Mechanism Of Caspase-1 Inhibitor In Improving Sepsis-associated Encephalopathy In Mice

Background: Sepsis-associated encephalopathy(SAE)is an encephalopathy caused by sepsis with high mortality.Clinical manifestations include cognitive dysfunction,memory loss,and emotional abnormalities,which seriously affect the quality of life of survivors..The mechanism of sepsis-associated encephalopathy is complicated.Previous studies have found that sepsis-associated encephalopathy is associated with the destruction of the blood-brain barrier,and is closely related to microglia activation.Studies have found that in traumatic brain injury,ischemic encephalopathy and intracranial infection and other cerebral diseases,cell pyroptosis in the brain exacerbates the development of the disease.However sepsis-associated encephalopathy still lacks relevant evidence.In our previous studies,we found that peripheral blood mononuclear cells happened to pyroptosis in sepsis patients.Therefore,in this study,animal experiments were conducted to observe whether cell pyroptosis happens in the brain during sepsis-associated encephalopathy,and the usage of cell pyroptosis inhibitors plays a protective role.Objective: To investigate the mechanism of Caspase-1 inhibitor in ameliorating sepsis-associated encephalopathy in mice.Methods : A sepsis model was established by CLP(cecal ligation and puncture).Male BALB/c mice,aged 8-12 weeks and weighing 20-25 g were selected and randomly divided into 3 groups: sham operation group(Sham group),cecal ligation and puncture group(CLP group),cecal ligation and puncture with VX-765 group(VX-765 group).The VX-765 group was intragastrically administered twice daily with VX-765(1 mg/ml)0.2 ml/per one.The survival rate of the mice was recorded for seven consecutive days after surgery.The following experiments were performed on 1 and 7 days after surgery:(1)Behavioral analysis: novel object recognition test,open field test,tail suspension test and elevated plus maze;(2)Cytometric Bead Assay(CBA)was used to determine whether there has correlation between peripheral blood and brain in the expression of inflammatory cytokines;(3)Transmission electron microscopy was used to observe changes in blood-brain barrier,brain neurons and mitochondrial ultrastructure;(4)Immunofluorescence was used to observe activation microglia in hippocampus and cortex;(5)Western blot was used to determine the expression of Caspase-1 and its active form,GSDMD and its fragment in the brain.Results:(1)VX-765 significantly improved the survival rate of septic mice and ameliorated cognitive dysfunction and mood changes;(2)The blood-brain barrier of the septic mice was destroyed,the tight junctions were interrupted,the number of mitochondria was reduced,and the nucleoli of the endothelial cell was marginalized.At the same time,expressions of pro-inflammatory cytokines IL-1?,TNF-? and MCP-1 in plasma were found to increase in parallel with those in brain tissue.Microglia in the brain is activated,the expression of Caspase-1 and its active forms,GSDMD and its cleavage are increased.(3)After the administration of Caspase-1 inhibitor(VX-765),the blood-brain barrier of septic mice was repaired and the number of mitochondria increased.Moreover,expressions of pro-inflammatory cytokines,pyroptosis-related proteins and the activation of microglia in plasma and brain tissue of septic mice decreased.Conclusions: Caspase-1 inhibitor can effectively improve sepsis-associated encephalopathy,protect the blood-brain barrier function by inhibiting the expression and release of peripheral pro-inflammatory cytokines,and effectively control the inflammatory storm in the brain which would further destroy the brain tissue and cause cognitive dysfunction.