Role of Necroptosis and Neuroinflammation in <scp>CSVD</scp> ‐Associated Cognitive Decline in db/db Mice

Diabetes-related cerebral small-vessel disease (CSVD) is an important causative factor of cognitive impairment, but its molecular mechanisms have not been clarified. The aim of this study was to investigate the role of the necrotic apoptotic pathway (RIP1/RIP3/MLKL) and the inflammatory response in diabetic CSVD. Wild-type C57BL/6 mice and leptin receptor-deficient db/db mice were categorized into six groups according to age (8-, 12-, and 16-week time points) and genotype. Cognitive function was assessed by the water maze experiment (escape latency, percentage of time spent in the target quadrant, and number of times through the table); cerebral atrophy and ventricular dilatation were detected by cranial MRI; cerebral microvascular structure, cortical neuronal damage, and ultrapathological changes in hippocampal mitochondria were observed by HE staining and transmission electron microscopy, respectively; and blood-brain barrier-associated proteins were detected by western blot and RT-qPCR (occludin, ZO-1, VEGFA) and necroptotic apoptotic pathway molecules (RIP1, RIP3, MLKL). Western blot and RT-qPCR were used to detect the protein and mRNA expression of blood-brain barrier-associated proteins (occludin, ZO-1, VEGFA) and necroptotic apoptotic pathway molecules (RIP1, RIP3, MLKL). Immunohistochemistry was used to localize the distribution of RIP1/RIP3/MLKL in the brain tissues; and plasma levels of inflammatory factors (IL-6, IL-10, TNF-α, NF-κB) were quantified by ELISA. In db/db mice: (1) spatial learning and memory abilities were reduced compared to wild-type (WT) mice; (2) at 16 weeks of age, db/db mice showed signs of temporal lobe atrophy and an enlarged fourth cerebral ventricle; (3) capillary proliferation and cortical injury were observed in the frontal cortex, along with mitochondrial swelling, degeneration, and nuclear membrane rupture in hippocampal cells; (4) the occludin and ZO-1 protein expression in the db/db-16W group decreased to 0.48- and 0.68-fold of the WT-8W group, respectively, and the VEGFA was elevated by 2.87-fold; the mRNA expression of RIP1/RIP3/MLKL was up-regulated to 3.02-, 3.12-, and 4.02-fold of the WT group, the relative expression of western blot proteins increased synchronously; the increase in the number of immunohistochemically positive cells increased synchronously, and (5) plasma inflammatory factors were significantly elevated in db/db mice: IL-6 ↑3.81-fold, TNF-α ↑4.23-fold, NF-κB ↑3.56-fold. This study reveals for the first time the molecular mechanism by which diabetes drives cerebral small vessel disease (CSVD) through the spatiotemporal-dependent activation of the necrotic apoptotic pathway (RIP1/RIP3/MLKL), and targeting the necrotic apoptotic pathway may serve as a potential therapeutic strategy for diabetes-associated cognitive deficits by concurrently protecting the blood-brain barrier and suppressing neuroinflammation.