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Abstract

Abstract Background Diabetic cognitive dysfunction (DCD) is a prevalent hyperglycaemia-induced central nervous system complication driven by hippocampal neuroinflammation. Glycoprotein non-metastatic melanoma protein B (GPNMB) exerts anti-inflammatory neuroprotection in neurodegenerative diseases, but its role and mechanism in DCD are undefined. SMAD4 restrains NF-κB-mediated microglial inflammation, and the physical and functional GPNMB–SMAD4 interaction in diabetic brain injury has not been explored. This study aimed to uncover GPNMB’s protective effects against DCD and its SMAD4/NF-κB-dependent regulatory pathway. Methods We established streptozotocin (STZ)-induced diabetic C57BL/6J mice and high-glucose (HG)-stimulated primary hippocampal microglia as in vivo and in vitro models. Stereotactic intracerebroventricular injection of adeno-associated virus-GPNMB (AAV-GPNMB) upregulated hippocampal GPNMB, with KGN (SMAD4 agonist) and A83-01 (SMAD4 inhibitor) utilized for rescue assays. Cognitive performance was evaluated via the Morris water maze. Immunofluorescence and immunohistochemistry detected microglial morphology, GPNMB colocalization and myelin integrity. Western blot and qPCR measured levels of GPNMB, SMAD4, p-NF-κB, M1/M2 markers and inflammatory cytokines. Co-immunoprecipitation (Co-IP) and molecular docking confirmed the GPNMB–SMAD4 protein interaction. Statistical comparisons were conducted using Student’s t-test and one-way ANOVA with Tukey’s post-hoc test. Results GPNMB mRNA and protein were downregulated in diabetic mouse hippocampi and HG-treated microglia. AAV-mediated GPNMB overexpression rescued cognitive impairment, inhibited excessive microglial M1 polarization, normalized inflammatory cytokine profiles, and restored myelin MBP/CNPase levels in vivo and in vitro. Bioinformatic prediction, molecular docking, Co-IP and immunofluorescence validated direct GPNMB-SMAD4 binding. Hyperglycemia depleted SMAD4 and triggered NF-κB activation, whereas SMAD4 stimulation suppressed NF-κB-driven neuroinflammation. SMAD4 inhibition by A83-01 fully eliminated GPNMB’s neuroprotective, anti-inflammatory and anti-demyelinating benefits. Conclusion Hyperglycemia suppresses microglial GPNMB, disrupts GPNMB-SMAD4 binding, and de-represses NF-κB signaling. This drives M1 microglial polarization, neuroinflammation, hippocampal demyelination and cognitive deficits. GPNMB overexpression mitigates DCD via SMAD4 stabilization and NF-κB suppression. The GPNMB/SMAD4/NF-κB cascade is a viable therapeutic target for DCD.