| Diabetes is a complex polygenic inherited disease.Maternal environmental changes are closely related to the susceptibility of the offspring to diabetes,and epigenetic modification is considered to be an important molecular basis linking the two,but the specific mechanism is currently not very clear.Objective:(1)To confirm that high-fat diet during pregnancy and lactation of female SD rats can lead to the disorder of glucose and lipid metabolism during weaning(3weeks old)of offspring SD rats.(2)To explore the changes of gene expression in liver tissue of offspring SD rats caused by high fat diet during pregnancy and lactation.(3)To explore the role of DNA methylation of differential gene expression in the liver of offspring SD rats caused by high fat diet during pregnancy and lactation.Methods:(1)Female SD rats were divided into two groups randomly.They were fed with normal diet and high-fat diet respectively.The model of high-fat diet during pregnancy and lactation was established.The offspring SD rats were measured body weight at birth,one week,two weeks and three weeks.(2)The oral glucose tolerance test was performed by gavage at the age of 3 weeks in the offspring rats(weaned).The offspring rats were killed,the blood of abdominal vein was taken,the fasting serum insulin content was measured,the area under the glucose tolerance curve and the insulin resistance index were calculated,the triglyceride concentration and the total cholesterol concentration were measured,the liver weight of each group of offspring rats was measured,the liver and pancreas tissues of each group of offspring rats were collected,and he staining was performed The histopathological changes of the liver and pancreas were observed under the light microscope.The insulin content of the islets was measured by Western blot.(3)The whole genome expression microarray and methylation sequencing microarray were used to find the differentially expressed genes and regions,and bioinformatics technology was used for correlation analysis,and then further molecular biology experiments were carried out for verification and in-depth analysis to detect the expression of genes.Results: High fat diet group(HFD group)and normal chow diet group(normal chow diet group),Compared with NCD group,the weight of HFD group was larger at birth and at weaning,and there were obvious glucose and lipid metabolism abnormalities at weaning:(1)compared with NCD group,HFD group had larger weight at birth and at weaning(3 weeks old);(2)compared with NCD group,HFD group had higher fasting blood glucose,higher blood glucose 120 minutes after taking glucose,larger area under glucose tolerance curve,and unclear fasting insulin content There was significant difference in HOMA-IR index;(3)compared with NCD group,the fasting triglyceride content and total cholesterol content of HFD group were significantly higher.Compared with NCD group,the weight of liver in HFD group increased,and he staining of liver showed obvious balloon like change of liver cells,and a large number of fat vacuoles with different sizes were observed;(2)compared with NCD group,he staining of pancreas in HFD group showed no significant difference,and insulin immunohistochemistry staining showed no significant difference.The Me DIP-seq and RNA-seq results showed that:(1)there were 46628 differential methylation sites(DMS),most of which were found on the X chromosome(8661,18.57%);DMS were mainly distributed in the intergenic region and intron region,and the proportion in transcription start site,transcription stop site,exon and intron element was 9.4%,3.33%,8.24% and 33.68%,respectively;(2)compared with NCD group,there were 501 hypomethylation regions(DMRs)and 46 hypermethylation regions(DMRs)in HFD group,most of them were found there are171 promoter regions(3 hypermethylation regions and 168 hypomethylation regions),152 introns(11 hypermethylation regions and 141 hypomethylation regions)and 143genes(30 hypermethylation regions and 113 hypomethylation regions)in HFD group;(3)compared with HCD group,there are 262 differential methylation genes(DMGs)in HFD group,including 248 hypomethylation genes and 14 hypermethylation genes;(4)DMGs Go enrichment analysis showed that the top three in the cell component were protein acetyltransferase complex,histone acetyltransferase complex and heterochromatin;the top three in the molecular function part were sulfhydryl ubiquitin specific protease activity,N-acetyltransferase activity and sulfhydryl ubiquitin hydrolase activity;the top three in the biological process part were histone H4 acetylation and histone Deacetylation of protein and deacetylation of protein;(5)using KEGG pathway analysis,to explore the related pathways of DMGs in steroid biosynthesis,nicotinine addition,citate cycle(TCA cycle),nuclear exercise repair and retrograde endocannabinoid Signaling;(6)compared with NCD group,385 genes were up-regulated and 491 genes were down regulated in HFD group;(7)the integration analysis of DMGs and DEGs showed that 9 genes simultaneously changed gene expression and DNA methylation,accounting for 3.44%(9 / 262)and 1.02% of DMGs respectively(9 / 876);compared with NCD group,TIMP1,STS,PLXNA3,GK and ap1s2 promoter regions in HFD group were hypermethylated,among which,STS,PLXNA3 and GK promoter regions all had Cp G islands,and the expression of STS,PLXNA3 and GK might be regulated by DNA methylation.BSSQ and q PCR were used to verify the MEDIP-seq data and RNA-seq data:(1)BSSQ results showed that compared with NCD group,the methylation level of PLXNA3 and GK promoter region in HFD group increased,which was consistent with the MEDIP-seq results;(2)q PCR results showed that compared with NCD group,TIMP1,SRPX and IL13RA1 m RNA expression level in HFD group increased,while STS,GK and PLXNA3 m RNA expression level increased The expression level of m RNA decreased,which was consistent with the result of RNA-seq.Conclusion:(1)The intake of high-fat diet during pregnancy and lactation in female SD rats can lead to significant increase of body weight,abnormal glucose tolerance,insulin resistance,mixed hyperlipidemia and fatty degeneration of liver.(2)Through the comprehensive analysis of DNA methylation group and transcriptome,we found that the influence of high-fat diet of pregnant and lactating mothers on the metabolism of glucose and lipid in offspring rats may be realized by changing the epigenetic modification of liver,and the abnormal methylation of key genes may be one of the factors.(3)Although the direct causal relationship and subsequent physiological consequences of these epigenetic changes are unclear,these findings may promote reproductive health and prevent the development of chronic diseases in the future. |
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