Purpose: To explore the mechanism underlying IL-8-induced neutrophil extracellular trap (NET) formation in patients with ocular-active Behçet's disease (BD) and the effect of inhibiting NET formation on the severity of inflammation in experimental autoimmune uveitis (EAU) mice. Methods: The serum extracellular DNA and neutrophil elastase (NE) and IL-8 levels in patients with ocular-active BD, the expression of myeloperoxidase, NE, and histone H3Cit in IL-8–induced neutrophils isolated from healthy controls, and the effects of NETs on HMC3 cells were detected. Female C57BL/6J mice were immunized with IRBP651–670 to induce EAU and EAU mice received intravitreal injection of the CXCR2 (IL-8 receptor) antagonist SB225002 or PBS. The serum levels of extracellular DNA, NE, and keratinocyte-derived chemokine (the mouse ortholog of human IL-8) and expression of myeloperoxidase, NE, and histone H3Cit in mouse retinas were detected. Disease severity was evaluated by clinical and histopathological scores. Results: Serum keratinocyte-derived chemokine expression levels in EAU mice and IL-8 expression levels in patients with ocular-active BD increased. IL-8 notably increased NET formation in a dose-dependent manner through an nicotinamide adenine dinucleotide phosphate oxidase and mitogen-activated protein kinase pathway dependent mechanism. IL-8–induced NET formation damaged HMC3 cells in vitro. Pretreatment with SB225002 notably ameliorated the production of NETs in EAU mice. Conclusions: Our data confirm that NET formation is induced by IL-8. IL-8–induced NET formation was found to be related to mitogen-activated protein kinase and nicotinamide adenine dinucleotide phosphate pathways. Pretreatment with the CXCR2 antagonist SB225002 alleviated neutrophil infiltration and suppressed NET formation in EAU mice.

Purpose: Among aging adults, age-related macular degeneration (AMD), is a prevalent cause of blindness. Nevertheless, its progression may be halted by antioxidation in retinal pigment epithelium (RPE). The primary effective constituent of Tripterygium wilfordii Hook. F., triptolide (TP), has demonstrated anti-inflammatory, antiproliferative, and antioxidant properties. The mechanics of the protective effect of triptolide against the oxidative damage in retinal pigment epithelial (RPE) were assessed in this study. Methods: ARPE-19 cells were pretreated with TP, and then exposed to sodium iodate (SI). First, cell viability was assessed using CCK-8. Subsequently, we measured indicators for cell oxidation including reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). Then, we used network pharmacological analysis and molecular docking to explore the signaling pathway of TP. Last, we used western blot, ELISA, and immunofluorescence assays to clarify the potential mechanistic pathways. Results: The network pharmacology data suggested that TP may inhibit AMD by regulating the PI3K/Akt signaling pathway. Experimental results showed that the potential mechanism is that it regulates the PI3K/Akt pathway and promotes Nrf2 phosphorylation and activation, thereby raising the level of antioxidant factors (HO-1, NQO1) and reducing the generation of ROS, which inhibit oxidative damage. Conclusion: Our findings suggested that the effect of TP on SI-exposed RPE cells principally relies on the regulation of oxidative stress through the PI3K/Akt/Nrf2 signaling pathway.

AIM: To investigate serum levels of soluble CD146 (sCD146) and vascular endothelial growth factor receptor 2 (VEGFR2) in patients with age-related macular degeneration (AMD). METHODS: Eighty-eight patients with exudative AMD and 45 sex- and age-matched healthy controls were enrolled in this study conducted in China. Serum samples was obtained from the patients with exudative AMD and from the controls. Serum sCD146 and VEGFR2 protein levels were measured using an enzyme-linked immunosorbent assay. RESULTS: We found that serum sCD146 and VEGFR2 protein levels were significantly higher in the patients with exudative AMD group than in the controls (t=3.859, P<0.001 and t=3.829, P<0.001, respectively). Serum sCD146 levels were significantly higher in patients with classic choroidal neovascularization (CNV) than in those with occult CNV (t=9.899, P<0.001). There was a significant difference in the trend for exudative AMD in the highest versus lowest quartile of circulating sCD146 levels (chi(2)=10.29, P=0.001). The receiver operating characteristic curve analysis showed that the area under the curve was 0.696 for sCD146 (95%CI: 0.601-0.791) with an optimum diagnostic cut-off value of 157.16 ng/mL, a sensitivity of 55.7%, and a specificity of 82.2%. CONCLUSION: The serum sCD146 level increases and may be a biomarker for exudative AMD.

