The Relationship And Mechanism Between White Matter Lesions And Diabetic Associated Cognitive Decline In Type 2 Diabetes Mellitus

Backround and ObjectivesType 2 diabetes mellitus(T2DM)has become a global chronic diseases.It can lead to diabetic ketosis,retinopathy,nephropathy,peripheral neuropathy and the other complications.However,with the global prevalence of DM,the cognitive dysfunction caused by T2DM has gradually attracted more and more attention.Diabetic associated cognitive decline(DACD)mainly showed mild to moderate cognitive impairment,characterized by acquired cognitive behavioral impairment,decreased ability of learning,memory and complex information processing.The degree of cognitive impairment varies from mild to moderate,but it will significantly reduce the quality of life.Neurophysiology and neuroradiology suggest that the brains of T2DM patients get old faster,especially the degeneration and metabolic disorder of hippocampal neurons.Studies have confirmed that DM significantly increased the risk of dementia and cognitive impairment.Memory,information processing,attention and executive function were decreased in different stages of T2DM and were not related to age.In recent years,it has been found that white matter damage(WML)may play a role in DACD.The information transmission among different gray matter regions depends on the integrity and health of white matter(WM).Once the integrity of WM structure is destroyed,information transmission obstacles may appear,resulting in the decline of cognitive function.Most of T2DM patients show extensive WML and WML appeared earlier than non-DM patients.WML may play a role in DACD.It has been found that executive function is significantly related to the integrity of WM in the left forelimb and the left internal capsule.The interruption of WM fiber network is also related to the slow down of information processing speed in T2DM patients.However,the relationship and mechanism between DACD and WML are still unclear,and the impact of WML on cognitive impairment in different stages of DM is also not clear.To explore the relationship between WML and DACD and the mechanism of how WML induced DACD,a study was first conducted to analyze the correlation between WML and DACD in T2DM patients.Due to many confounding factors,T2DM rat model was further established.We further explored the relationship between WML and T2DM and the relationship between WML and DACD in animal models.Finally,label-free protein quantitative technique was used to analyze the differential proteome in the WM of T2DM group and healthy control group,and then the differential proteins that may be related to the DACD in WM were screened out.After bioinformatics analysis,some differential proteins were selected for verification.Our research aimed to provide a new treatment strategy to the DACD caused by WML.Part I:Correlation analysis between white matter lesions and diabetic associated cognitive decline in type 2 diabetes mellitus patientsMaterials and Methods1.We analyzed 519 T2DM inpatients.WML was diagnosed by white matter hyperintensity(WMH)in magnetic resonance imaging(MRI).According to the results of MRI,the patients were divided into non-WMH group(n=139)and WMH group(n=380).The Montreal Cognitive Assessment(MoCA)was used to evaluate the cognitive function of patients.Compared the incidence of cognitive dysfunction between the two groups,and analyzed the main risk factors of WML,the risk factors of cognitive dysfunction and the correlation between them.2.Statistical analysis:All the data were analyzed by the SPSS 20.0 software.The measurement data was expressed as mean ± standard deviation((?)).The counting data was expressed as case(%).The t-test or Chi-square test were used for the comparison between the two groups.The related risk factors were analyzed by Logistic regression.Spearman test was used to analyze the correlation.Statistical significance was determined at P<0.05 level.Results1.The comparison of demographic data:There were significant differences in the duration of diabetes,the prevalence of hypertension,total cholesterol(TC)and MoCA scores between the WML group and non-WML group(P<0.05).2.Risk factors analysis of WML:The age,duration of diabetes,hypertension and glycosylated hemoglobin(HbAlc)were significantly correlated with WML(?=0.146?0.110?3.532?0.448).3.Risk factors and correlation analysis of cognitive dysfunction:There were 203 patients with cognitive dysfunction in WML group and 52 patients in non-WML group.The number of patients with cognitive dysfunction in WML group was significantly higher than that in non-WML group(P<0.05).WML patients mainly showed a decline in visual space,executive function,attention,computational power and delayed recall function.The WML,age and body mass index(BMI)were significantly correlated with cognitive dysfunction in diabetic patients(?