Effects Of Docosahexaenoic Acid Combined With Folic Acid And Vitamin B₁₂ On Diabetic Cognitive Dysfunction And Its Mechanism

Background and objective:Diabetic nervous system complications are one of the common complications of diabetes.Diabetic cognitive dysfunction（DCD）refers to the impairment of cognitive function caused by diabetes.The main clinical manifestations are the decline of learning ability,memory ability,language expression ability and judgment ability.At present,there is no effective drug for the treatment of DCD.In addition to controlling blood glucose,folic acid and vitamin B₁₂(VB₁₂)are often recommended to prevent and treat DCD.However,more and more studies have found that folic acid and VB₁₂ supplementation can only prevent and improve some DCD patients.In addition,randomized controlled trials in different populations have shown that docosahexaenoic acid（DHA）,folic acid and VB₁₂ have a synergistic effect in the prevention and treatment of cognitive impairment,but the mechanism is not clear.Based on this,the purpose of this study was to study the relationship between DHA,folic acid,VB12 and DCD in patients with diabetes mellitus（Chapter 1）.Then the diabetic mouse model was established,and DHA-rich deep-sea fish oil and/or folic acid and VB₁₂ were used to explore the effect and molecular mechanism of DHA,folic acid and VB₁₂ in preventing DCD.Methods:The first chapter is the cross-sectional study.All diabetic patients over 65 years old in 10 natural villages in Licha Town,Jiaozhou City,Qingdao were selected.The general demographic characteristics of the subjects were collected by questionnaire,and the cognitive function of the subjects was evaluated by mini mental state examination（MMSE）.At the same time,the peripheral blood of the patients was collected to detect the serum biochemical indexes such as fasting blood glucose,total cholesterol（TC）,triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）,low density lipoprotein cholesterol（LDL-C）and homocysteine（HCY）,and then the fatty acid composition of erythrocyte phospholipid membrane was detected by gas chromatography.Multivariate logistic regression was used to analyze the correlation between erythrocyte phospholipid membrane DHA,serum HCY and DCD,and the predictive value of DHA combined with HCY in DCD was evaluated by receiver operator characteristic curve（ROC）.The second chapter is animal experiment.Eight-week-old male C57BL/6J mice were induced into diabetic model mice by streptozotocin（STZ）.Then they were randomly divided into diabetic model group（DM）,metformin group（MET,150 mg/kg）,deep-sea fish oil group（DHA,150 mg/kg）,folic acid and VB₁₂ group(VB,folic acid 100μg/kg and VB₁₂ 65μg/kg),DHA plus folic acid and VB₁₂ group(DHA+VB,DHA 150 mg/kg+folic acid 100μg/kg and VB₁₂ 65μg/kg),and normal control group（NC）was set as well.Intervention through intragastric administration.After continuous intervention for 12weeks,the behavioral tests of mice were performed,including new object recognition,Y maze and Morris water maze.After the behavioral test,mice were normally fed for 5 days,then the mice were killed,and the samples of serum and brain tissue were collected.Then serum biochemical indexes,brain fatty acid composition,brain histopathology,DNA epigenetic modification and related proteases,tricarboxylic acid cycle metabolites and related catalytic enzymes in brain tissue were detected.Results:A total of 287 elderly patients with diabetes were included in the cross-sectional study.According to the level of education and MMSE score,99 patients with cognitive impairment were found,that is,the prevalence rate of DCD was 34.5%.Compared with diabetic group（DM）,the level of DHA in erythrocyte membrane in DCD group was significantly lower（P=0.013）.The serum HCY level of DCD group was higher than DM group,while the difference was not statistically significant.After adjusting for possible confounding factors such as age,sex,marital status,living status,education,smoking,drinking,body mass index（BMI）,history of hypertension,etc.,the DCD risk of the DHA T3 quantile was reduced by about 53%compared with the T1 quantile（OR=0.427,95%CI:0.223,0.818）,and the DCD risk of the HCY T2 quantile was about 2.2 times that of the T1 quantile（OR=2.175,95%CI:1.126,4.199）.The ROC curve shows that,compared with the inclusion of DHA alone（AUC=0.705,95%CI:0.642,0.768;sensitivity:0.796,specificity:0.516;Youden index:0.312）or HCY alone（AUC=0.696,95%CI:0.631,0.761;sensitivity:0.582,specificity:0.750,Youden index:0.322）,including DHA and HCY at the same time can better predict DCD risk（AUC=0.722,95%CI:0.659,0.786;sensitivity:0.653,specificity:0.745:0.398,Youden index:0.398）.The results of animal experiments are as follows:in the Y maze,the autonomous alternation percentage and recognition memory index of DM group were significantly lower than those of NC group,but there was no significant difference between DHA group,VB group and DM group.The autonomous alternation percentage and recognition memory index of DHA+VB group was significantly higher than that of DM group.In the water maze,the escape latency of DM group was significantly higher than that of NC group,the platform crossover number and the percentage of the time spent in the target quadrant of DM group were significantly lower than those of NC group.There was no significant difference in