Role Of MTHFR,HCY And BDNF In The Development Of Neural Tube Defects In Rats

Objectives: To study the effects of methylenetetrahydrofolate reductase(MTHFR),homocysteine(HCY)and brain-derived neurotrophic factor(brain-derived neurotrophic factor)in the folic acid-homocysteine metabolic pathway derived neurotrophic factor(BDNF)in the development of neural tube defects in rats.Methods: 1.1 Experimental animals and groupsSex Mature female rats purchased from Ningxia Medical University Experimental Animal Center were randomly divided into three groups: normal control group,neural tube defects group and folic acid intervention group.The rats in normal group and neural tube defects were fed with common diet,and the folic acid group was fed with folic acid compound diet(60μg · kg-1).Female and male rats 1: 1 ratio of cage at night,to cage the first 2 days after see female feminine suppository confirmed as 0.5d(E0.5d).On day 10.5 of pregnancy(E10.5d),all-trans retinoic acid AT-RA(105mg · kg-1)was given intragastrically to the neural tube defects group and the folic acid group.At the same time,Continue feeding to E15.5d.1.2 The general morphology of embryos observed and statistics of each group deformity rateThe pregnant female mice were anesthetized by intraperitoneal injection of 10%chloral hydrate(0.3mL / 100g)on day E15.5d.The pregnant mice were routinely disinfected,the abdominal cavity was opened,the double horn-shaped uterus was dissected,the gestational sac and placenta were separated,Neural tube general morphology.Embryonic appearance of encephalocele,high spina bifida,sacrococcygeal swelling as dilated neural tube defects.Records and statistics of live births,stillbirths,absorption of fetal numbers,dominant neural tube defects.1.3 pregnant rats plasma HCY concentration testPregnant mice at 15.5 days of pregnancy,the use of enzyme-linked immunosorbent assay(ELASA)determination of normal group,neural tube defects and folic acid group plasma HCY concentrations.1.4 H.Embryonic neural tube tissue H.E.stainingThe fresh embryos of rats were embedded in paraffin,and the morphology of neural tube in HE staining and the location of neural tube were observed under microscope.1.5 embryonic neural tube tissue MTHFR,BDNF factor immunohistochemical staining and image analysisTissue sections of pregnant rats were obtained.The neural tube tissues of embryonic tissues were stained with immunohistochemistry technique.The average immune response of MTHFR and BDNF expressed in embryonic neural tube was calculated by Image-Pro Plus 6.0(IPP)Optical density(MOD)value.1.6 Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling(TUNEL)staining and imaging of embryonic neural tube tissue analysisTissue sections of pregnant rats were taken and TUNEL technique was used to stain the neural tube tissues of embryos.The apoptosis rate was calculated using Image-Pro Plus 6.0 image analysis software.2 results:2.1 The general morphology of embryos observed and statistics of each group of deformity rate of statistical analysisThree groups of rats were harvested embryos in 241 cases.Under the dissecting microscope,the embryos of rats were observed.In the normal group,the zona pellucida and continuous neural tube were visible on the dorsal surface of the embryo.In the deformity group,the dominant neural tube deformities such as encephalocele,spina bifida and sacrococcygeal swelling were observed.The normal development of the tube,even see the tail deformity.Compared with the normal group,the rate of neural tube defects in the deformity group increased(P <0.05)and the rate of neural tube defects in the folic acid group increased(P <0.05).Compared with the deformitygroup,the deformity rate of the folate group decreased(P <0.05).2.2 embryonic neural tube tissue HE stainingThe normal group of neural tube tissue closed completely.Spinal deformity group with spondylollar bulging,vertebral pedicle fusion.Folic acid intervention group less dominant spina bifida,more volume deformity,sacrococcygeal general structure of the intact,near the skin with small tearing bulging.2.3 three groups of pregnant rats plasma HCY concentration comparisonELASA test showed that plasma HCY levels in pregnant rats were(1.752 ±0.005)nmol / ml in normal group,(2.034 ± 0.101)nmol / ml in normal group and(1.775 ± 0.016)nmol in folic acid group / ml.Compared with the normal group,the concentration of plasma HCY in the pregnant group was higher than that in the normal group(P <0.05).There was no difference in the concentration of HCY among the pregnant women in the folic acid group(P> 0.05).Compared with the deformity group,the concentration of HCY in the pregnant rats in the folic acid group decreased(P <0.05)2.4 Embryonic neural tube tissue MTHFR,BDNF immunohistochemical staining products MOD valueMOD values of MTHFR in normal embryonic neural tube tissue,neural tube abnormal embryonic tissue and folic acid embryonic neural tube tissue were 0.2295 ±0.0217,0.056 ± 0.0146,0.08825 ± 0.0133.MOD values of BDNF in normal embryonic neural tube,neural tube abnormal embryo and folic acid embryonic neural tube were 0.6158 ± 0.0077,0.1655 ± 0.0249,0.357 ± 0.0444.In deformity group,MOD values of immunostaining products of MTHFR and BDNF in embryonic neural tube cells were lower than those in normal group and folic acid group(P <0.05).The MOD values of intracellular immunostaining products expressed by MTHFR and BDNF in the folic acid group were lower than those in the normal group(P <0.05).2.5 TUNEL staining analysis of neural tube in embryonic neural tube: The apoptosis rate of neural tube in normal group was(1.02 ± 0.15)%,that of neural tube in abnormal group was(20.24 ± 2.28)%,Nerve cell apoptosis rate(2.31 ± 0.65)% in the tissue.The apoptosis rate of neural tube in deformity group was higher than that innormal group and folic acid group(P <0.05).The apoptosis rate of neural tube in normal group and folic acid group had no significant change(P> 0.05).Conclusion: 1.The expression of MTHFR in neural tube defects reduces HCY accumulation,resulting in downregulation of BDNF expression and increased apoptosis in embryonic neurons,resulting in blocked neural tube.2.In the mouse with neural tube defects,the MTHFR factor with weaker enzyme activity can be activated to a certain extent after folic acid supplementation.The neurotoxic substance HCY can be effectively degraded,inhibiting the apoptosis of neural tube cells and promoting the expression of BDNF in neural tube tissue Normal expression.