Trimethylamine-N-Oxide Promotes High-Glucose-Induced Dysfunction and NLRP3 Inflammasome Activation in Retinal Microvascular Endothelial Cells.

Introduction: Along with blood glucose levels, diabetic retinopathy (DR) development also involves endogenous risk factors, such as trimethylamine-N-oxide (TMAO), a product of intestinal flora metabolic disorder, which exacerbates diabetic microvascular complications. However, the effect of TMAO on retinal cells under high-glucose conditions remains unclear. Therefore, this study examined the effects of TMAO on high-glucose-induced retinal dysfunction in the context of NLRP3 inflammasome activation, which is involved in DR. Materials and Methods: TMAO was assessed in the serum and aqueous humor of patients using ELISA. Human retinal microvascular endothelial cells (HRMECs) were treated for 72 h as follows: NG (normal glucose, D-glucose 5.5 mM), NG + TMAO (5 µM), HG (high glucose, D-glucose 30 mM), and HG + TMAO (5 µM). The CCK8 assay was then used to assess cell proliferation; wound healing, cell migration, and tube formation assays were used to verify changes in cell phenotype. ZO-1 expression was determined using immunofluorescence and western blotting. Reactive oxygen species (ROS) formation was assessed using DCFH-DA. NLRP3 inflammasome complex activation was determined using a western blot. Results: The serum and aqueous humor from patients with PDR contained higher levels of TMAO compared to patients with nontype 2 diabetes (Control), non-DR (NDR), and non-PDR (NPDR). TMAO showed significant acceleration of high-glucose-induced cell proliferation, wound healing, cell migration, and tube formation. ZO-1 expression decreased remarkably with the combined action of TMAO and a high glucose compared to either treatment alone. TMAO also promoted high-glucose-activated NLRP3 inflammasome complex. Conclusion: The combination of TMAO and high-glucose results in increased levels of ROS and NLRP3 inflammasome complex activation in HRMECs, leading to exacerbated retinal dysfunction and barrier failure. Thus, TMAO can accelerate PDR occurrence and development, thus indicating the need for early fundus monitoring in diabetic patients with intestinal flora disorders.

Apoptosis of Lens Epithelial Cells and Expression of NLRP3-related Proteins in Patients with Diabetes and Cataract.

AIM: To compare the expression of apoptosis-related factors and Nlrp3-related proteins in the lens epithelial cells (LECs) of patients with diabetes and cataract and patients with age-related cataract (ARC) alone. METHODS: All patients were divided into four groups according to the presence or absence of diabetes mellitus (DM) and the degree of diabetic retinopathy (DR). LECs were obtained during cataract surgery. The expression levels of cleaved caspase-3, caspase-7, ASC, caspase-1and Nlrp3 in LECs were determined. And analyzed by age, course of DM, and HbA1c levels. RESULTS: The incidence of LEC apoptosis and positive rates of cleaved caspase-3 and caspase-7 expression were significantly higher in the groups with DM (P<0.05).The positive expression rates of ASC, caspase-1, and Nlrp3 increased with longer duration of DM, increased HbA1c level, or advanced DR (P<0.05). CONCLUSION: In cataract patients with DM, the expression of apoptosis-related factors in LECs increased. Nlrp3-related protein expression levels, diabetes duration, HbA1c levels, and extent of DR may be potential risk factors for diabetic cataract formation.

MSC-derived exosomal lncRNA SNHG7 suppresses endothelial-mesenchymal transition and tube formation in diabetic retinopathy via miR-34a-5p/XBP1 axis.

AIMS: Diabetic retinopathy (DR) is the most common complication of type 2 diabetes mellitus, which could result in visual impairment. Accumulating studies have shown the implication of long non-coding RNAs (lncRNAs) in the pathogenesis of DR. Our aims are to investigate whether lncRNA SNHG7 plays a role during DR pathogenesis. MAIN METHODS: Human retinal microvascular endothelial cells (HRMECs) were treated with high glucose (HG) to build cell model. Relative expression of RNAs were examined using qPCR, and western blot or immunofluorescence analysis was adopted to detect the protein expression. Cell viability, migration and angiogenic capacity of HRMECs were estimated through CCK-8, transwell and tube formation experiments, respectively. Dual-luciferase reporter and RNA pull down assays were employed to verify the interplay between miR-34a-5p and SNHG7 or XBP1. Mesenchymal stem cells (MSCs) were identified by examining typical surface makers using flow cytometry and the differentiation abilities via Alizarin red, Oil red O and Alcian blue staining. MSC-derived exosomes were verified by transmission electron microscopy and western blot. KEY FINDINGS: LncRNA SNHG7 sponged to and negatively regulated miR-34a-5p. SNHG7 overexpression repressed HG induced endothelial-mesenchymal transition (EndMT) and tube formation of HRMECs, while miR-34a-5p overexpression could reverse this effect. miR-34a-5p targeted and negative regulated XBP1. Knockdown of miR-34a-5p repressed HG induced EndMT and tube formation, which were partially blocked by XBP1 inhibition. MSC-derived exosomes could transfer SNHG7 to HRMECs and modulated EndMT and tube formation. SIGNIFICANCE: The MSC-derived exosomal lncRNA SNHG7 suppresses EndMT and tube formation in HRMECs via miR-34a-5p/XBP1 axis.

The FoxM1-ABCC4 axis mediates carboplatin resistance in human retinoblastoma Y-79 cells.

Carboplatin is the most commonly used drug in the first-line treatment of human retinoblastoma (RB), but its clinical application is greatly limited due to acquired drug resistance upon the long-term treatment. Forkhead box protein M1 (FoxM1) is the transcription factor aberrantly expressed in various types of human cancers, which plays an essential role in the regulation of tumorigenesis, tumor metastasis and drug resistance. However, little is known about the role of FoxM1 in chemo-resistance of human RB. In this study, we investigated the regulatory effect of FoxM1 on carboplatin resistance in human RB Y-79 cells and carboplatin-resistant Y-79 (Y-79CR) cells, as well as the possible mechanism. Our results showed that FoxM1 was up-regulated in Y-79CR cells and silencing of FoxM1 promoted carboplatin sensitivity and accumulation, while overexpression of FoxM1 in Y-79 cells performed oppositely. Our study further revealed that FoxM1 enhanced carboplatin resistance in Y-79CR cells through directly up-regulating the transcription of ATP-binding cassette transporter C4 (ABCC4), an important drug efflux transporter. Overall, our study demonstrated the novel role of FoxM1-ABCC4 axis in human RB, which provides insights into the prevention of carboplatin resistance in human RB.