ITPR2 regulates microglial pyroptosis by Ca2+ transfer through mitochondria-ER contacts in Diabetic Retinopathy

Diabetic retinopathy (DR) is a chronic diabetes-induced retinal microvascular complication that can lead to blindness. While vascular dysfunction and inflammation are recognized contributors, the regulatory mechanisms governing pyroptosis in DR remain poorly defined. Inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) has been implicated in calcium signaling and inflammatory responses, yet its role in DR pathogenesis is unclear. This study aimed to investigate whether ITPR2 regulates mitochondria-associated endoplasmic reticulum membrane formation, NLRP3 inflammasome activation, and microglial pyroptosis in DR. A diabetic rat model was established by intraperitoneal streptozotocin injection, and an advanced glycation end products (AGEs) -treated microglial cell model was used for in vitro studies. ITPR2 expression was manipulated by siRNA knockdown and plasmid overexpression. Mitochondrial morphology and function were assessed by transmission electron microscopy, MitoTracker staining, oxygen consumption rate, mitochondrial permeability transition pore opening, and membrane potential assays. Endoplasmic reticulum-mitochondria contacts were evaluated by imaging and Pearson correlation analysis. Pyroptosis was measured by flow cytometry, Western blotting of NLRP3, GSDMD-N, caspase-1, and IL-1β, and ELISA of inflammatory cytokines. Blood-retinal barrier integrity was examined by tight junction protein expression, FITC-dextran permeability, and transendothelial electrical resistance. ITPR2 expression was elevated in diabetic rat retinas and AGEs stimulated microglia. ITPR2 knockdown suppressed AGEs induced mitochondria-associated endoplasmic reticulum membrane formation, reduced mitochondrial calcium overload, and ameliorated mitochondrial structural damage and dysfunction. These changes attenuated NLRP3 inflammasome activation and microglial pyroptosis, thereby reducing proinflammatory cytokine release. Consequently, endothelial tight junction protein degradation was alleviated and blood-retinal barrier permeability decreased. In vivo, intravitreal injection of adeno-associated virus shRNA targeting ITPR2 mitigated diabetes induced retinal capillary leakage, reduced acellular capillary formation, preserved tight junction protein expression, and suppressed microglial activation and pyroptosis in diabetic rats. ITPR2 promotes DR progression by enhancing mitochondria-associated endoplasmic reticulum membrane formation, driving mitochondrial calcium overload and dysfunction, and activating NLRP3-mediated microglial pyroptosis, which ultimately disrupts the blood-retinal barrier. Targeting the ITPR2 signaling pathway may represent a promising therapeutic strategy for alleviating DR.