Exploring The Mechanism Of Cognitive Changes In ALDH2 Deficient Mice After Chronic Acohol Intake Based On Multi-Omics

Objectives:To establish the chronic alcohol intake model based on Aldh2 ^E487K mice.To evaluate the changes of cognitive function,alcohol,acetaldehyde,blood lipid and inflammatory factors in mice after chronic alcohol intake.To explore the lipidomics and transcriptomics in mice after chronic alcohol intake and to explore the potential pathway of cognitive impairment mediated by ALDH2.Methods:Part Ⅰ.Aldh2 ^E487K homozygous,heterozygous and wild-type mice were randomly selected and fed with alcohol liquid diet for 60 days to establish chronic alcohol intake mice model.Y-maze and new object recognition experiments were carried out to evaluate the cognitive function of mice by free alternation rate and recognition index.In addition,blood alcohol and acetaldehyde concentrations,blood lipid levels and inflammatory factors were detected in homozygous and wild-type mice.Part Ⅱ.After 60 days of alcohol liquid diet,the cerebral cortex of Aldh2 ^E487Khomozygous and wild-type mice were collected,and non targeted lipomics analysis was carried out in positive and negative ion mode based on LC-MS.Principal component analysis（PCA）was used to evaluate the lipid components.At the same time,mass spectrometry was used to investigate the distribution of different lipids in mouse brain.Part Ⅲ.After 60 days of alcohol liquid diet,the cerebral cortex of Aldh2 ^E487Khomozygous and wild-type mice were collected,and the RNA expression in the cerebral cortex of the experimental mice was examined by full transcriptome sequencing.The differentially expressed genes were screened based on the differentially expressed genes,including the comparison of differentially expressed genes,functional annotation and pathway enrichment analysis.Combined lipomics and transcriptomics analysis and obtained the metabolic pathway related to chronic alcohol intake in Aldh2 ^E487K homozygous mice.Results:Part Ⅰ.A chronic alcohol intake model in mice was established by 60 day liquid alcohol diet.The alcohol and acetaldehyde concentrations in mice with Aldh2 ^E487Khomozygous mice were significantly higher than those in wild-type mice（P<0.05）.Y-maze test showed that after chronic alcohol intake,the free alternation rates of Aldh2^E487K homozygous,heterozygous and wild-type mice were significantly lower than those of normal diet group（P<0.05）,the free alternation rate of Aldh2 ^E487Khomozygous mice was more significantly lower than wild-type mice（P<0.05）,and there was no significant difference between wild-type mice and heterozygous mice（P>0.05）.New object recognition test showed that after chronic alcohol intake,the recognition indexes of Aldh2 ^E487K homozygous,heterozygous and wild-type mice were significantly lower than those of normal diet group（P<0.05）,the recognition indexe of Aldh2 ^E487K homozygous mice was more significantly lower than wild-type mice（P<0.05）,and there was no significant difference between wild-type mice and heterozygous mice（P>0.05）.After chronic alcohol intake,there was no significant difference in total cholesterol（CHOL）,high density lipoprotein（HDL-C）and triglyceride（TG）between the homozygous mice and wild-type mice（P>0.05）,but the low density lipoprotein（LDL-C）concentration decreased significantly（P<0.05）,and Aldh2 ^E487Khomozygous mice decreased more significantly than wild-type mice（P<0.05）.After chronic alcohol intake,the concentration of inflammatory factors in Aldh2 ^E487Khomozygous mice and wild-type mice increased significantly after chronic alcohol intake（P<0.05）;the concentration of inflammatory factors in Aldh2 ^E487Khomozygous mice was higher than that in wild-type mice（P<0.05）.Part Ⅱ.After chronic alcohol intake,there were 114 different lipid substances were expressed in positive ion mode,93 of which were up regulated and 21 of which were down regulated;there were 120 different lipid substances in negative ion mode,68 of which were up regulated and 52 of which were down regulated;these differential lipids are glycerophospholipids,glycerides and sphingolipids.After chronic alcohol intake,there were 57 differentially expressed lipid substances in the positive mode,47 of which were up-regulated and 10 of which were down regulated;there were43 different lipid substances in negative ion mode,33 of which were up-regulated and 10 of which were down regulated;these differential lipids are glycerophospholipids,glycerides and sphingolipids.The results of mass spectrometry showed that the distribution of lipid substances in the cortex of experimental mice was also different（P<0.05）,and some lipid substances in other parts of the brain,such as the hippocampus,were also different（P<0.05）.Part Ⅲ.KEGG analysis showed that 14 pathways were significantly enriched after chronic alcohol intake in wild-type mice（P<0.05）;there are 17 differentially expressed genes in the Neuroactive ligand-receptor interaction pathway,9 of which are up-regulated and 8 down regulated.Aldh2 ^E487K homozygous mice were significantly enriched to 10 after chronic alcohol intake（P<0.05）;four differentially expressed genes involved in fat digestion and absorption pathway were down regulated.Glycerol phospholipid metabolism and arachidonic acid metabolism were strongly associated with chronic alcohol intake in wild-type mice;Aldh2 ^E487K homozygous mice after chronic alcohol intake were strongly associated with glycerol phospholipid metabolism,linolenic acid metabolism and arachidonic acid metabolism.The selected lipid substances were phosphatidylcholines,which were regulated by PLA2G.After chronic alcohol intake,PLA2G was down regulated in both homozygous and wild-type mice.Conclusions:Part Ⅰ.The chronic alcohol intake model of Aldh2 ^E487K mice was successfully established.The cognitive ability of experimental mice was investigated by Y-maze test and new object recognition test.The results showed that chronic alcohol intake reduced the cognitive ability of mice,and ALDH2 deficiency aggravated the effect of alcohol on cognitive ability of mice.The results of serum lipid level and inflammatory factors showed that chronic alcohol intake led to the decrease of LDL-C concentration and the increase of inflammatory factors concentration.ALDH2 deficiency aggravated the effect of alcohol on LDL-C and inflammatory factors concentration in mice.Part Ⅱ.After chronic alcohol intake in wild-type mice,a variety of major lipid substances such as phosphatidylcholine,ceramide,triacylglycerol,and phosphatidyl-ethanolamine,phosphatidylserine,etc.were changed with statistical differences,which may be related to the effect of alcohol on the cerebral cortex of mice.After chronic alcohol intake,a variety of major lipid substances such as phosphatidylcholine,phosphatidylethanolamine,phosphatidylglyceride,triacylglycerol,diacylglycerol and sphingomyelin were changed with statistical differences.It is related to the aggravation of ALDH2 deficiency and the effect of alcohol on the cerebral cortex of mice.The results of mass spectrometry confirmed the different lipid substances obtained by lipomics,and further obtained their distribution differences in the mice brain.Part Ⅲ.The accumulation pathway of wild-type mice after chronic alcohol intake was 14.HTR1,DRD2 and DRD1 may be the cause of significantly lower cognitive ability after chronic alcohol intake in mice.The accumulation pathway of Aldh2 ^E487Kmice after chronic alcohol intake was 10.Apolipoprotein A1 and p LA2G may be the cause of the significant decrease of cognitive ability of Aldh2 ^E487K homozygotes mice in the condition of chronic alcohol intake.There were two pathways that had strong correlation between lipid substance and difference genes after chronic alcohol intake in wild-type mice,and three in Aldh2 ^E487K homozygotes mice.The different lipid substances were phosphatidylcholines,which were regulated by PLA2G and showed down regulation.The metabolic pathway of linolenic acid may be related to the lipid changes after chronic alcohol intake in mice with glyoxaldehyde deficiency.