AbstractBackgroundSepsis‐associated encephalopathy (SAE) always manifests with severe inflammatory symptoms and cognitive impairment. High mobility group box 1 (HMGB1) is a pro‐inflammatory cytokine. In this study we investigated the role of HMGB1 in SAE.MethodsAn SAE mouse model was established through cecal ligation and puncture surgery and then injected with adenovirus short hairpin RNA (Ad‐sh)‐HMGB1 or Ad‐sh‐myeloid differentiation protein (MD‐2). The cognitive impairment and pathological injury in mice of different groups were evaluated using the Morris water maze experiment, Y‐maze test, tail suspension test, fear conditioning test, and haematoxylin‐eosin staining. The expressions of HMGB1 (fully reduced and disulfide (ds)HMGB1), MD‐2, and NLRP3 in SAE mice were determined. Then, levels of inflammatory cytokines were measured. The binding relation between HMGB1 and MD‐2 was predicted and certified. Additionally, MD‐2 was downregulated to verify the role of the binding of HMGB1 and MD‐2 in neuroinflammation and cognitive impairment in SAE.ResultsExpressions of HMGB1, MD‐2, NLRP3, and inflammatory cytokines were enhanced in the SAE mouse model, which were in parallel with impaired cognitive function. HMGB1 silencing resulted in downregulated NLRP3 expression and alleviated neuroinflammation and cognitive impairment in SAE mice. Mechanically, dsHMGB1 bound to MD‐2 to activate NLRP3, thereby exacerbating neuroinflammation and cognitive impairment in SAE mice. The limited binding of HMGB1 and MD‐2 downregulated NLRP3 expression to alleviate neuroinflammation and cognitive impairment in SAE mice.ConclusionHMGB1 was overexpressed in SAE, and dsHMGB1 bound to MD‐2 to activate NLRP3 inflammasome, inducing neuroinflammation and cognitive impairment in SAE.