A Preliminary Study Of Relationship Between Maternal High Folic Acid Supplementation And Ednrb Gene Expression In Fetus Mouse Colon On The Effect Of Pathogenesis Of Hirschsprung Disease

Objective: We firstly establish and evaluate high folate acid (HFA) induced pregnant mouse model by supplementing RHAA, which can reveal the relationship between environment (HFA intaking) and some disease during embro development, it can also provide some pre-experiment data.Methods: 60, 8-10 week-old female C57BL/6 mice with weight ranging from 18g to 20g were selected as study objectives.They were sorted by weight and randomly divided into five groups. One group was supplemented with low dose folate (0mg FA/kg diet) and the other 3 groups were treated with high dose FA supplentment (40,160 and 320mg FA/kg diet), the last goup was normal dosage FA diet (2mg FA/kgdiet).Copulate with male mice after two weeks of FA treatment. Paunch the 20 days pregnant mice and collect embryo for studying. Record the weight and diet dosage twice every week. Measure the blood serum FA concentration of pre-pregnant female mice,advanced stage mice and embryo。Evaluate this mouse model and the effect of different FA supplement levels on embro during development.Results: 1, There are no significant statistics differences in avavage diet intaking,weight gain and food efficacy compared with normal FA supplement goup before and after copulation. High dosage FA supplements have no significant effect on mice growth.2, High dose FA treated groups are less in avevage weight and nose- buttocks length compared with control group (p<0.05) and it succesufully establish lower dosage FA induced embro deficiency mouse model, which suppresses embro development. Both of all manifest noticeable restraint in embro development.3, As FA supplements increase, blood serum FA concentration of mother and embro show consistent increasement. The 160mg FA /kgdiet treated group has the highest blood FA concentration and blood serum concentration of mother of folate deficiency group before pregnancy of two weeks , combined low folate diet group in the cage before, mean serum folate concentration of animals were significantly lower than before the experiment, to SFA of the late third trimester dropped to the lowest animal, up to about 37% before the experiment, together about 50% before the cager. In this goup, the embro show higher blood FA concentration than their mother.4, because of dietary folate supply, Blood serum concentration of the control group before cage was slightly higher than its before the expriment (P>0.05), to SFA of the late third trimester dropped to 78%(p<0.01), shows the blood serum concentration of maternal mice may be due to physiological pregency decreased significantly.Conclusion: 40-160mg FA/Kgdiet can successfully induce high FA dosage mouse model, which can inhibit embro development and maturation. This study provides a clue that environments can induce some gene's specific site methylation and regulate expression through epigenetics mechanism, which finally causes variated phenotype. It has established the basement for disease pathogenesy prediction. Objective: To study the effect of mother's FA diet on offspring's EDNRB/EDN3/ ECE1 signal transduction way and HD phenotype.Explore intestinal tract's high FA factor's potential fuction and application in preveting Hirschsprung diseaseMethods: younged (8-10 week-old, n 60) female C57BL/6 mice with weight ranging from 18g to 20g were selected as study objectives. They were sorted by weight and randomly divided into five groups. One group was supplemented with low dose FA (0mg FA/kgdiet) and the other 3 groups were treated with high dose FA supplentment (40,160,320mg FA/kgdiet), the last goup was normal dosage FA diet (2mg FA/kg diet).Copulate with male mice after two weeks of FA treatment. Paunch the 20 days pregnant mice and collect embryo for studying. Check the embro's colon tissue for HD positive phenotype. HPLC-MS/MS,BSP,PCR and western-blot were also used to whole genome DNA and EDNRB promoter methylation status.Results: 1,Megacolon phenotype are detected in high FA dosage groups (40,160 and 320mg FA/kg diet), Positive incidences are 1/42(2.4%),2/33(6.1%)and 1/36(2.8%)respectively.2, High FA supplement groups'genome methylation levels are higher than control group, but it has no significant statistcs differences.but for folate deficiency group (p<0.05).3, EDNRB promoter methylation levels in all FA supplent groups are 20.1%,18.8%,25.1%,22.1% and 22.6% respectively. The major methylation sites are 594,597 and 652, which in consistent with embro's blood serum FA concentration.4, High FA supplement groups have a lower CT (EDNRB gene mRNA expression level), compared with control group (p<0.01). EDNRB expression can detected in all groups, while it lessens as FA supplement increases with no significant differences.Conclusion: The dilated conlon phenotypes confirm that high dose FA intaking during pregnancy could induce hyperhmethylation in EDNRB promoter and suppress its expression. It establishs the base that acceptable FA supplement could prevent some Hirschsprung's disease.