To Explore The Role Of SS-31 In Regulating Drp1 In Sepsis-associated Encephalopathy

Background:Neuroinflammation is one of the main pathogenesis of sepsis-associated encephalopathy(SAE),involving pathological processes such as microglia activation and mitochondrial damage.Increased inflammatory factors and uncontrolled inflammation caused by microglia activation have been demonstrated.Mitochondrial dysfunction can activate microglia,which play an important role in innate immunity and are closely related to the activation of the NLRP3 inflammasome.This paper focuses on the role of mitochondrial protective peptide SS-31 in regulating dynamo-related protein Drp1 in SAE.Objective: 1.To investigate the effect of SS-31 on the cognitive function of SAE mice.2.To explore the relationship between mitochondria and NLRP3 inflammasome-mediated neuroinflammation,and the role of SS-31.Method: 1.After the mice underwent cecal ligation and perforation,they were given intraperitoneal administration the mitochondrial targeting peptide SS-31 for 7 consecutive days,and the concealed platform experiment was performed for 6 days,the space exploration experiment was performed on the 7th day,and the mice were sacrificed on the 8th day.2.Prepared frozen sections of mouse brain,and detected the expression level of interleukin-1β(IL-1β)in the hippocampus and the expression of NLRP3,Caspase1 and p-Drp1(ser616)in Iba1-labeled cells by immunofluorescence assay;3.Use ROS kit to detect the content of ROS in the hippocampus of mouse brain;4.Electron microscope to detect the morphological changes of mitochondria in hippocampus;5.The mouse microglia cell line BV-2 cells were pretreated with different concentrations(0,0.1,0.5,1,3,5 and 10μmol/L)of SS-31 for 24 hours,and then BV-2 cells were detected using CCK8 kit;6.BV-2 cells were pretreated with SS-31,followed by LPS/ATP induction for 24 hours,Western Blot method to detect the protein NLRP3,Caspase1,cleaved-Caspase1,GSDMD,GSDMD-N,Drp1 and p-Drp1(ser616)expression level;7.The size of mitochondria of BV-2 cells after treatment was observed by electron microscope;8.The cytoplasm and mitochondria of BV-2 cells were obtained by sucrose gradient centrifugation,and then the levels of Drp1,GSDMD and GSDMD-N in cytoplasm and mitochondria were detected by Western Blot method.9.Use small interfering RNA to knock down Drp1 gene in BV-2 cells,and then induce LPS/ATP for 24 hours.Western Blot method was used to detect the expression levels of proteins NLRP3,Caspase1,cleaved-Caspase1,GSDMD,GSDMD-N and Drp1.Results: 1.SS-31 improves the survival rate of CLP mice and improves the spatial memory function of mice;2.SS-31 inhibits the production of ROS in the hippocampus of CLP mice,excessive mitochondrial fission,and the protein expression of IL-1β,NLRP3 and p-Drp1 in Iba1-labeled cells;3.SS-31 attenuates LPS/ATP-induced NLRP3 inflammasome activation,Drp1 mitochondrial recruitment and mitochondrial over-fission in BV-2 cells;Knockdown of Drp1 inhibits LPS/ATP-induced NLRP3 inflammasome activation in BV-2 cells.Conclusion: SS-31 inhibits Drp1-mediated excessive mitochondrial fission,thereby attenuating the hippocampal neuroinflammatory response induced by NLRP3 inflammasome activation in microglia and improving cognitive function in SAE mice.