Combination Effect And Mechanism Of Folic Acid And N-3 Polyunsaturated Fatty Acid On Neural Tube Defects Induced By Diabetic Pregnancy

Objective Previous studies found that supplementation of folic acid and n-3 PUFA can effectively reduce the incidence of NTD,but cannot completely prevent NTD.Pregnant mice were divided into five groups according to whether they were fed n-3 PUFA fortified diet and whether they were intraperitoneal injected with folic acid.The aim of the present study was to investigate the combination effect and mechanism of n-3 PUFA and FA on neural tube defects induced by diabetic pregnancy.Method1.Establishment of animal models and intervention measures: Two hundred8-week-old ICR mice(160 females and 40 males)were housed in the environment of 12 h light/dark cycle,constant temperature(21-23 ℃)and 50-60% constant humidity.After one week diet adaptation,female mice were caged with males overnight at a female/male ratio of 4/1 and examined for a vaginal plug in the next morning.The day when a plug was found was termed day 0.5 of gestation(E0.5d).Previous studies have found that intraperitoneal folic acid injection and dietary n-3 PUFA supplementation can effectively reduce the incidence of NTD,but cannot completely prevent the occurrence of NTD.So pregnant mice were randomly allocated into 5 groups: Healthy control group(HC,AIN-93 G standard chow,daily intraperitoneal injection of PBS buffer),diabetic mellitus control group(DMC,AIN-93 G standard chow,daily intraperitoneal injection of PBS buffer),diabetic mellitus + FA intervention group(DMFA,AIN-93 G standard chow,daily intraperitoneal injection of PBS dissolved FA solution,3mg/kg/d),diabetic mellitus+ n-3PUFA intervention group(DMn-3,50% fat in AIN-93 G chow was replaced with fish oil,daily intraperitoneal injection of PBS buffer),diabetic mellitus + FA and n-3 PUFA intervention group(DMFA+n-3,50% fat in the AIN-93 G chow was replaced with fish oil by intraperitoneal injection of PBS dissolved FA solution 3mg/kg/d).At E6.5d,pregnant mice in the DMC,DMFA,DMn-3 and DMFA+n-3 were intraperitoneally injected with230 mg/kg STZ,and the HC was intraperitoneally injected with equivalent sodium citrate buffer.At E8.5d,blood was collected from the caudal vein to detect the fasting blood glucose level of the mice.Mice with fasting blood glucose ≥ 11.1 mmol/L were considered as diabetic pregnant mice.Some mice in each group were sacrificed at E10.5d to detect parameters concerning the molecular mechanism,and the remaining were sacrificed at E12.5d to examine NTD.Blood samples,liver,embryo and placenta were collected and weighed,frozen in liquid nitrogen and stored at-80℃ for further analysis.2.Laboratory analysis: Fasting blood glucose level of pregnant mice was measured by glucometer.Embryos at E12.5d were examined for NTD by a stereomicroscope.The fatty acid composition in serum,embryo,placenta of E12.5d pregnant rats were detected by gas chromatography.TUNEL assay was used to detect the apoptosis level of E12.5d embryonic neuroepithelial cells.The m RNA level of key genes in the Pax3-P53 apoptosis signaling pathway and FA-Hcy metabolic pathway in E10.5d embryos were detected by quantitative RT-PCR.Western blotting assay was used to detect the expression levels of key proteins in Pax3-P53 apoptosis signaling pathway and FA-Hcy metabolic pathway in E10.5d embryos.DNA methyltransferase 3b(Dnmt3b)enzyme activity was detected by ELISA.The methylation of Pax3 gene promoter region in E10.5d embryos was determined by sequenom time-of-flight mass spectrometry.The concentrations of FA,Hcy and their metabolites in E10.5d embryos were detected by UPLC-QTOFMS.Results1.There was no significant difference in chow intake during pregnancy among the five groups.At E8.5d and E12.5d,the fasting blood glucose concentration of pregnant diabetic mice in the four groups was higher than that in HC.There was no significant difference in the number of implantation beds among the five groups.The number of survival embryos in DMC group was significantly lower than that in HC,and the number of resorptions was significantly higher than that in HC.There was no significant difference in the number of survivals and resorptions between HC and DMFA+n-32.The ratio of DHA and total n-3 PUFA in serum and embryo in DMC group was significantly lower than that in HC.The ratio of EPA,DHA,n-3 PUFA in serum,embryo,palcenta in DMn-3 and DMFA+n-3 group was significantly higher than that in DMC.Supplementation with FA alone had no effect on the ratio of n-3 PUFA.3.The incidence of NTD in DMC(19.41%),DMFA(8.57%)and DMn-3(7.82%)was higher than that in HC(0%).Neuroepithelial cell apoptosis rate in DMFA,DMn-3and DMFA+ n-3 embryos was significantly lower than that in DMC.Neuroepithelial cell apoptosis rate in DMFA+n-3 was significantly lower than that in DMFA and DMn-3.4.At E10.5d embryo,the m RNA and protein expression levels of Pax3 and Bcl-2 in DMC were significantly lower,while the m RNA and protein levels of P53 and Bax in DMC were significantly higher than that in HC.The m RNA and protein levels of Pax3 and Bcl-2 in DMFA+ n-3 group were significantly higher,while the m RNA and protein expression levels of P53 and Bax were significantly lower than that in DMC.5.The concentration of FA,THF and 5-MTHF in DMC were significantly lower than that in HC,while the content of Hcy in DMC was significantly higher than that in HC.The concentration of FA,THF and 5-MTHF in DMFA,DMFA+n-3 embryos were significantly higher than that in DMC,while the concentration of Hcy in DMFA,DMFA+n-3 were significantly lower than that in DMC.6.At E10.5d embryo,the Dnmt3 b enzyme activity in DMC was significantly higher than that in HC.The Dnmt3 b enzyme activity in DMFA+n-3 was significantly lower than that in DMFA and DMFA+n-3.7.At E10.5d embryo,the m RNA and protein levels of MTHFR,CBS,CSE in DMC group were significantly lower than that in HC,while the m RNA and protein levels of MTR,MAT,Dnmt3 b and SAHH were significantly higher than that in HC.The m RNA and protein levels of MTHFR and CBS in DMFA+n-3 group were significantly higher than that in DMC,while the m RNA and protein levels of MTR,MAT,Dnmt3 b and SAHH were significantly lower than DMC.8.The average methylation level of Pax3 gene promoter region in DMC was significantly higher than that in HC.Among the 45 Cp G sites detected in the promoter region of DMC group,16 Cp G sites showed significantly higher methylation level compared with HC group,while no site showed lower methylation level.The methylation level of DMFA+n-3 group was significantly lower than that of DMC group in 14 sites.Conclusion Both FA and n-3 PUFA can effectively reduce the incidence of NTD caused by diabetic pregnancy.The combination effect of them is better than one alone.Its mechanism may be related to the regulation of Pax3-P53 apoptosis signaling pathway and FA-Hcy metabolism pathway.These findings provide new evidence for nutritional intervention of NTD induced by diabetic pregnancy.