escape latency,platform crossover number and percentage of the time spent in the target quadrant between DHA group,VB group and DM group.The escape latency of DHA+VB group was significantly lower than that of DM group and DHA group,while the platform crossover number and the percentage of the time spent in the target quadrant were significantly higher than those of DM group.Fasting insulin and HOMA-IR in DM group were significantly higher than those in NC group.There was no significant difference in fasting insulin and HOMA-IR between DHA group and DM group.HOMA-IR in VB group and DHA+VB group was significantly lower than that in DM group.The levels of TC,TG,HDL-C and LDL-C in DM group were significantly higher than those in NC group.The levels of TG and LDL-C in DHA group and VB group were significantly lower than those in DM group.The levels of TC,TG,HDL-C and LDL-C in DHA+VB group were significantly lower than those in DM group.The levels of DHA in brain tissue of DHA group,VB group and DHA+VB group were significantly higher than those of DM group,and the levels of DHA in brain tissue of DHA+VB group were significantly higher than those of DHA group and VB group.The serum HCY level in DM group was significantly higher than that in NC group,and the HCY levels in DHA group,VB group and DHA+VB group were significantly lower than that in DM group.Statistical analysis of TUNEL staining showed that the apoptosis rate of neurons in DM group was significantly higher than that in NC group,and the apoptosis rate of neurons in DHA group,VB group and DHA+VB group was significantly lower than that in DM group.There was no significant difference in the expression of neuron marker protein Neu N between DHA group and VB group and DM group,but the expression of Neu N protein in DHA+VB group was significantly higher than that in DM group.The expression of antiapoptotic protein Bcl-XL in DM group,DHA group and VB group was significantly lower than that in NC group,while the expression of Bcl-XL in DHA+VB group was similar to that in NC group.The expression of pro-apoptotic protein Bax in DM group was significantly higher than that in NC group,while the expression of Bax protein in DHA group and VB group was not significantly different from that in DM group,while the expression of Bax protein in DHA+VB group was significantly lower than that in DM group.Dot blot showed that the overall level of DNA 5-methylcytosine（5m C）in DM group and VB group was higher than that in NC group,while the overall level of 5m C in DHA+VB group was lower than that in VB group,although there was no significant difference between groups.The overall level of DNA5-hydroxymethylcytosine in DM group was significantly lower than that in NC group,while the overall level of 5hm C in DHA group,VB group and DHA+VB group was significantly higher than that in DM group.The 5m C/5hm C ratio in DM group was significantly higher than that in NC group,while the 5m C/5hm C ratio in DHA group,VB group and DHA+VB group was significantly lower than that in DM group,but similar to that in NC group.There was no significant difference in protein expression of DNA methyltransferase（DNMTs）family among different groups.The concentration of S-adenosylmethionine（SAM）in VB group was significantly higher than that in NC group,DM group,DHA group and DHA+VB group.The concentration of S-adenosylhomocysteine（SAH）in VB group and DHA+VB group was significantly lower than that in DM group.The concentration of SAH in VB group was significantly lower than that in DHA group,and the SAM/SAH ratio in VB group was significantly higher than that in NC group.The protein expression of 10-11 translocation protease 2（TET2）in DM group was significantly lower than that in NC group.The expression of TET2 protein in DHA group and VB group was higher than that in DM group,but there was no significant difference.The expression of TET2 protein in DHA+VB group was significantly higher than that in DM group.The level ofα-ketoglutaricacid（α-KG）in DM group was significantly lower than that in NC group,while the level ofα-KG in DHA group,VB group and DHA+VB group was significantly higher than that in DM group.The ratio ofα-KG/（succinic acid+fumaric acid）in DM group was significantly lower than that in NC group,while the ratio ofα-KG/（succinic acid+fumaric acid）in DHA group,VB group and DHA+VB group was significantly higher than that in DM group.The p-AMPK/AMPK ratio in DM group was significantly lower than that in NC group,while the p-AMPK/AMPK ratio in DHA group and DHA+VB group was significantly higher than that in DM group.The p-AMPK/AMPK ratio in VB group increased compared with DM group,but there was no significant difference.Conclusion:Diabetic patients with higher erythrocyte membrane DHA levels had a lower risk of DCD,while diabetic patients with higher serum HCY levels had a higher risk of DCD.And erythrocyte membrane DHA combined with serum HCY modeling can better predict the risk of DCD.Folic acid and VB₁₂promote DNA methylation by increasing methyl donors;DHA promotes TET activity by enhancing tricarboxylic acid cycle homeostasis,and enhances TET2 protein stability by activating p-AMPK,thus promoting DNA demethylation;folic acid and VB₁₂ and DHA supplement together to maintain the balance of DNA methylation and demethylation,thus preventing the occurrence of DCD.