| Folic acid is one of water-soluble B vitamins. Human body can not directly synthesize folic acid and only get it from foods or drugs. Folic acid mainly functions in various one-carbon transfer reactions, which are crucial for cell growth and tissue repair and essential for fetal growth and development. Quantities of clinical investigations manifested that periconceptional administration of folic acid could reduce the occurrence and recurrence of neural tube defects (NTDs). Therefore, in1992, the United States Public Health Service suggested that women of childbearing age should take400μg folic acid daily for three months before and after conception. From the year of1998, many countries, including the United States, Canada, began to generalize folic acid-fortifed cereals and flour. Moreover, in our country, the government decided to offer folic acid to reproductive women from countryside for free for three months from the year of2009. Epidemiological investigations in recent years showed that the incidence of NTDs was reduced about50%-70%since application of folic acid supplement and fortified food. Based on these data, it was regarded as an important public health measure to take folic acid for prevention of NTDs before and during pregnancy.It was well established that maternal diabetes was associated with an increased risk of fetal malformations and growth disturbance, including NTDs. But the incidence of NTDs was obviously decreased through effective control of maternal blood glucose level, indicating that more healthy-like babies were born as a result of medical improvement. However, results of tracing studies showed that there was different extent of defects in the central nervous system (CNS) of diabetic offsprings, although they appeared normal. Therefore, it is significant to research on the CNS development of these healthy-like offsprings of diabetic women. Both of clinical and experimental studies showed that folic acid supplementation decreased the incidence of NTDs in diabetic pregnancy. In the light of the above, some experts recommended that diabetic women should take high dose of folic acid (4-5mg/d) during their pregnancy. However, it remained unclear about the effects of high dose of folic acid on the development of CNS in diabetic embryos.Many experts focused on exploring the mechanisms of folic acid for prevention of NTDs in light of folic acid absorption, methylation and so on. Besides, folic acid acted as an antioxidant for easing oxidative stress in diabetic offsprings. However, the mechanisms of folic acid on the development of CNS remain to be investigated. The CNS is derived from neural tube, which is mainly composed of neural stem cells (NSCs). During pregnancy, nutrients may affect the proliferation, apoptosis and differentiation of embryonic NSCs and, consequently, exert an impact on the development of CNS. It was shown that folic acid affected the proliferation, apoptosis and differentiation of NSCs in vitro. Therefore, we speculate that folic acid may influence the development of CNS by regulating the proliferation, apoptosis and differentiation of NSCs. However, these findings are derived mainly from in vitro experiments and they may not fully reflect the actual scenario in vivo during CNS development.In the present study, we established diabetic pregnant mice model and explored the effects and mechanisms of folic acid, especially high dose of folic acid on the development of CNS from mice embryos without obvious malformations in light of proliferation, apoptosis, differentiation of NSCs and neuronal migration.Part I Effects of folic acid supplementation on blood glucose levels of pregnant mice and embryonic developmentFirst of all, we established diabetic mice model by introperitoneal injections of streptozotocin and then these mice were divided into hyperglycemic (HG,6.7mM<blood glucose level<16.7mM) and diabetic (DM, blood glucose leve1≥16.7mM) groups according to their blood glucose levels. After caged with healthy adult male mice, these pregnant mice were administrated with different doses of folic acid (0,3and15mg/kg body weight) from embryonic day0.5(E0.5) to designated day. Then we determined the effects of folic acid on the blood glucose levels of pregnant mice and the development of embryos, especially on the development of CNS. It was shown that maternal blood glucose levels of the HG and DM group for each checkpoint were all higher than that of the control group, whereas there was no significant difference between non-folic acid and folic acid supplementation groups. Both doses of folic acid improved the rate of viable embryos in the HG and DM groups. More importantly, the incidence of NTDs was decreased with folic acid supplementation, notably when a high dose was administered to HG and DM pregnant mice. In addition, administration of folic acid increased fetal body weight and crown-rump length of the DM group. On the basis of hematoxylin-eosin-stained sections, there was no evidence of structural abnormalities in groups with or without folic acid supplementation.These results manifested that folic acid exerted no obvious effects on blood glucose levels of pregnant mice, but improved the rate of viable and NTD embryos of HG and DM pregnancy and embryomic body weight and crown-rump length of DM pregnancy. In addition, there were no obvious alterations in the brains of control, HG and DM embryos without brain malformations after folic acid supplementation.Part II Effects of folic acid supplementation on proliferation and apoptosis