Abstract Objectives To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR). Method The expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1β, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1β, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1β and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA). Results Compared with the surrounding normal tissues, the expression of caspase-1, IL-1β, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05). Conclusions MCP can promote the development of PVR lesions.

RESUMO Objetivo: Atualmente, a retinopatia diabética é considerada uma doença inflamatória crônica envolvendo a ativação de inflamassomas NLRP3 e piroptose da micróglia da retina. Neste estudo, objetivamos investigar se a sinalização de inflamassomas NLRP3 induz a morte da micróglia da retina sob condições de alta glicose. Métodos: A micróglia da retina foi estimulada por altos níveis de glicose durante 24 horas. A viabilidade celular, a liberação de LDH e a atividade da caspase1 foram analisadas in vitro. Avaliou-se a expressão de citocina pró-inflamatória (IL1β), de marcador de micróglia ativado (Iba1), de NLRP3, de caspase1 clivada e de GSDMD clivada. Subsequentemente, a micróglia da retina foi pré-tratada com inibidores da sinalização de inflamassomas NLRP3 antes da estimulação com altos níveis de glicose e suas alterações moleculares e funcionais foram avaliadas. Resultados: A estimulação com altos níveis de glicose (25 mM, 50 mM ou 100 mM) diminuiu a viabilidade celular, mas aumentou a liberação de LDH e a atividade da caspase1 de forma dependente da dose. Além disso, os altos níveis de glicose aumentaram a expressão das proteínas IL1β, Iba1, NLRP3, caspase1 clivada e GSDMD clivada. No entanto, o pré-tratamento com inibidores da sinalização de inflamassomas NLRP3 e a posterior estimulação com altos níveis de glicose (25 mM) induziu citotoxicidade, a ativação de inflamassomas NLRP3 e a piroptose da micróglia da retina. Conclusão: A sinalização de inflamassomas NLRP3 pode modular a inflamação e a piroptose da micróglia da retina na presença de altos níveis de glicose.

ABSTRACT Purpose: Diabetic retinopathy is currently considered a chronic inflammatory disease involving NOD-like receptor family pyrin domain containing 3 inflammasome activation and retinal microglial pyroptosis. In this study, we aimed to investigate whether NOD-like receptor family pyrin domain containing 3 inflammasome signaling induces pyroptotic death of retinal microglia under high-glucose conditions. Methods: Retinal microglia were stimulated by high glucose levels for 24 h. Cell viability, lactate dehydrogenase release, and caspase-1 activity were detected in vitro. The expression of pro-inflammatory cytokine (interleukin-1β, activated microglia marker ionized calcium-binding adapter molecule-1), NOD-like receptor family pyrin domain containing 3, cleaved caspase-1, and cleaved gasdermin D were examined. Subsequently, retinal microglia were pretreated with the inhibitors of NOD-like receptor family pyrin domain containing 3 inflammasome signaling prior to stimulation with high glucose, and their molecular and functional changes were evaluated. Results: High-glucose (25, 50, or 100 mM) stimulation decreased cell viability, but enhanced lactate dehydrogenase release and caspase-1 activity in a dose-dependent manner. Moreover, high glucose upregulated the protein expression of interleukin-1β, ionized calcium-binding adapter molecule-1, NOD-like receptor family pyrin domain containing 3, cleaved caspase-1, and cleaved gasdermin D. However, pretreatment with the inhibitors of NOD-like receptor family pyrin domain containing 3 inflammasome signaling inhibited high glucose (25 mM)-induced cytotoxicity, NOD-like receptor family pyrin domain containing 3 inflammasome activation, and pyroptosis of retinal microglia. Conclusions: NOD-like receptor family pyrin domain containing 3 inflammasome signaling may modulate retinal microglia-related inflammation and pyroptosis under high-glucose conditions.

