The Role And Mechanism Of TREM2 In Vascular Dementia

Part I The correlation between serum soluble TREM2 andcognitive function of Chinese Han vascular dementiapatientsObjective:Vascular dementia(VD)is the second largest type of dementia in the world.It has a high fatality rate and disability rate,seriously affects the quality of life of patients,and brings a heavy economic burden to society and families.At present,the pathogenesis of VD has not been fully elucidated,and there is no effective intervention for VD.Triggering receptor expressed on myeloid cells 2(TREM2)is a novel type of transmembrane cell immune receptor.In recent years,a lot of evidence has shown that TREM2 is closely related to the pathogenesis of Alzheimer’s disease(AD).However,there is no report about TREM2 participating in the pathogenesis of VD.This study aims to explore the role and mechanism of TREM2 in VD.Methods:A total of 120 patients with VD and 120 healthy volunteers were enrolled in this study.They were divided into vascular dementia(VD)group and healthy control(Normal control,NC)group.Clinicians collect demographic indicators of all study controls including age,gender and education,and record clinical examination results such as systolic blood pressure,diastolic blood pressure,body mass index(BMI),glycated hemoglobin,fasting blood glucose,total Cholesterol,triglycerides,high-density lipoprotein and low-density lipoprotein clinical statistics.The trained physicians evaluate the cognitive function and use the mini-mental state examination(MMSE)method.The evaluating physicians are not clear about the grouping of the subjects.All the subjects collected peripheral blood on the morning of enrollment,and the level of serum soluble TREM2(solution TREM2,sTREM2)was detected by Enzyme-linked immunosorbent assay(ELISA).Results:(1)Demographic indicators:age(70.4±6.0 years VS 70.9 ± 6.7 years),gender(male/female;74/46 VS 67/53)and years of education(7.4 ±2.1 years VS 7,9 ± 1.9 years)in the VD and NC respectively,and there is no statistical difference(p>0.05);(2)Clinical statistics:systolic blood pressure(149.7 ± 12.1 mmHg VS 147.2 ± 10.3 mmHg),diastolic blood pressure(95.1±8.5 mmHg VS 93.6 士 9.7 mmHg),height and body mass index(24.7±1.4 VS 24.4 ±1.2),glycated hemoglobin(6.51 ± 0.74mm/L VS 6.39 士 0.82 mm/L),fasting blood glucose(6.65±0.80 mm/L VS 6.53±0.91 mm/L),total cholesterol(4.56±0.73 mm/L VS 4.70±0.88 mm/L),triglycerides(1.63±0.22 mm/L VS 1.60±0.19 mm/L),high density lipoprotein(1.25±0.18 mm/L VS 1.28±0.14 mm/L)and low density compared with lipoprotein(2.62±0.18 mm/L VS 2.58±0.23 mm/L),there was no statistically significant difference(p>0.05);(3)MMSE score:The MMSE scores of the VD group and the NC group were(22.8±1.7)points and(27.8±1.0)points respectively,the difference was statistically significant(p<0.001);(4)Serum sTREM2 levels;The serum sTREM2 levels in the VD group and the NC group were(214.2±16.9)pg/ml and(220.5±14.7)pg/ml,respectively.Compared with the NC group,the serum sTREM2 level of the VD group was significantly reduced,and the difference was statistically significant(p=0.002);(5)Correlation analysis:In the patients in the VD group,the MMSE score was analyzed by Pearson correlation analysis with demographic,clinical statistical indicators and serum sTREM2 levels.The results showed that the MMSE score of patients in the VD group was positively correlated with serum sTREM2 levels(r=0.387,P=0.008),and with age(r=-0.271,p=0.081),gender(r=0.293,p=0.167),Years of education(r=0.245,p=0.193),systolic blood pressure(r=0.306,p=0.528),diastolic blood pressure(r=0.264,p=0.456),height and body mass index(r=-0.312,p=0.304),Glycosylated hemoglobin(r=0.229,p=0.240),fasting blood glucose(r=0.275,p=0.339),total cholesterol(r=0.428,p=0.645),triglycerides(r=0.333,p=0.461),High-density lipoprotein(r=-0.251,p=0.702)and low-density lipoprotein(r=0.270,p=0.326)were not significantly related(p>0.05);(6)Regression analysis:In patients in the VD group,linear regression analysis was performed on the MMSE