=0.759?0.161?0.221).Spearman correlation analysis showed that there was a significant correlation between WML and MoCA score(r=0.738,P=0.001).Part ?:Correlation analysis between white matter lesions and diabetic associated cognitive decline in male rat models of type 2 diabetesMethods1.Choosed sixty Sprague-Dawley male rats and divided into four groups after adaptive feeding for 1 week:control group(NC),normal control+metformin group(NC+MET),T2DM group,and T2DM+metformin group(T2DM+MET).Group feeding:Rats in T2DM group and T2DM+met group were fed with high fat and high sugar diet,NC group and NC+met group were fed with normal diet.After 8 weeks of group feeding,The rats in T2DM group and T2DM+MET group were intraperitoneally injected with streptozotocin to establish T2DM model.2.The cognitive function of rats in each group was evaluated for three times by Morris water maze before group feeding,after 8 weeks of group feeding and 8 weeks after intraperitoneal injection of streptozotocin.After each water maze examination,diffusion tensor imaging(DTI)was used to measure the fractional anisotropy(FA)of bilateral thalamus in each group to evaluate the degree of WML.After the third DTI,the rats were killed and immunofluorescence was used to quantitatively detect myelin basic protein(MBP),oligodendrocyte transcription factor-1(OLIG1)and oligodendrocyte transcription factor-2(OLIG2).3.Statistical analysis:All the data were analyzed by the SPSS 20.0 software.The Shapiro-Wilk test was used to assess the continuous data for normal distribution.Normally distributed data were expressed as means ± standard error and analyzed using analysis of variance(ANOVA).Non-normally distributed data were expressed as median(P25,P75)and analyzed using the Kruskal-Wallis test.Pearson and Spearman correlation analyses were used to analyze the correlations.Statistical significance was determined at P<0.05 level.Results1.Before group feeding:There was no significant difference in escape latency and crossing platform times of rats among each group(P>0.05),and there was no significant difference in FA value of bilateral thalamus among each group(P>0.05).2.After 8 weeks of group feeding:The rats in T2DM group and T2DM+MET group showed IFG,but did not meet the diagnostic criteria of T2DM.In this stage,there was no significant difference in escape latency and crossing platform times among the groups(P>0.05).But the fractional anisotropy(FA)values of the right thalamus area were significantly lower in both T2DM groups compared with controls(P<0.05).3.8 weeks after intraperitoneal injection of streptozotocin:T2DM model was successfully established.The maze escape latency was longer and the number of passing through the platform was smaller in the T2DM and T2DM+MET groups than in the NC and NC+MET group(P<0.05).The FA values of the thalamus were lower in the T2DM(bilateral thalamus)and T2DM+MET(left thalamus)groups than in controls,while the FA values in the left thalamus area were lower in the T2DM+MET group than in NC and NC+MET groups(P<0.05).4.Correlation analysis of WML and cognitive impairment in T2DM rats:the escape latency of T2DM rats was significantly correlated with the FA value of right thalamus(r=0.996,P=0.001),and the number of passing through the platform were significantly correlated with FA value of right thalamus(r=0.831,P=0.001).5.Immunofluorescence results:MBP in T2DM group and T2DM+MET group was significantly lower than that in NC group and NC+MET group(P<0.05),while the expression levels of OLIG1 and OLIG2 were significantly higher than that in NC group and NC+MET group(P<0.05).Part III:Differential proteomics analysis of white matter from type 2 diabetic ratsMaterials and Methods1.Choosed twenty Sprague-Dawley male rats and divided into two groups:control group(10 rats)and T2DM group(10 rats).T2DM models were established by high fat and high glucose diet combined with intraperitoneal injection of streptozotocin.2.The differential proteomics analysis of white matter was performed through the Label-free comparative proteomics technology,and then the changed proteomes that may be related to the molecular mechanism of WML in T2DM rats were screened.After bioinformatics analysis of the differential proteins,some of the differential expressed proteins were verified by Western blot.3.Statistical analysis:Statistical analysis:All the data were analyzed by the SPSS 20.0 software.The measurement data was expressed as mean ± standard deviation((?)).The t-test were used for the comparison between the two groups.Statistical significance was determined at P<0.05 level.Screening criteria for differential proteins among different sample:FC<-2.0 or FC>2.0 and P<0.05 were considered as differential proteins.Results1.A total of 38 differential proteins were screened,including 24 up-regulated proteins and 14 down-regulated proteins.2.The differential proteins mainly distributed in cell membrane,cytoplasm,exosomes,and synapses.Most of them were proteins with molecular binding activity.And they were mainly involved in biological processes such as nervous system development,response to drugs,hypoxia,negatively regulating apoptosis of neural cells,and chemical synaptic transmission.3.Analysis of the protein domains revealed that the differential expressed proteins mainly had EF-hand-like domains,EF-hand domains,PDZ domains,immunoglobulin subtype 2 domains,and S100 class calcium-binding protein domains.The varied proteins were mainly concentrated on the four KEGG signal pathways,and there may be direct or indirect interactions between most of the differential proteins.4.These differential proteins contained BDNF,NOS1,GAP43,SLC17A7,DNM1,GRM5,and SLC1A2 would be core backbones of the protein complexes to form interactive network.5.Western blot experiments verified that the expression levels of proteins were consistent with the results of proteomics experimental results.Conclusions1.is significantly correlated with DACD in T2DM patients.2.appeared before the onset of T2DM and DACD,and DACD gradually appeared with the aggravation of WML.The WML of right thalamus was significantly correlated with DACD in rat model.Metformin may reduce WML,but has no effect on DACD.3.The differential proteins related to WML of T2DM rats can be effectively screened by using lab el-free technology,which may be related to synaptic transmission or signal transduction regulation of nervous system cells.