of neuroepithelial cells of mouse embryosIn the present study, the proliferation of neuroepithelial cells in the El1.5forebrain was detected by BrdU incorporation assay. Results showed that the percentage of BrdU-positive cells were higher in the control group supplied with folic acid. A higher percentage of BrdU-postive cells were found in the HG and DM groups supplemented with both doses of folic acid. Our results suggested that folic acid promoted the proliferation of neuroepithelial cells of embryos from normal, HG and DM pregnancy.We next examined the apoptosis of neuroepithelial cells in the El1.5forebrain with TUNEL assay. It was shown that there was no obvious difference for the percentage of TUNEL-positive cells in the control group after folic acid supplementation. The percentage of apoptotic cells in the forebrain from the HG and DM groups was decreased significantly after folic acid supplementation. Our results suggested that folic acid decreased the apoptosis of neuroepithelial cells of embryos from normal, HG and DM pregnancy.Part Ⅲ Effects and mechanisms of folic acid supplementation on differentiation of neural stem cells of mouse embryos To explore the effects of folic acid on the development of neurons in vivo, we examined the expression of neuronal specific class Ⅲ β-tubulin (Tuj1), an early neuronal marker, in the telencephalons of E13.5embryos. We found that a higher incidence of Tuj1-positive cells was detected in the control, HG and DM groups with folic acid supplementation. The protein and mRNA levels of Tuj1in the telencephalons of the control, HG and DM groups were also increased after folic acid supplementation, especially at high dose. These results suggested that folic acid promoted the differentiation of NSCs into neurons of embryos from normal, HG and DM pregnancy.In order to determine the causes of folic acid supplementation that led to prematuration of neurons in the telencephalons, we examined mRNA expression of basic helix-loop-helix (bHLH) transcription factors, such as Neurogl, Neurog2, Mashl, NeuroDl and NeuroD2, which were correlated with neurogenesis. We found that the mRNA levels of Neurogl, Neurog2, Mashl and NeuroD2were increased in E13.5telencephalons with folic acid supplementation, especially at high dose. The mRNA expression of NeuroD1appeared to be unaltered in all groups. Our results indicated that neurogenesis-related bHLH transcription factors might be involved in the differention of NSCs into neurons under normal, HG and DM conditions.Next, we mainly compared the difference of glial fibrillary acidic protein (GFAP) expression in groups with or without folic acid supplementation. A few GFAP-positive cells were detected in the subventricular zone and hippocampus of E18.5telencephalons, but they existed as immunostained dots or lines with no obvious characteristic cell shape. Administration of folic acid, especially at high dose, markedly increased the GFAP-positive cells in the telencephalons of the control, HG and DM groups. Results of western blot and real-time RT-PCR showed that GFAP expression was elevated in the telencephalons of the control, HG and DM groups with folic acid supplementation. These results suggested that folic acid promoted the differentiation of NSCs into astrocytes of embryos from normal, HG and DM pregnancy.In order to search for the mechanisms of folic acid on the development of astrocytes in the telencephalons of the control, HG and DM groups, we examined the mRNA levels of Hesl, Hes5, Id1and Id2, whose expression in coordination determines the astrogliogenesis. It was found that the mRNA levels of Hes5, Id1and Id2were significantly increased in E18.5telencephalons of the control group with folic acid supplementation. Moreover, folic acid supplementation, especially at high dose, enhanced the elevated mRNA levels of Hes5, Idl and Id2in the HG and DM groups. However, the mRNA expression of Hesl remained relatively unaltered in all groups. Our results indicated that astrogliogenesis-related bHLH transcription factors might participate in the differention of NSCs into neurons,Part IV Effect of folic acid supplementation on neuronal migration of mouse embryosIn this study, we determined the effect of folic acid on neuronal migration in the telencephalons of embryos at El3.5. Doublecortin (DCX) is one kind of microtubule-associated proteins and mainly expressed in migrating neurons. Immunostaining results showed that the expression of DCX in folic acid supplementation groups was markedly increased. The protein level of DCX was obviously increased in the HG and DM groups with high dose of folic acid supplementation. These results indicated that folic acid might affect neuronal migration of embryos from normal, hyperglycemic and diabetic pregnancy.In conclusion, folic acid not only promotes the proliferation and decreases the apoptosis of neuroepithelial cells, but also promotes neuronal migration in embryos from the control, HG and DM pregnancy. These may be mechanisms of folic acid benefit for the development of CNS. However, folic acid, especially at high dose, can aggravate the premature differentiation of NSCs in embryos exposed to hyperglycemic and diabetic conditions, which may perturb the internal environment and be detrimental to the normal development of CNS. Arising from the present results, it is suggested that folic acid, especially at high dose, may be a double-edged sword in its periconceptional use in hyperglycemic and diabetic pregnancy. |
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