Role Of TREM2 In The Learning And Memory Behavior And Neuro-injury Induced By Hypercholecterol In SH-SY5Y Cells

Triggering receptors expressed on myeloid cells(TREM)are a class of immunoglobulin super family expressed in myeloid cells,and TREM2 is one of its main members.Studies have shown that TREM2 is not only involved in inflammation and immune response,but also closely related to the regulation of synaptic plasticity,and the imbalance of TREM2 expression in hippocampus may be related to the pathogenesis of Alzheimer's Disease(AD).Cholesterol is a major component of the cell membrane.Hypercholesterolemia is an important risk factor for neuropsychiatric diseases such as AD.Abnormal accumulation of cholesterol in the brain can lead to?-amyloid accumulation and neuroinflammation to induce nerve damage.This study intends to observe and compare the differences in neuropsychology and hippocampus TREM2 expression in SD female rats at different ages,and to explore the possible effects of TREM2 in neuropsychiatric behavior,especially learning and memory behavior;by the cell model to observe the role of TREM2 in high cholesterol-induced neuronal injury in order to provide experimental evidence for the role of aging and high cholesterol stimulation in the pathogenesis of AD.This study is divided into two parts:1.The effects of different ages on learning and memory related behaviors and hippocampus TREM2 expression in female SD rats;2.The role of TREM2 in high cholesterol-induced neuronal damage.Objective: The aim of this study was to investigate the role of TREM2 in the learning and memory behaviors and high cholesterol-induced neuronal injury.Method:1.Animal model:Female Sprague-Dawley rats aging 2 months and 6 months were enrolled with 9rats in each group.The behavioral tasks were carried out after touching and adaptive feeding for 1 week,including the open field test,the elevated plus maze test,the Morris water maze test,the Y maze,and forced swimming test.The protein expression of TREM2 in the hippocampus was detected using Western blot technique.2.Cell model:The SH-SY5 Y cells were cultured and stimulated with different concentrations of cholesterol.MTT method was used to observe the time-and dose-related changes of cell injury induced by high cholesterol,based on which the experimental conditions of cholesterol stimulation was setThe SH-SY5 Y cells were routinely cultured and stimulated with cholesterol according to the results of time and dose-effect experiments.The morphology and function of SH-SY5 Y cells were observed by morphological observation,Oil Red O stain and flow cytometry.The protein expression of BDNF,Copine6,TREM1,TREM2 and key molecules in the Wnt signaling pathway were detected by Western blot;The expression of TREM2 in SH-SY5 Y cells was interfered by over-expression or siRNA interference,and the morphology and function of SH-SY5 Y cells were observed.Western blot was used to detect the expression of proteins involved in inflammation and synaptic plasticity regulation.Result:1.In vivo study:There was no significant difference between the two groups as regard to the moving distance,the duration in the center,and the frequencies of rearing and grooming in the open field test and the immobility in the forced swimming test.However,the sucrose preference index in the sucrose preference test,the duration in the target quadrant in the test phase of the Morris water maze,and the preference index of the novel arm in the Y maze were increased in the 6 month group as compared with the 2 month group,while the escaping latency in the Morris water maze was decreased.The hippocampal protein expression of TREM2 in the 6 monthgroup was less intensive than that in the 2 month group.Result of the Pearson correlation test showed that the expression of hippocampal TREM2 was negatively correlated with the target quadrant swimming distance in the Morris water maze.2.In vitro study:2.1 Compared with the control group,cholesterol(0.1~1000?M)could inhibit the proliferation of SH-SY5 Y cells.Moreover,under the condition of cholesterol(0.1-100 ?M),the proliferation ability of SH-SY5 Y cells gradually decreased with the increasing cholesterol stimulation concentration,and reached about 50% at 100?M,but the proliferation ability increased with the cholesterol concentration 1000 ?M.The proliferative capacity of SH-SY5 Y cells decreased with the increaing cholesterol stimulation time,reaching the plateau at 24 h.Therefore,the experimental conditions selected in the subsequent experiments were: SH-SY5 Y cells stimulated with cholesterol 100 ?M and 24 h.2.2 Cultured with cholesterol 100 ?M and 24 h,the percentage of G0/G1 in SH-SY5 Y cells were increased significantly,indicating an increase of SH-SY5 Y cells in quiescent phase and pre-DNA synthesis phase.Results of morphological and molecular biological experiments confirmed cell injury and lipid accumulation in SH-SY5 Y cells,together with the up-regulated expression of SREBP-1 and SREBP-2.Moreover,the expression of BDNF,Copine6,TREM2,and p-?-catenin/?-catenin were decreased in SH-SY5 Y cells after stimulation with cholesterol 100 ?M and 24 h,while the expression of TREM1,Nesfatin-1,and p-GSK-3?/GSK-3? was up-regulated.2.3 Overexpression of TREM2 reversed the expression of BDNF,Copine6,Nesfatin-1 and p-GSK in SH-SY5 Y cells induced by cholesterol stimulation,but had no significant effect on the expression of p-?-catenin.The expression of BDNF and Copine6 was attenuated after silencing of TREM2,and the protein expression of Nesfatin-1 and p-GSK-3? was up-regulated.However,the expression of the corresponding proteins in the negative siRNA control group was also statistically significant compared with the normal control group.Therefore,this result needs to be explored and verified in details method using a larger sample size inthe future studies.Conclusion:1.The female SD rats aging 6 month have more exploring activity and better learning and memory ability than 2 month group,the mechanism of which might be associated with the decreased expression of TREM2 in the hippocampus.2.High cholesterol stimulation can induce the proliferation of SH-SY5 Y cells,intracellular lipid deposition and metabolic disorders,and lead to the imbalanced expression of BDNF,Copine6,TREM2 and key molecules in the Wnt/?-catenin+signaling pathways.Among them,TREM2 might be an important factor mediating high cholesterol-induced SH-SY5 Y cell injury.