Study On The Mechanism Of Soluble TREM-1 Protein Induced Hippocampus Damage In Mice

Background: Soluble triggering receptor expressed on myeloid cells-1(sTREM-1)is a member of the immunoglobulin superfamily.It is mainly expressed by myeloid cells and is elevated in a variety of neurodegenerative diseases.As the main immune cells in the brain,microglia are involved in almost all brain diseases.When the homeostasis of the brain is unbalanced,microglia microglia will proliferate rapidly,be activated by M1/M2 type,and respond to nerve damage,phagocytosis and clear damaged neuron cells to maintain the stability of the central nervous system.A number of studies have shown that the PI3K-AKT pathway not only plays an important role in various physiological functions of the brain,is also a key pathway for multiple members of the TREM family to play a regulatory role.Up to now,it is unclear whether the effect of high levels of sTREM-1 in the brain on brain function is related to the abnormal function of microglia,and whether it depends on the PI3K-AKT pathway to exert its regulatory mechanism.Objective: The purpose of this study was to analyze the effect of sTREM-1 on hippocampus of mice and to explore the specific mechanism.Methods: 1.Inject recombinant sTREM-1 protein into the lateral ventricle.Female C57BL/6J mice that aged 6-8 weeks were randomly divided into 6 groups: CON groups(24 h,72 h,120 h),sTREM-1 groups(24 h,72 h,120 h),12 animals in each group.The mice in the sTREM-1 group were injected with recombinant sTREM-1 protein into the lateral ventricle,while the mice in the CON group were treated with sterile saline as a control.H&E,TUNEL and FJB stained sections were used to evaluate the damage of the mouse hippocampus;QPCR and Western Blot to detect the expression of synaptic-related markers in the hippocampus;flow cytometry to detect the proportion of microglia in the hippocampus;immunofluorescence to detect the changes in phagocytic of microglia in the hippocampus.2.Culture BV2 mouse microglia and HT22 mouse hippocampal neuron cell lines in vitro.After treating BV2 with sTREM-1,CCK-8 and RTCA detected the changes of the number;flow cytometry and Western Blot were used to detect its apoptosis and phenotypic changes.Gene Sequencing detectd the changes in the function of BV2 cells;flow cytometry detected the phagocytic function of BV2 cells on fluorescent beads;BV2 cells treated with recombinant sTREM-1 protein were co-cultured with HT22 cells,Q-PCR and Western Blot detected the expression of synapse-related markers in HT22 cells.3.Detect the effect of sTREM-1 on PI3K-AKT pathway in vivo and in vitro.Q-PCR and Western Blot were used to detect PI3 K and AKT genes and phosphorylated PI3 K and phosphorylated AKT protein expression levels in mouse hippocampus and BV2 cells.4.Inhibited PI3K-AKT pathway and detected the changes of microglia phagocytic function in vivo and in vitro.Female C57BL/6J mice that aged 6-8 weeks were randomly divided into 3 groups: control group(CON group),sTREM-1 reatment group(sTREM-1 group),PI3K-AKT inhibitor treatment group(sTREM-1+LY group),9 animals in each group.PI3K-AKT inhibitor-LY294002 was injected into the lateral ventricle for 15 min,and then sTREM-1 was injected as described above.Q-PCR and Western Blot were used to detect the expression of synapse-related markers in the hippocampus;immunofluorescence was used to detect changes in the phagocytic function of microglia in the hippocampus.BV2 cells and HT22 cells were cultured in vitro,flow cytometry was used to detect the phagocytic ability of BV2 cells pretreated with LY294002 on fluorescent beads;Western Blot was used to detect its effect on the expression of synaptic markers in HT22 cells.5.Inhibit the PI3K-AKT pathway,H&E,TUNEL and FJB stained sections were used to evaluate the damage of the hippocampus.All data were analyzed using Graph Prism8 statistical software.The comparison between the two groups was performed by Student’s t test.One-way ANOVA was used to analyze the differences between multiple groups.P<0.05 indicated that the differences were statistically significant.Results: 1.sTREM-1 induced damage to the hippocampus of mice: First,the results of H&E stained sections showed that compared with the CON group,the structure of the nerve cells in the sTREM-1 group was blurred,the number was reduced,the arrangement was disordered,and the size and shape were irregular.Secondly,the results of TUNEL staining showed that the number of apoptotic cells in the sTREM-1 group was significantly increased。Finally,we found that the number of FJB-positive dying neurons in the sTREM-1 group increased significantly.These results indicated that the sTREM-1 had an inducing effect on hippocampus damage in mice.2.sTREM-1 promoted neuronal phagocytosis of microglia in the hippocampus of mice: In order to explore the cause of the hippocampus damage caused by sTREM-1,we tested the neuron phagocytosis functional changes of microglia in the brain tissue and found that the expression of synapse-related markers in pre-synaptic and post-synaptic neurons in sTREM-1 group was significantly lower than that in the CON group.Due to the neuron phagocytic characteristics of microglia are related to their activation,we further examined the activation state of microglia in the hippocampus and found that the proportion of resting and activated microglia in the sTREM-1 group was significantly increased.which indicated that the phagocytic function of microglia was enhanced.In order to clarify the effect of sTREM-1 on microglia,by treating BV2 cells with sTREM-1 in vitro,we found that sTREM-1 could promote the proliferation of BV2 cells,reduce apoptosis,induce M1 polarization,and reduce M2 phenotypes and promote their phagocytic function.These results indicated that sTREM-1 could promote the neuronal phagocytosis of microglia.3.Inhibition of PI3K-AKT pathway in microglia could improve hippocampus damage and abnormal phagocytosis caused by sTREM-1: In view of the important role of the PI3K-AKT pathway in brain tissue damage and the TREM family,we first detected the expression of PI3 K and AKT in different treatment groups,and found that compared with the CON group,the phosphorylation levels of PI3 K and AKT in sTREM-1 group both were significantly increased.In vitro studies had shown that after sTREM-1 treatment,the expression of phosphorylated PI3 K and phosphorylated AKT in BV2 cells was significantly up-regulated,indicating that sTREM-1 can directly promote the activation of the PI3K-AKT pathway in microglia.In order to clarify the role of PI3K-AKT pathway in sTREM-1-induced hippocampal damage and abnormal phagocytosis of neurons,the PI3K-AKT pathway inhibitor-LY294002 was injected into the lateral ventricle for pretreatment.We found that in vivo and in vitro studies confirmed that LY294002 reversed the excessive phagocytosis of microglia caused by sTREM-1,and the results of H&E,TUNEL,and FJB staining suggested that brain tissue damage was also improved accordingly.These results indicated that abnormal phagocytosis and brain damage caused by sTREM-1 depended on the PI3K-AKT pathway.Conclusion: This study found that sTREM-1 could induce damage to the mouse hippocampus by promoting the abnormal neurons phagocytosis of microglia,and its underlying mechanism was related to the activation of the PI3K-AKT pathway.The results of this study suggested that sTREM-1 might be a key target for inducing brain lesions.