Effect Of TAM Receptor On TLR4/MYD88 Signaling Pathway Expression In Acute Carbon Monoxide Poisoning Delayed Encephalopathy Mice Model

Objective:To establish a mouse model of delayed encephalopathy after carbon monoxide poisoning(DEACMP),To explore the expression of TLR4/MYD88 signaling pathway in the hippocampus of DEACMP model mice.To explore the effect of TAM receptor deletion on the expression of TLR4/MYD88 signaling pathway under this condition,so as to provide more evidence and ideas for the study of the pathogenesis of DEACMP.Methods:Wild type,Axl and Mer double knockout mice were bred,and gene determination was performed on the knockout mice.Male mice aged 8 to 10 weeks with qualified cognitive function were selected by water maze experiment and randomly divided into control group(exposed to air only)and model group(CO poisoning group),and were further divided into wild-type control group(WT control),Axl and Mer double gene knockout control group(AM-/-control)according to genotype.Wild-type DEACMP model group(WT model group)and Axl and Mer double-gene knockout DEACMP model group(AM-/-model group)were further divided into 3 subgroups,and the time nodes of each subgroup were 7,14 and 21 days after CO poisoning,respectively.The static inhalation method was used to establish the DEACMP mouse model.Morris water maze experiment was used to observe the behavioral changes of each group of mice.HE staining method was used to observe the morphological and pathological changes of neurons in the hippocampus of mice.Western Blot was used to detect the expression of TLR4 and MYD88 protein in the hippocampus of mice in each group.The expression of inflammatory cytokines IL-6,TNF-α and IL-1β in the hippocampal tissue of mice in each group was quantitatively detected by Elisa.Results:1.Morris Water Maze experiment:there was no significant difference in the escape latency of water maze in the control group at different times;In the model group,there was no significant difference in escape latency on the 7th day after CO poisoning compared with before modeling(P>0.05),but the escape latency on the 14th and 21st days after CO poisoning was gradually prolonged(P<0.05).Compared with WT model group,the escape latency of mice in AM-/-model group was significantly prolonged on the 14th and 21st days after CO poisoning(P<0.05).2.HE staining results:There were no obvious structural changes in hippocampal neuron cells of mice in the control group,while the number of cells in the hippocampal region of mice in the model group was reduced,the arrangement was loose,and some necrotic cells with nuclear pyrosis and nuclear fragmentation could be seen,among which the pathological changes of hippocampal tissue were more obvious in the AM-/model group.3.Western Blot:Compared with WT control group,TLR4 and MYD88 protein expressions in hippocampus of WT model group were significantly increased at 7,14 and 21 days after CO poisoning(P<0.05).Compared with AM-/-control group,the protein expressions of TLR4 and MYD88 in the hippocampus of mice in AM-/-model group were significantly increased at 7,14 and 21 days after poisoning(P<0.05).The protein expressions of TLR4 and MYD88 in AM-/-model group were significantly higher than those in WT model group(P<0.05).In the WT and AM-/-model groups,the expression of TLR4 and MYD88 protein firstly increased and then decreased,and reached the peak on the 14th day after CO poisoning.4,Elisa:Compared with WT control group,the expressions of IL-6,IL-1βand TNF-α in hippocampus of WT model group were significantly increased at 7,14 and 21 days after poisoning,with statistical significance(P<0.05);Compared with AM-/-control group,the expression of inflammatory factors in AM-/-model group was significantly increased at 7,14 and 21 days after poisoning(P<0.05),and the expressions of IL-6,IL-1βand TNF-α in AM-/-model group were higher than those in WT model group(P<0.05).Conclusion:1.Static inhalation poisoning method can be used as an ideal method to establish DEACMP mouse model,which can well simulate clinical poisoning process,and has the characteristics of simple operation,high safety and consistent poisoning dose.2.The successful preparation of the model can be judged according to the behavioral changes of Morris water maze and histopathological changes of hippocampus after carbon monoxide poisoning in mice.3.In the pathogenesis of DEACMP,the deletion of Axl and Mer receptors in TAM family may aggravate the impairment of cognitive function.4.TLR4,MYD88,and downstream inflammatory factors IL-6,IL-1β,and TNF-α were continuously highly expressed in the hippocampus of DEACMP model mice,and the expression levels increased first and then decreased,suggesting that TLR4/MYD88 signaling pathway may be involved in the occurrence and development of DEACMP.5.In the pathogenesis of DEACMP,Axl and Mer deletion in TAM family will lead to increased expression of TLR4/MYD88 signaling pathway.