This study aims to investigate the mechanism by which resveratrol(RES) alleviates cerebral vascular endothelial damage in sepsis-associated encephalopathy(SAE) through network pharmacology and animal experiments. By using network pharmacology, the study identified common targets and genes associated with RES and SAE and constructed a protein-protein interaction( PPI) network. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed to pinpoint key signaling pathways, followed by molecular docking validation. In the animal experiments, a cecum ligation and puncture(CLP) method was employed to induce SAE in mice. The mice were randomly assigned to the sham group, CLP group, and medium-dose and high-dose groups of RES. The sham group underwent open surgery without CLP, and the CLP group received an intraperitoneal injection of 0. 9% sodium chloride solution after surgery. The medium-dose and high-dose groups of RES were injected intraperitoneally with 40 mg·kg-1 and 60 mg·kg~(-1) of RES after modeling, respectively, and samples were collected 12 hours later. Neurological function scores were assessed, and the wet-dry weight ratio of brain tissue was detected. Serum superoxide dismutase(SOD), catalase( CAT) activity, and malondialdehyde( MDA) content were measured by oxidative stress kit. Histopathological changes in brain tissue were examined using hematoxylin-eosin(HE) staining. Transmission electron microscopy was employed to evaluate tight cell junctions and mitochondrial ultrastructure changes in cerebral vascular endothelium. Western blot analysis was performed to detect the expression of zonula occludens1( ZO-1), occludin, claudins-5, optic atrophy 1( OPA1), mitofusin 2(Mfn2), dynamin-related protein 1(Drp1), fission 1(Fis1), and hypoxia-inducible factor-1α(HIF-1α). Network pharmacology identified 76 intersecting targets for RES and SAE, with the top five core targets being EGFR, PTGS2, ESR1, HIF-1α, and APP. GO enrichment analysis showed that RES participated in the SAE mechanism through oxidative stress reaction. KEGG enrichment analysis indicated that RES participated in SAE therapy through HIF-1α, Rap1, and other signaling pathways. Molecular docking results showed favorable docking activity between RES and key targets such as HIF-1α. Animal experiment results demonstrated that compared to the sham group, the CLP group exhibited reduced nervous reflexes, decreased water content in brain tissue, as well as serum SOD and CAT activity, and increased MDA content. In addition, the CLP group exhibited disrupted tight junctions in cerebral vascular endothelium and abnormal mitochondrial morphology. The protein expression levels of Drp1, Fis1, and HIF-1α in brain tissue were increased, while those of ZO-1, occludin, claudin-5, Mfn2, and OPA1 were decreased. In contrast, the medium-dose and high-dose groups of RES showed improved neurological function, increased water content in brain tissue and SOD and CAT activity, and decreased MDA content. Cell morphology in brain tissue, tight junctions between endothelial cells, and mitochondrial structure were improved. The protein expressions of Drp1, Fis1, and HIF-1α were decreased, while those of ZO-1, occludin, claudin-5, Mfn2, and OPA1 were increased. This study suggested that RES could ameliorate cerebrovascular endothelial barrier function and maintain mitochondrial homeostasis by inhibiting oxidative stress after SAE damage, potentially through modulation of the HIF-1α signaling pathway.
Animals ; Mice ; Network Pharmacology ; Resveratrol/administration & dosage* ; Male ; Sepsis-Associated Encephalopathy/genetics* ; Signal Transduction/drug effects* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Endothelium, Vascular/metabolism* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Humans ; Sepsis/complications* ; Oxidative Stress/drug effects*

Microglial pyroptosis and neuroinflammation have been implicated in the pathogenesis of sepsis-associated encephalopathy (SAE). OGT-mediated O-GlcNAcylation is involved in neurodevelopment and injury. However, its regulatory function in microglial pyroptosis and involvement in SAE remains unclear. In this study, we demonstrated that OGT deficiency augmented microglial pyroptosis and exacerbated secondary neuronal injury. Furthermore, OGT inhibition impaired cognitive function in healthy mice and accelerated the progression in SAE mice. Mechanistically, OGT-mediated O-GlcNAcylation of ATF2 at Ser44 inhibited its phosphorylation and nuclear translocation, thereby amplifying NLRP3 inflammasome activation and promoting inflammatory cytokine production in microglia in response to LPS/Nigericin stimulation. In conclusion, this study uncovers the critical role of OGT-mediated O-GlcNAcylation in modulating microglial activity through the regulation of ATF2 and thus protects against SAE progression.
Animals ; Microglia/metabolism* ; Pyroptosis/physiology* ; Mice ; Sepsis-Associated Encephalopathy/prevention & control* ; Activating Transcription Factor 2/metabolism* ; N-Acetylglucosaminyltransferases/genetics* ; Mice, Inbred C57BL ; Male ; Mice, Knockout

OBJECTIVES: Sepsis-associated encephalopathy (SAE) is a common complication of sepsis, which can lead to long-term cognitive impairment and anxiety in patients, and may even contribute to mortality in septic individuals. There is substantial individual variability in the incidence and severity and susceptibility of SAE, but the mechanisms regulating susceptibility remain unclear. Previous studies have shown that hippocampal damage is directly associated with cognitive and emotional disturbances in SAE. This study aims to explore the impact of hippocampal differentially expressed genes on SAE susceptibility in a mouse model. METHODS: Male specific pathogen-free (SPF)-grade C57BL/6 mice (6-8 weeks old) were randomly divided into a saline control group (Con group) and an SAE model group. SAE was induced by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS), while control mice received an equivalent volume of saline. Cognitive and anxiety-like behaviors were assessed using the open field test (OFT), novel object recognition (NOR), and Y-maze test. Based on mean±standard deviation of behavioral results from the Con group, SAE mice were further classified into high-sensitivity (HS) and low-sensitivity (LS) subgroups. Immunohistochemistry was performed to detect the expression of immediate early gene c-Fos and neuronal marker neuronal nuclei (NeuN). Nissl staining was used to assess neuronal injury in the dentate gyrus (DG), cornu ammonis 1 (CA1), and cornu ammonis 3 (CA3) regions of the hippocampus. RNA sequencing (RNA-seq) was conducted on hippocampal tissues from HS and LS mice to identify differentially expressed genes, followed by pathway enrichment analysis. RESULTS: No significant behavioral susceptibility differences were observed between the overall SAE group and controls. However, HS mice showed severer cognitive deficits and anxiety-like behavior compared to LS mice. Immunohistochemistry revealed significantly higher expression of c-Fos in the hippocampus of LS mice (P<0.05), while Nissl and NeuN staining revealed milder neuronal damage in the hippocampus of LS mice than that of HS mice (both P<0.05). RNA-seq analysis identified 130 upregulated and 142 downregulated DEGs in LS and HS mice, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that upregulated genes in LS mice were primarily involved in pluripotency regulation, cyclic adenosine monophosphate (cAMP) signaling, and Wnt signaling pathways, in contrast, the downregulated genes were mainly related to cell adhesion, neuroactive ligand-receptor interaction, and calcium signaling pathways. CONCLUSIONS Differential gene expression in the hippocampus may contribute to individual susceptibility to cognitive and emotional dysfunction in SAE, suggesting potential genetic targets for individualized intervention.
Animals ; Sepsis-Associated Encephalopathy/genetics* ; Male ; Hippocampus/pathology* ; Mice, Inbred C57BL ; Mice ; Anxiety/genetics* ; Lipopolysaccharides ; Genetic Predisposition to Disease ; Disease Models, Animal ; Sepsis/genetics*