score and demographic,clinical statistical indicators,and serum sTREM2 levels.The results showed that the MMSE score and age(β=-0.228,p=0.071),gender(β=0.309,p=0.113),years of education(β=0.270,p=0.196),systolic blood pressure(β=0.261,p=0.414),diastolic blood pressure(β=0.237,p=0.372),height and body mass index(β=-0.385,p=0.229),glycated hemoglobin(β=0.409,p=0.318),fasting blood glucose(β=0.325,p=0.421),total cholesterol(β=0.283,p=0.679),triglycerides(β=0.362,p=0.506),high-density lipoprotein(β=-0.413,p=0.437)and low density Lipoprotein β=0.334,p=0.658)has no obvious causal relationship,and there is a clear causal relationship with serum sTREM2 levels(β=0.396,p<0.001),this correlation is independent of demographic indicators and clinical statistics index.Conclusion:(1)The serum sTREM2 level of VD patients was significantly reduced;(2)Serum sTREM2 level is an independent risk factor for cognitive impairment in VD patients and can be used as a potential biomarker for predicting cognitive function in VD patients.Part 2 Mechanism of overexpression of TREM2 to improvecognitive function in mice with vascular dementiaObjective:Vascular dementia(VD)is the second largest type of dementia after Alzheimer’s disease(AD).With the advent of an aging society,its incidence rate has increased year by year,and it has become an important public health problem.Although research on vascular dementia has made some progress in the past few decades,its pathogenesis is still not completely clear,and neuroinflammation is considered to play an important role in the pathogenesis of vascular dementia.Triggering receptor expressed on myeloid cells 2(TREM2)is a transmembrane receptor mainly expressed on microglia.Recent studies have shown that TREM2 has anti-inflammatory properties during the immune response effect.However,the anti-inflammatory effect of TREM2 in vascular dementia has not been reported so far.Therefore,this study aims to explore the role and potential mechanism of TREM2 in vascular dementia.Methods:Thirty 8-week-old C57BL/6J mice were used to create a vascular dementia model by transient bilateral common carotid artery occlusion(BCCAO)method,and were divided into a sham control group and blood vessels.In the sexual dementia(VD)group,mice in the Sham group were isolated from both common carotid arteries without ligation.Reverse transcription-polymerase chain reaction(RT-PCR)and Western Blots were used to detect the Sham group and the 1st,3rd and 5th days after modeling,7th,14th and 28th day of the VD group TREM2 gene and protein expression,clear TREM2 expression changes in VD.The mice in the VD group were divided into TREM2 overexpression lentivirus control VD group(LV-control)group and TREM2 overexpression lentivirus VD according to whether they were injected with TREM2 overexpression lentivirus(LV-TREM2).In the(LV-TREM2)group,mice in the Sham group and LV-control group were injected with blank control vector in the lateral ventricle.Four weeks after injection of the lentiviral vector into the lateral ventricle of each group of mice,Morris water maze test was performed to test their spatial learning and memory abilities.After the Morris water maze test,mice were anesthetized and sacrificed.Paraffin sections of the hippocampal CA1 area of the mice were made Immunohistochemical methods were used to detect the M1 type microglia marker iNOS and M2 type microglia in the hippocampus tissues of each group.The expression level of cell marker Arg-1 and the total protein of hippocampus were extracted at the same time,and the protein concentration was measured by BCA(Bicinchonininc acid,BCA)method,and the expression changes of iNOS and Arg-1 were further confirmed by Western blots;RT-PCR Method to detect pro-inflammatory factors(IL-1β,IL-6 and TNF-α),anti-inflammatory factors(IL-4,IL-10 and TGFβ)and inflammatory chemokines(MIP-1αand MCP-1)Gene level;Finally,Nissl staining was performed in paraffin sections of the hippocampal CA1 area of each group of mice to detect the apoptosis level