Effect Of Polyunsaturated Fatty Acids On Offspring Neural Tube Defects Induced By Diabetic Pregnancy

Objective To explore the effect and mechanism of n-3 polyunsaturated fatty acids(PUFA)on neural tube defects(NTD)in offspring induced by diabetic pregnancy.Method1.Animal groups: Two hundred 8-week-old ICR mice(150 females and 50 males)were used.After one week of diet adaptation,the female and male mice were mated at night in a ratio of 3:1.The female mice were found to have vaginal plug in the morning of the next day,which was recorded as the 0.5th day of pregnancy(E0.5d).Pregnant mice were randomly divided into five groups: healthy control group(AIN-93 G standard diet),diabetic control group(AIN-93 G standard diet),diabetic low dose n-3 PUFA group(25%fat in AIN-93 G diet was replaced with fish oil(rich in C20:5n-3 PUFA,C22:5n-3 PUFA and C22:6n-3 PUFA),diabetic high dose n-3 PUFA group(50% fat in AIN-93 G diet was replaced with fish oil)and diabetic n-6 PUFA group(all fat in AIN-93 G diet was replaced with corn oil(rich in C18:2 n-6 PUFA).On E6.5d,pregnant mice in diabetic control group,diabetic low dose n-3 PUFA group,diabetic high dose n-3 PUFA group and diabetic n-6 PUFA group were intraperitoneally injected with 200 mg / kg streptozocin(STZ)(dissolved in sodium citrate buffer).The healthy control group was intraperitoneally injected with equal volume of sodium citrate buffer.The drinking water of pregnant mice was changed to 0.5% glucose solution to prevent the death of pregnant mice due to hypoglycemia during the modeling process.After E8.0d,the drinking water was replaced by ordinary pure water,and the fasting blood glucose level of mice was detected by tail vein blood sampling at E8.5d,if it was more than 11.1 mmol / L,the diabetic pregnant mice model was established successfully.2.During the experiment,the health status of mice was observed daily,and the average daily food intake of pregnant mice in each group were measured and weighed daily.3.Through the extraction,concentration,methyl esterification and separation and purification of lipids,the effects of different dietary intake on the levels of fatty acids in serum and embryo of E12.5d pregnant mice were analyzed by gas chromatography.4.Observed the viable fetus in mice by stereoscopic microscope(20X)to determine whether NTD was present,and recorded the number of NTD embryos and healthy embryos to calculate the incidence rate of NTD.5.The apoptosis level of neural epithelial cells in E12.5d embryo was detected by fluorescence TUNEL assay.6.The protein expression levels of Pax3,P53,Bax and Bcl-2 in E10.5d embryos were detected by Western blot.7.Ultra performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-QTOFMS)was used to analyze the non targeted metabonomics of liver and embryo samples of E12.5d pregnant mice.Result1.There was no significant difference in the average daily dietary intake of pregnant rats in each group during pregnancy.2.On E8.5d,the fasting blood glucose level of the four groups of diabetic pregnant mice was significantly higher than that of the healthy control group(P < 0.05),and the average fasting blood glucose level of the four groups of diabetic pregnant mice was more than11.1 mmol / L without significant difference.On E12.5d,the blood glucose level of the four groups of diabetic control group was significantly higher than that of the healthy control group(P < 0.05).3.In the healthy control group,the bedding material was dry,the hair was glossy,the reaction was sensitive and the mental state was good.After E8.5d,diabetic pregnant mice appeared polydipsia,polyuria,erect hair,slow reaction and mental depression.4.The levels of C20:5n-3,C22:6n-3 and total n-3 PUFA in serum and embryo of pregnant mice in diabetic high dose group were significantly higher than those in healthy control group,diabetic control group and diabetic n-6 PUFA group(P < 0.05).The levels of C20:4n-6 and total n-6 PUFA in serum and embryo of pregnant mice in diabetic n-6PUFA group were significantly higher than those in healthy control group,diabetic control group,diabetic low dose n-3 PUFA group and diabetic high dose n-3 PUFA group(P < 0.05).5.There was no significant difference in the number of implantation beds among the five groups.The number of live fetuses in the four groups of diabetic pregnant mice was significantly lower than that in the healthy control group(P < 0.05),and the number of absorbed fetuses in the four groups of diabetic pregnant mice was significantly higher than that in the healthy control group(P < 0.05).The weight of embryo and placenta in diabetic high dose n-3 PUFA group and diabetic n-6 PUFA group was significantly higher than that in diabetic control group(P < 0.05).6.The incidence rate of NTD in diabetic control group(12.50%),low dose n-3 PUFA group(11.11%)and diabetes n-6 PUFA group(12.03%)was significantly higher than that in healthy control group(1.33%)(P < 0.05).The incidence rate of NTD in high dose n-3PUFA group(4.44%)was significantly lower than that in diabetic control group(P <0.05),and there was no significant difference compared with healthy control group.7.The embryonic neuroepithelial cells of diabetic control group,diabetic low dose n-3PUFA group and diabetic n-6 PUFA group showed excessive apoptosis.The apoptosis rate of the three groups was significantly higher than that of the healthy control group(P< 0.05),and the apoptosis rate of the diabetic high-dose n-3 PUFA group was significantly lower than that of the diabetic control group(P < 0.05).There was no significant difference between diabetic high dose n-3 PUFA group and healthy control group.8.At E10.5d,the protein expression levels of Pax3 and Bcl-2 in diabetic control group were lower than those in healthy control group,and the protein expression levels of P53 and Bax were higher than those in healthy control group.The protein expression levels of Pax3 in diabetic low dose n-3 PUFA group,diabetic high dose n-3 PUFA group and diabetic n-6 PUFA group were higher than those in diabetic control group,and the protein expression levels of P53 and Bax were lower than those in diabetic control group.The protein expression level of Bcl-2 in diabetic high dose n-3 PUFA group was higher than that in diabetic control group.9.Compared with healthy control group,11 kinds of liver metabolites were significantly increased and 10 kinds of liver metabolites were significantly decreased in pregnant rats with diabetes control group.There were 6 metabolites significantly increased and 6metabolites significantly decreased in diabetic control embryos compared with healthy control embryos.Most of these metabolites are involved in the metabolic pathways of sphingolipids.Ceramide,sphingosine and 1-phosphate sphingosine play important roles in regulating cell proliferation and apoptosis.The levels of ceramide and sphingosine in liver and embryo of pregnant mice in diabetic control group were higher than those in healthy control group.In the livers of pregnant mice,levels in diabetic high dose n-3PUFA group were lower than those in the diabetic control group,while levels of diabetic n-6 PUFA in the livers of pregnant mice and embryos were higher than those in the diabetic control group.Therefore,n-3 PUFA may reduce the level of embryonic neuroepithelial cell apoptosis by regulating the level of key metabolites in the pathway of sphingolipid metabolism.Conclusion1.n-3 PUFA can reduce the risk of NTD in offspring due to diabetic pregnancy.2.High dose n-3 PUFA supplementation during pregnancy in diabetic pregnant mice can significantly reduce the apoptosis level of embryonic neuroepithelial cells.3.n-3 PUFA may reduce the apoptosis level of embryonic neuroepithelial cells by regulating the expression level of key genes in Pax3-P53 cell apoptosis signaling pathway and the level of key metabolites in sphingolipid metabolism pathway,so as to prevent NTD caused by diabetic pregnancy.