Assessment of methylene blue and NAD on LPS-induced acute brain injury: effects on neuroinflammation, oxidative stress, and blood–brain barrier integrity in a rat endotoxemia model

Sepsis-induced brain dysfunction remains a significant challenge with limited therapeutic options, warranting exploration of multi-target approaches. Methylene blue (MET) is a phenothiazine derivative with mitochondrial-enhancing and electron transport chain regulatory properties, while nicotinamide adenine dinucleotide (NAD) serves as a crucial cofactor in cellular energy metabolism and redox reactions. This study investigated the therapeutic effects of MET and NAD, individually and in combination, in lipopolysaccharide (LPS)-induced sepsis model. Male Sprague–Dawley rats (n = 35) were randomized into five groups: control, LPS (8 mg/kg), LPS + NAD (250 mg/kg), LPS + MET (15 mg/kg), and LPS + NAD + MET. Brain tissues were analyzed for oxidative stress, inflammation, and blood–brain barrier integrity. The combination therapy demonstrated the most pronounced improvements across clinical parameters, antioxidant capacity evidenced by significantly elevated superoxide dismutase levels (p < 0.001) and cerebral histopathological outcomes. It also significantly reduced myeloperoxidase activity (p < 0.001) compared to the LPS group. Pro-inflammatory cytokines IL-6 and IL-8 were significantly decreased in both MET and NAD monotherapy groups (p < 0.05), while TNF-α showed a decreasing trend without statistical significance. Brain-derived neurotrophic factor levels were maintained in the combination group, comparable to controls, while TGF-β showed differential modulation across treatment groups. Immunohistochemical analysis revealed LPS-induced blood–brain barrier disruption, evidenced by reduced occludin expression (p < 0.01), increased aquaporin-4 expression in perivascular astrocytic end-feet (p < 0.05), and elevated glial fibrillary acidic protein reactivity in protoplasmic astrocytes (p < 0.01). In conclusion, MET and NAD individually provided significant protection against LPS-induced brain injury by reducing neuroinflammation, oxidative stress, and neuronal damage. The combined treatment offered incremental benefits in certain outcomes. These findings suggest that both agents may have therapeutic potential in SAE. © The Author(s) 2026.