DE-mRNAs were mainly involved in the following biological processes: IL-17 signalling pathway, viral protein interaction with cytokine and cytokine receptor, cytokine-cytokine receptor interaction, ECM-receptor interaction, and focal adhesion. The GSEA results revealed that the epithelial mesenchymal transition (EMT) process was significantly enriched in upregulated mRNAs. The pterygium-associated circRNA-miRNA-mRNA network was established based on the top 10 DE-circRNAs, 4 validated miRNAs (upregulated miR-376a-5p and miR-208a-5p,downregulated miR-203a-3p and miR-200b-3p), and 31 DE-mRNAs. We found that miR-200b-3p, as a regulator of FN1, SDC2, and MEX3D, could be regulated by 5 upregulated circRNAs. In addition, we screened out EMT-related DE-mRNAs, including 6 upregulated DE-mRNAs and 6 downregulated DE-mRNAs. The EMT-related circRNA-miRNA-mRNA network was established with the top 10 circRNAs, 8 validated miRNAs (upregulated miR-17-5p, miR-181a-5p, and miR-106a-5p, downregulated miR-124-3p, miR-9-5p, miR-130b-5p, miR-1-3p, and miR-26b-5P), and 12 EMT-related DE-mRNAs. We found that hsa_circ_0002406 might upregulate FN1 and ADAM12 by sponging miR-26b-5p and miR-1-3p, respectively, thus promoting EMT in pterygium. Briefly, the study provides a novel viewpoint on the molecular pathological mechanisms in pterygium formation. CircRNA-miRNA-mRNA regulatory networks participate in the pathogenesis of pterygium and might become promising targets for pterygium prevention and treatment.

Doxycycline Suppresses Vasculogenic Mimicry in Human Pterygium Fibroblasts.

PURPOSE: To explore the effect of doxycycline on vasculogenic mimicry (VM) formation and the potential mechanism in human pterygium fibroblasts in order to find novel targets for pterygium therapy. METHODS: First, we demonstrate the existence of VM in 73 pterygium specimens by CD31 and periodic acid Schiff (PAS) dual staining. Then we used cell counting kit-8, clone formation assay and flow cytometry to prove the inhibitory effect of doxycycline on cell proliferation and apoptosis. The VM formation was evaluated through wound healing assay, cell transwell assay and three-dimensional cell culture combined with PAS staining. Finally, we used Western blot to testify the correlation of the VM and the factors in protein level preliminarily. RESULTS: Our results showed that VM existed in human pterygium specimens exactly. Otherwise, in human pterygium fibroblasts, doxycycline induced a dose-dependent inhibitory effect on cell proliferation and apoptosis induction. Besides, doxycycline significantly suppressed vasculogenic mimicry tube formation, cell migration and invasion. Furthermore, doxycycline impaired the expression of MMP-9, MMP-2 and VEGF which may related to pterygium VM formation. CONCLUSIONS: Doxycycline decelerated pterygium progression might be through inhibiting VM formation according to the downregulation of MMP-9, MMP-2 and VEGF, which may provide the basis of further studies involving doxycycline for pterygium treatment.

Expression profile analysis to identify potential gene changes induced by dexamethasone in the trabecular meshwork.

AIM: To investigate potential gene changes in trabecular meshwork (TM) induced by dexamethasone (DEX) in steroid-induced glaucoma (SIG). METHODS: The expression data of 24 cases from a public functional genomics data were sorted to identify the mechanisms of action of DEX on the TM. The relationships of the differentially expressed genes (DEGs) were enriched using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, the hub genes were screened by the Search Tool for the Retrieval of Interacting Genes Database (STRING) and Cytoscape tools. Finally, human TM cells (HTMCs) were treated with DEX to preliminarily explore the function of hub genes. RESULTS: Totally 47 DEGs, including 21 downregulated and 26 upregulated genes were identified. The primary enriched results of the DEGs consisted of inflammatory response, extracellular matrix (ECM), negative regulation of cell proliferation, TNF signalling pathway and the regulation of tryptophan channels by inflammatory mediators. Subsequently, pro-melanin-enriched hormone (PMCH) and Bradykinin B1 receptor (BDKRB1) were screened as hub genes. It is verified in GSE37474 data set. Western blot and quantitative real-time polymerase chain reaction (qPCR) results showed that protein and RNA expression levels of BDKRB1 were significantly decreased after DEX treatment, while PMCH was not significantly changed. CONCLUSION: BDKRB1 may be a key gene involved in SIG onset, providing a suitable therapeutic target for improving the prognosis of SIG patients.