The Mechanism Of Amitriptyline Alleviating Central Nervous System Inflammation By Suppressing Microglia Pyroptosis In Sepsis Survivor Mice

Objectives:To study the mechanism of Amitriptyline(AMT)alleviating inflammation in the central nervous system by constructing the sepsis mouse model using cecum ligation and puncture..Methods:Thirty C57BL/6 mice were divided into two groups randomly:SHAM group and CLP group.The tissue protein of hippocampus and amygdala were taken to Western Blot for detecting the protein expression of NLRP3 and c-caspase-1.The open field,new object recognition and other behavioral test were used to test cognitive impairment,anxiety and depression of sepsis survivor mice after CLP surgery.In addition,BV2 cells were stimulated in vitro by LPS and pretreated by AMT by Flow cytometry to test the number of microglial activation and Western Blot to detect the protein expression of NLRP3 and c-caspase-1 and CCK8 kit for cytotoxicity.Eighty C57BL/6 mice were randomly divided into 4 groups:SHAM group,SHAM+AMT group,CLP group and CLP+AMT group.We recorded the survival rate,body weight and temperature of mice in each group within 14 days after surgery.On the twentyeighth day after CLP surgery,we used Western Blot tests to detect the protein expression of NLRP3 and c-caspase-1 in hippocampus and amygdala and ELISA kit to test the expression of IL-1β cytokines,the paraffin slice Nissl staining technique was used to observe neuronal injure in hippocampus and amygdala.New object recognition and open field tests were used to detect cognitive impairment,anxiety and depression disorder.Results:1.Compared with the SHAM group mice,the expression of pyroptosis related proteins was high in the hippocampus and amygdala on 3d,7d,14d and 28d after surgery in the CLP group mice.On fourteenth day after CLP,open field test showed that the total distance of mice in CLP group was lower than the SHAM group mice.In twenty-eighth day after CLP,compared to the SHAM group mice,the CLP group mice spent less time on central region and had lower frequency in crossing line.In new object recognition test,the recognition index in CLP group mice decreased after surgery compared with SHAM group.So,behavior tests showed that sepsis survivor mice had cognitive impairment and depression-like behavior in twenty-eighth day after surgery.2.BV2 cell experiments showed that the LPS+AMT group had the lower number of the BV2 cells activation compared with the LPS group and AMT could alleviate the activation of BV2 cells induced by LPS.Compared with the LPS+ATP group,the protein expression of NLRP3 and c-caspase-1 was less and the number of living cells was increased in the LPS+ATP+AMT group,so AMT could suppress the pyroptosis in BV2 cells.3.Compared with the CLP group mice,the survival rate was improved and the sepsis clinical score was decreased in the CLP+AMT group mice.The protein expression levels of NLRP3,c-caspase-1 and IL-1β in the hippocampus and amygdala were decreased in the CLP+AMT group mice after 28 days compared with the CLP group mice.The paraffin slice Nissl staining showed that hippocampal and amygdala tissues were deeply stained in the CLP+AMT group mice,so the AMT could alleviate the central nervous system inflammation damage in sepsis survivor mice.The behavior tests showed that the CLP+AMT group mice spent more time on central region and had higher frequency in crossing line compared with CLP group mice,and recognition index were increased,and immobility time in forced swimming test and tail suspension test were decreased in the CLP+AMT group mice.So AMT alleviated cognitive dysfunction and depression-like behavior in sepsis survivor mice.Conclusions:By suppressing the NLRP3-c-caspase-1-IL-1β signaling pathway in microglia,AMT alleviated inflammation in the hippocampus and amygdala,and alleviated cognitive impairment and depression-like behavior in sepsis survivor mice.