Protective effects of triptolide against oxidative stress in retinal pigment epithelium cells via the PI3K/AKT/Nrf2 pathway: a network pharmacological method and experimental validation.

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.

IL-8 Triggers Neutrophil Extracellular Trap Formation Through an Nicotinamide Adenine Dinucleotide Phosphate Oxidase- and Mitogen-Activated Protein Kinase Pathway-Dependent Mechanism in Uveitis.

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.

Dermatophagoides Pteronyssinus 1 (DerP1) May Trigger NLRP3-Mediated Corneal Epithelial Cell Pyroptosis by Elevating Interleukin-33 Expression Levels.

PURPOSE: To characterize the in vivo effects of Dermatophagoides pteronyssinus 1 (DerP1) in mice and determine the underlying NLRP3 inflammasome-mediated pyroptosis signaling mechanisms in the human corneal epithelial cells (HCECs). METHODS: DerP1 was used to induce allergic conjunctivitis in C57 mice. HCECs were sensitized with DerP1 in vitro to mimic their condition observed in allergic conjunctivitis in vivo. Transmission electron microscopy was used to evaluate pyroptosis in the HCECs, enzyme-linked immunosorbent assays to assess interleukin (IL)-33, IL-1ß and IL-4 levels, flow cytometry to detect the proportion of Th2 cells, MTT assays to assess cell metabolic activity, immunofluorescence to evaluate the effects of DerP1 on functional HCEC phenotypes, and Western blot assays to detect the expression of NOD-like receptor family pyrin domain-containing 3 (NLRP3), gasdermin D (GSDMD), N-terminal fragment of GSDMD (GSDMD-N), pro-caspase-1, cleaved caspase-1, IL-1ß, and IL-33. IL-33 expression in the HCECs was knocked down via lentivirus transfection. RESULTS: In vivo, DerP1 promotes pyroptosis, production of Th2 inflammatory cytokines and IL-33, and NLRP3 activation in mouse corneas. In vitro, pyroptotic bodies were found in the HCECs after sensitization with DerP1. Various concentrations of DerP1 increased the expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved caspase-1, and IL-1ß in the HCECs, with the largest increase observed after exposure to 20 µM DerP1. In vitro, recombinant human IL-33 mediated the expression of pyroptotic biomarkers in the HCECs, whereas IL-33 silencing diminished 20 µM DerP1-induced increase in their expression levels. CONCLUSIONS: DerP1 induces pyroptosis and allergic conjunctivitis, the expression of Th2 inflammatory cytokines, NLRP3 activation, and IL-33 in mouse corneas in our model. These effects would attribute to its activating NLRP3-GSDMD signaling pathway axis via enhancing IL-33 expression in HCECs.

Causal analysis of serum polyunsaturated fatty acids with juvenile idiopathic arthritis and ocular comorbidity.

BACKGROUND & OBJECTIVE: To investigate the causal effects of plasma Polyunsaturated fatty acids (PUFAs) on the risk of juvenile idiopathic arthritis (JIA) and ocular comorbidity through Mendelian randomization (MR) analysis. METHODS: Genetic variants (formerly single nucleotide polymorphisms, SNPs) that are strongly associated with PUFAs levels (P < 5×10-8) were selected as instrumental variables. Summary-level MR was performed with outcome estimates for JIA (n = 31,142) and JIA associated iridocyclitis (n = 94,197). The inverse variance-weighted (IVW) method was employed as the main approach to combine the estimation for each SNP. Two set of models with summary statistics were conducted and multiple sensitivity analyses were applied for testing of pleiotropic bias. RESULTS: In model 1, genetically predicted n-6 PUFAs linoleic acid (LA) and arachidonic acid (AA) were associated with lower and higher risk of JIA associated iridocyclitis using IVW (ORLA = 0.940, 95% CI: 0.895-0.988, P = 0.015; ORAA = 1.053, 95% CI: 1.007-1.101, P = 0.024). No such association was observed between each plasma PUFAs and JIA susceptibility (P > 0.05). In further MR analysis, results from model 2 also showed a consistent trend. Besides, multiple sensitivity analyses revealed that there was no obvious evidence for unknown pleiotropy (P > 0.05). CONCLUSIONS: Our MR study provides genetic evidence on the possible causality that plasma LA level might protect against JIA associated iridocyclitis, whereas AA was responsible for opposite effect.

Aß1-40 Oligomers Trigger Neutrophil Extracellular Trap Formation through TLR4- and NADPH Oxidase-Dependent Pathways in Age-Related Macular Degeneration.

Neutrophils participate in the advancement of the human innate immune system and respond to perceived endogenous and exogenous threats. As a response mechanism, neutrophil extracellular traps (NETs) form near pathogens and surrounding tissues during an immune response. Drusen is an important marker of Age-Related Macular Degeneration (AMD) and plays an important role in the course of AMD. Aß1-40 is the main component of drusen. However, the relationship between NETs and AMD or Aß1-40 is unclear. Here, we found elevated levels of NETs in the serum of AMD patients and elevated levels in the serum of mouse models. We also observed the accumulation of neutrophils in the mouse retina. In addition, the production of NETs was inhibited by PAD4 inhibitors, which can alleviate chronic inflammation. Moreover, we confirmed that Aß1-40 can induce NETs formation via the Toll-like receptor 4 (TLR4) and neutrophil NADPH oxidase (NOX) pathways. Our study confirmed that the formation of NETs is induced by Aß1-40, and the results suggest that NETs may play a vital role in AMD pathogenesis.