Introduction: Oxidative stress-prompted degeneration of the retinal pigment epithelium (RPE) notably contributes to the onset of age-related macular degeneration (AMD). However, the pathways leading to RPE deterioration and possible preventative strategies are not yet completely comprehended.Methods: Ferroptosis was assayed through the evaluation of lipid peroxidation (C11-BODIPY and MDA), reactive oxygen species (ROS), transmission electron microscopy (TEM), iron content measurement, q-PCR, western blotting, and immunofluorescence. To assess the structure and retinal function of RPE in mice, ERG (electroretinography), OCT (optical coherence tomography), and H&E (hematoxylin and eosin) staining were employed. Network pharmacology methods were utilized to elucidate the potential mechanisms underlying melatonin's protective effects against ferroptosis in RPE cells in AMD. Genetic engineering techniques were applied to investigate the regulatory relationships among phosphatidylinositol 3-kinase (PI3K), protein kinase-B (AKT), murine double minute-2 (MDM2), protein 53 (P53), and solute carrier family 7 member 11 (SLC7A11). In vitro knockdown experiments of MDM2 were conducted to explore its regulatory role in ferroptosis within RPE cells.Results: Aβ1-40 can trigger ferroptosis in RPE cells. Melatonin can inhibit the oxidative stress and ferroptosis induced by Aβ1-40 in RPE cells. Melatonin exhibits a protective effect on Aβ1-40-induced AMD, significantly improving the structure of the mouse retina and RPE layer, and facilitating the restoration of visual function. Network pharmacology methods revealed that the potential targets of melatonin in AMD are closely related to ferroptosis, and indicated that the predominant pathways are significantly associated with the PI3K/AKT/MDM2/P53 signaling pathway. Knocking down the specific expression of MDM2 can significantly weaken the inhibitory effect of melatonin on oxidative stress and ferroptosis.Discussion: Melatonin can suppress cell death by ferroptosis in RPE via the PI3K/AKT/MDM2/P53 pathway, thereby preventing and decelerating the progression of AMD.

Age-related macular degeneration (AMD) is a common blindness diseases. Retinal pigment epithelium (RPE) dysfunction due to smoking is an essential environmental factor in the pathogenesis of AMD. Ferroptosis is a novel type of iron-dependent programmed cell death (PCD). However, the relationship between cigarette smoke extract (CSE)-induced RPE damage and ferroptosis remains unclear. In our study, we extracted CSE using a modified device to explore the optimal concentration of CSE, and observed the expression of proteins and molecules after CSE exposure for ARPE-19 cells by protein immunoblotting and assay kits for iron ions and mitochondrial membrane potential (MMP). At the same time, CSE was injected into the vitreous cavity of mice with a microsyringe for AMD modeling to observe the morphology of the retina-RPE-choroid complex and the differences expression of proteins. In addition, the protective effects of ferroptosis inhibitors on CSE-induced RPE cell damage were also investigated by in vivo and in vitro experiments. In this study, we observed that CSE induced cellular damage in a human retinal pigment epithelial cell line (ARPE-19), resulting in ferrous ion (Fe²⁺) accumulation, an increas in reactive oxygen species (ROS) and lipid peroxidation (LP), a reduction in GSH levels, and the inhibition of Gpx4 expression. In addition, transmission electron microscopy (TEM) of in vivo and in vitro samples showed that after exposure to CSE, the mitochondria of RPE cells were wrinkled, the membrane density was increased, and the number of cristae decreased or cristae were not observed. The results of this study indicate that the ferroptosis inhibitors ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) protect RPE cells from CSE-induced ferroptosis, and this evidence paves the way for AMD studies.