Effects Of NLRP3 Inhibitor MCC950 On Cognitive Function In Sepsis-associated Encephalopathy Or Isoflurane-anesthesia Mouse

Section 1: Role of NLRP3 inflammasome in cognitive impairments in a mouse model of sepsis-associated encephalopathyObjective: Sepsis-associated encephalopathy(SAE)is a common complication that is associated with long-term cognitive impairments and increased mortality in sepsis survivors,yet the underlying mechanisms remain unclear and thus an effective intervention is still lacking.Nod-like receptor protein 3(NLRP3)inflammasome is the most widely investigated inflammasome.This study aimed to investigate the role of Nod-like receptor protein 3 inflammasome in cognitive impairments in a mouse model of SAE.Methods: Sixty adult male C57BL/6 mice were equally(random number)divided into two groups: sham group and CLP group.Mouse model of SAE was established by cecal ligation and puncture(CLP)surgery.Saline(10 m L/kg)was intraperitoneally injected 30 min before surgery and on day 1,2,4 and 6 after surgery according the grouping.Seven days after surgery,six mice in each group were deeply anesthetized and decapitated.The brain was rapidly removed to determine hippocampal contents of NLRP3,apoptosis-associated speck-like protein(ASC),interleukin-1?(IL-1?)and IL-18 by Western Blot.The remaining mice in each group were used in behavioral tests 14 days after surgery.Results: Compared with the sham group,the freezing time of context test was significantly decreased and the hippocampal contents of NLRP3,IL-1? and IL-18 were significantly increased in the CLP group.The results of open field,the freezing time of tone test and the content of ASC have no significant difference.Conclusion: The SAE mice showed significant cognitive impairments after CLP operation,which is probably due to the over activation of NLRP3 inflammasome and releasing of downstream inflammatory cytokines.Section 2: Effects of NLRP3 inhibitor MCC950 on cognitive function in a mouse model of sepsis-associated encephalopathyObjective: MCC950 is a newly developed,selective,small-molecule NLRP3 inflammasome inhibitor that can freely cross the blood-brain barrier.To further investigate the role of Nod-like receptor protein 3 inflammasome in cognitive impairments in a mouse model of SAE,we applied the Nod-like receptor protein 3 inhibitor MCC950 to observe its effects on cognitive function in the SAE mice.Methods: One hundred adult male C57BL/6 mice were randomly(random number)divided into three groups: sham + saline group(n = 20,Sham group),CLP + saline group(n = 40,CLP group)and CLP + MCC950 group(n = 40,MCC950 group).Mouse model of SAE was established by cecal ligation and puncture(CLP)surgery.Saline(10 m L/kg)or MCC950(10 mg/kg)was intraperitoneally injected 30 min before surgery and on day 1,2,4 and 6 after surgery according the grouping.Seven days after surgery,six mice in each group were deeply anesthetized and decapitated.The brain was rapidly removed to determine hippocampal contents of NLRP3,ASC,IL-1? and IL-18 by Western Blot analysis and ELISA,and observe the NLRP3-immunoreactive cells in CA1 region by immunohistochemical analysis.The remaining mice in each group were used in behavioral tests 14 days after surgery.Results: When compared MCC950 group with the CLP group,the freezing time of context test was significantly increased,the hippocampal content of NLRP3,IL-1? and IL-18 were significantly reduced,and the number of NLRP3-immunoreactive cells per mm2 in CA region were significantly decreased.The results of open field,the freezing time of tone test and the content of ASC have no significant difference.Conclusion: Activation of NLRP3 inflammasome is supposed to be involved in the mechanism of SAE and associated cognitive impairments.MCC950 administration can improve cognitive function in a mouse model of SAE,which is probably due to the inhibiting of NLRP3 inflammasome and downstream inflammatory cytokines IL-1? and IL-18.Section 3: Effects of NLRP3 inhibitor MCC950 on cognitive function in a mouse model of isoflurane anesthesiaObjective: Isoflurane anesthesia can induce cognitive deficits in aged mammals.This section was to investigate the effects of NLRP3 inhibitor MCC950 on hippocampal pyroptosis and cognitive function in isoflurane-anesthesia aged mice.Methods: Seventy two aged male C57BL/6 mice were randomly divided into four groups(n=18/each group): oxygen(control)+ normal saline group(CN group),oxygen + MCC950 group(CM group),isoflurane + normal saline group(IN group)and isoflurane + MCC950 group(IM group).Normal saline or MCC950(10 mg/kg)was intraperitoneally injected 30 min before gas inhalation.Twenty four hours after the 2 h-gas inhalation,six mice in each group were decapitated and the brain was rapidly removed to determine the hippocampal contents of NLRP3,Pro-caspase-1,Cleaved caspase-1,Pro-Gasdermin-D(Pro-GSDMD),Cleaved GSDMD,interleukin-1?(IL-1?)and IL-18,and observe the neuronal morphological damage in CA1 region.The remaining mice in each group were used in behavioral tests 1 week after the gas inhalation.Results: Compared with the IN group,the hippocampal contents of NLRP3,Cleaved caspase-1,Cleaved GSDMD,IL-1? and IL-18 were decreased(P < 0.05),the neuronal morphological damage was reversed and the behavior in MWM tests was significantly improved in the IM group.Conclusion: Administration of NLRP3 inhibitor MCC950 can reverse isoflurane-induced neuronal damage and cognitive dysfunction,which is probably due to the inhibiting of NLRP3-dependent pyroptosis in the hippocampus of aged mice.