of the hippocampal neurons of each group of mice.Results:(1)TREM2 gene and protein levels:The levels of TREM2 gene and protein in VD group mice on day 1,day 3,day 5,day 7,day 14,and day 28 were significantly higher Sham group(p<0.05),the highest peak of TREM2 appeared on the third day after modeling;(2)The results of the Morris water maze show that the swimming speed of mice in the Sham group,LV-control group and LV-TREM2 group were(12.34±0.96)cm/s,(12.23±1.01)cm/s and(12.29±1.05)cm/s,there was no significant difference in the swimming speed of the three groups of mice(p>0.05);the escape latency of the mice in the Sham group,LV-control group and LV-TREM2 group on days 1-4 were respectively[(62.30±3.12)cm/s,(41.50±2.34)s,(23.40±2.10)s and(16.23±1.28)s],[(62.42±3.18)cm/s,(56.89±2.97)s,(46.79±2.54)s and(35.17±1.95)s]and[(62.51±3.16)cm/s,(50.90 ± 2.61)s,(38.05 ± 2.03)s and(26.72±1.52)s],the results indicate training On day 1,the escape latency of the three groups of mice was not significantly different(p>0.05),while the escape latency of mice in the LV-TREM2 group on days 2-4 was significantly shorter than that of the LV-control group.The difference was statistically significant(p<0.05);the original platform quadrant time of mice in Sham group,LV-control group and LV-TREM2 group were(65.33±3.25)cm/s,(42.16±2.31)cm/s and(54.67±2.78)s,mice in LV-control group the original platform quadrant time was significantly shorter than that of the LV-TREM2 group.The three groups of mice had significant differences in the original platform quadrant time(p<0.05);The number of mice crossing the original platform quadrant in the Sham group,LV-control group and LV-TREM2 group were(6.23±0.45)cm/s,(2.36 ± 0.39)cm/s and(4.28±0.41)s respectively.Compared to the mice in LV-control group,the number of crossing the original platform was significantly increased in the mice in LV-TREM2 group.The number of crossing the original platform were significantly different among the three groups(p<0.05);(3)Microglial phenotype detection:The results of immunohistochemistry and Western blots showed that compared with the LV-control group,the expression of iNOS,a M1 type microglia marker,was significantly reduced in the LV-TREM2 group.The expression level of Arg-1,a marker of M2 microglia,increased significantly,indicating that TREM2 can promote the polarization of microglia to M2.The comparison of iNOS and Arg-1 expression levels among the three groups of mice was statistically significant(p<0.05);(4)Inflammatory factors:RT-PCR results show that compared with LV-control group mice,the gene levels of proinflammatory factors(IL-1β IL-6 and TNF-α)in hippocampal brain tissue of LV-TREM2 group mice are obvious Decreased,anti-inflammatory factors(IL-4,IL-10 and TGFβ)and inflammatory chemokines(MIP-1α,MCP-1)gene levels increased significantly,indicating that TREM2 can inhibit Ml-type microglia-mediated Inflammatory factor expression,and promote the expression of anti-inflammatory factor mediated by M2 microglia.The comparison of gene levels among pro-inflammatory factors,anti-inflammatory factors and inflammatory chemokines among the three groups of mice was statistically significant(p<0.05);(5)Apoptosis of hippocampal neurons:The results of Nissl staining showed that the proportion of hippocampal staining-positive neurons in the Sham group,LV-control group and LV-TREM2 group were(0.84±0.06),(0.37 ± 0.03)and(0.72 ± 0.05)respectively.Compared with the LV-control group,the apoptosis of hippocampal neurons in the LV-TREM2 group was significantly reduced.The proportion of hippocampus staining-positive neurons in the three groups was statistically different(p<0.05).Conclusion:(1)The expression of TREM2 in VD mice was significantly increased after modeling;(2)Overexpression of TREM2 can significantly improve the spatial learning and memory function of VD mice;(3)TREM2 may play a neuroprotective role by promoting the polarization of microglia to the M2 type,reducing inflammatory damage and neuronal apoptosis in VD.