RESUMO Objetivos: Avaliar a eficácia do antagonista de prostaglandinas no rompimento da barreira hemato-retiniana induzida por cirurgia simulada intraocular do segmento anterior. Métodos: Os ratos foram divididos aleatoriamente em grupo controle negativo, grupo modelo, grupo de tratamento profilático com drogas anti-inflamatórias não esteroides, grupo de tratamento com anti-inflamatórias não esteroides, grupo de tratamento profilático com corticosteroides e grupo de tratamento com corticosteroides. Quatro e 48h após a modelagem, as concentrações de PGE1, PGE2 e PGF2 α no humor aquoso e no corpo vítreo em modelo em ratos foram detectadas através de Elisa. A integridade da barreira hemato-retiniana foi quantitativamente mensurada utilizando o azul de Evans como marcador. Resultados: Quatro horas após a modelagem, as concentrações de PGE1, PGE2 e PGF2α no humor aquoso e no corpo vítreo no grupo controle negativo e no grupo de tratamento profilático com anti-inflamatórias não esteroides foram significativamente menores do que as do grupo modelo. As concentrações de PGE1, PGE2 e PGF2α no humor aquoso e no corpo vítreo no grupo de tratamento profilático com corticosteroides foram maiores do que as observadas no grupo controle negativo e no grupo de tratamento profilático com anti-inflamatórias não esteroides. 48h após a modelagem, as concentrações de PGE1, PGE2 e PGF2α no humor aquoso e no corpo vítreo no grupo de tratamento profilático com anti-inflamatórias não esteroides, no grupo de tratamento com anti-inflamatórias não esteroides, no grupo de tratamento profilático com corticosteroides e no grupo de tratamento com corticosteroides foram menores do que as observadas no grupo modelo e maiores que as observadas no grupo negativo. O extravasamento retinal de azul de Evans no grupo de tratamento profilático com anti-inflamatórias não esteroides foi maior que no grupo controle negativo e menor que nos grupos de tratamento com anti-inflamatórias não esteroides, de tratamento profilático com corticosteroides, de tratamento com corticosteroides e no grupo modelo. O extravasamento retinal de azul de Evans observado nos grupos de tratamento com anti-inflamatórias não esteroides, de tratamento profilático com corticosteroides e de tratamento com corticosteroides foi inferior ao observado no grupo modelo. Conclusões: Este estudo valida que o antagonista das prostaglandinas pode aliviar a ruptura da barreira hemato-retiniana em um modelo em ratos e que o tratamento profilático com anti-inflamatórias não esteroides pode alcançar melhor eficácia.

ABSTRACT Purpose: To evaluate the efficacy of prostaglandin antagonists on blood-retinal barrier breakdown induced by anterior segment intraocular simulated surgery. Methods: Rats were randomly assigned to a negative control group, model group, nonsteroidal anti-inflammatory drugs prophylactic treatment group, nonsteroidal anti-inflammatory drugs treatment group, corticosteroid prophylactic treatment group, and corticosteroid treatment group. Four hours and 48h after modeling, the concentrations of PGE1, PGE2, and PGF2 α in the aqueous humor and vitreous body of the rat model were visualized using ELISA. The integrity of the blood-retinal barrier was quantitatively measured using Evan’s blue as a tracer. Results: Four hours after modeling, the concentrations of PGE1, PGE2, and PGF2α in the aqueous humor and vitreous body in the negative control group and the nonsteroidal anti-inflammatory drugs prophylactic treatment group were significantly lower than those in the model group. The concentrations of PGE1, PGE2, and PGF2α in the aqueous humor and vitreous body in the corticosteroid prophylactic treatment group were higher than those in the negative control group and the nonsteroidal anti-inflammatory drugs prophylactic treatment group. Forty-eight hours after modeling, the concentrations of PGE1, PGE2, and PGF2α in the aqueous humor and vitreous body in the nonsteroidal anti-inflammatory drugs prophylactic treatment group, nonsteroidal anti-inflammatory drugs treatment group, corticosteroid prophylactic treatment group, and corticosteroid treatment group were lower than those in the model group, but higher than those in the negative group. Retinal Evan’s blue leakage in the nonsteroidal anti-inflammatory drugs prophylactic treatment group was higher than that in the negative control group, and lower than those in the nonsteroidal anti-inflammatory drugs treatment group, corticosteroid prophylactic treatment group, corticosteroid treatment group, and model group. Retinal Evan’s blue leakage in the nonsteroidal anti-inflammatory drugs treatment group, corticosteroid prophylactic treatment group, and corticosteroid treatment group were lower than those in the model group. Conclusions: This study confirms that prostaglandin antagonists can relieve blood-retinal barrier breakdown in a rat model and that nonsteroidal anti-inflammatory drugs prophylactic treatment can achieve better efficacy.