Mechanism Of Neural Tube Defects Induced By Maternal Diabetes Through β-hydroxybutyric Acid

Objective:1.To construct a model of neural tube defects(NTDs)caused by diabetes and detect content of β-hydroxybutyrate(BHB)in serum and fetal brain tissue of pregnant mice.2.To test whether BHB can induce NTDs.3.To investigate whether BHB can cause the increase of H3K27 ac by inhibiting the expression of HDAC1 and HDAC3,then cause NTDs in mouse embryos by increasing the expression level of MEST and interfere with the maturation of LRP6.Methods:1.Maternal diabetes-induced NTDs model was established by intritoneal injection of streptozotocin,and the closure of neural tube of E10.5 embryos was observed and detected by stereomicroscopy and HE staining.2.The content of β-hydroxybutyric acid(BHB) in serum of normal pregnant mice and diabetic pregnant mice,and homogenate of brain tissue of normal pregnant mice embryos and neural tube defects of diabetic pregnant mice were determined by Elisa.3.The effect of BHB on NTDs was detected by whole embryo culture technology,and the effect of serum BHB on the content of BHB in embryonic brain tissue was detected by Elisa.4.The expression of HDAC1,HDAC3 and H3K27ac in the brain tissues of normal embryos of normal pregnant mice and neural tube defects of diabetic pregnant mice were detected by Western blotting and immunohistochemistry.5.The expression of HDAC1,HDAC3 and H3K27ac in HT-22 cells treated with different concentrations of BHB were detect by Real-time PCR,Western blotting and immunofluorescence.6.The expression of Mest in the brain tissues of normal embryos of normal pregnant mice and neural tube defects of diabetic pregnant mice were detected by Real-time PCR.7.The expression of MEST and mature LRP6 in the brain tissues of normal embryos of normal pregnant mice and neural tube defects of diabetic pregnant mice were detected by Western blotting.8.Protein expression changes of MEST and mature LRP6 in BHB-treated HT-22 cells were detected by Western blotting.9.Western blotting was used to detect protein expression changes of H3K27 ac,MEST and mature LRP6 after Hdac1 and Hdac3 overexpression vectors were transfected into BHB-treated HT-22 cells.10.Statistical analysis: SPSS16.0 was used for analysis,data were expressed as mean±standard deviation(X±S)and P<0.05 indicated statistically significant difference.Results:1.Maternal diabetes-induced neural tube defects model was successfully constructed by intraperitoneal injection of streptozotocin at 200 mg/kg into E5.5 pregnant ICR mice.the incidence of NTDs was 17.2%.The main manifestations of NTDs were neural tube exposure and forebrain underdevelopment.HE staining showed unclosed neural tube.2.The serum β-hydroxybutyric acid of diabetic pregnant mice was apparently higher than untreated mice (E8.5: P<0.05;E9.5: P<0.001;E10.5: P<0.001).The content of β-hydroxybutyric acid in the brain homogenate of diabetic pregnant mice embryos with neural tube defects was higher than normal embryos(P<0.001).3.E8.5 embryos were cultured with normal serum for 2 days,morphology is normal.With β-hydroxybutyric acid was added at a concentration of 2mM,E8.5 embryos were cultured for 2 days,embryos development were delayed.With β-hydroxybutyric acid was added at a concentration of 4mM.E8.5 embryos were cultured for 2 days,embryos development were delayed and neural tube defects were observed.Elisa showed that the increase of BHB concentration in serum caused the increase of BHB content in embryonic brain tissue.4.Real-time PCR showed that the mRNA expression of Hdac1 and Hdac3 in embryos with neural tube defects were lower than control group(Hdac1: P<0.05;Hdac3:P<0.05).Western blotting showed that the protein expression of HDAC1 and HDAC3 in the brain tissues of neural tube defects were lower than control group(HDAC1: P<0.01;HDAC3: P<0.01)and the expression of H3K27 ac in the brain tissues of neural tube defects increased(P<0.05).5.Real-time PCR showed that Hdac1 expression in cells treated with 2mM and4 mM BHB was lower than control group(2mM: P<0.001;4mM: P<0.001),Hdac3 expression was also low(2mM: P<0.05;4mM: P<0.01).These results indicated thatβ-hydroxybutyric acid decreased the expression of Hdac1 and Hdac3.Western blotting and immunofluorescence results showed that the expression of H3K27ac was increased in cells treated by BHB (4mM: P<0.001).6.Real-time PCR showed that the m RNA expression of Mest in the brain tissues of embryos with neural tube defeets was higher than control group(P<0.05).7.Western blotting showed that the expression of MEST in neural tube defects brain tissues was higher than control group,the expression level of mature LRP6 decreased in neural tube defects brain tissues(MEST: P<0.05;Mature LRP6: P<0.05).8.Western blotting showed that the expression level of MEST increased(P<0.01),and the expression level of mature LRP6 decreased in BHB-treated cells(P<0.01).9.Western blotting showed that the expression of H3K27ac(HDAC1: P<0.05;HDAC3: P<0.05)and MEST(HDAC1: P<0.05;HDAC3: P<0.01)decreased,while the protein expression of mature LRP6 increased(HDAC1: P<0.01;HDAC3: P<0.01),after Hdac1 and Hdac3 overexpression vectors were transfected into BHB-treated HT-22 cells.Conclusion:1.Maternal diabetes-induced NTDs model was established by intritoneal injection of 200mg/Kg streptozotocin.The serum β-hydroxybutyric acid content of diabetic pregnant mice was increased.The β-hydroxybutyric acid content of the brain tissues of the neural tube defects of diabetic pregnant mice was higher than control group,which was positively correlated with the change of β-hydroxybutyric acid in serum..2.Increased serum β-hydroxybutyric acid content can induce neural tube defects and the high concentration of BHB in serum can cause the increase of BHB content in embryonic brain tissue.3.The experiment showed that diabetic pregnant mice may inhibit the expression levels of HDAC1 and HDAC3 and increase the level of H3K27ac by increasing the content of BHB in the embryonic brain tissue,and then increase the expression level of MEST,which interferes with the maturation of LRP6 and causes NTDs.