| ObjectiveGestational diabetes mellitus (GDM) is a common complication of perinatal period, which refers that women have diabetes symptoms during pregnancy. In recent years, the global prevalence of GDM is increasing year by year and getting more and more attention. The epidemiological investigations and animal experiments showed that GDM can cause embryonic malformations, including central nervous system malformations, cardiovascular system abnormalities, digestive system malformations, urinary system malformations, reproductive system abnormalities and skeletal malformations. Among these, eye malformations is getting more and more attention. However, the mechanism is still unclear. Currently, there is no particularly good measurement to treat GDM. The main treatment is hypoglycemic therapy, such as insulin, which has side effects. So finding an effective drug to prevent GDM is very important. In this topic, based on preliminary studies, chick embryo model was used to investigate the mechanism of hyperglycemia induced eye malformation and, as well as the interventional effect of Proanthocyanidins. This research was designed to reveal the mechanism of GDM-induced embryonic eye malformation and to offer scientific basis of the safe and effective drug during pregnancy.MethodsAccording to preliminary results, hyperglycemia-induced eye malformation in chick embryo was established. And then, we screened out the most serious damaged gene by quantitative PCR. Pax6 plasmid was constructed and transfected into chicken embryo. Test embryo mortality, eye malformation rate, eye Pax6 gene and protein expression levels,Otx2 and Six3 gene expression levels. Study the protective effect of proanthocyanidins:The fertilized eggs were randomly divided into 5 groups, normal control (0.72% normal saline), model (glucose, 0.2 mmol/egg), Proanthocyanidins groups (1 nmol/egg and 10 nmol/egg) and edaravone group (0.1 nmol/egg). Proanthocyanidins and edaravone were injected into the air chamber respectively on EDD 0. Glucose was injected into the air chamber on EDD 1 (proanthocyanidins group and edaravone group were also injected glucose). Record embryo mortality, the incidence of eye abnormalities, weight and number of somites. Embryo morphology of all groups were observed by stereo microscope. Use kits to determine glucose content and oxidative stress levels. GLUT1 gene and Pax6 gene were measured by fluorescence quantitative PCR. Pax6 protein level was determined by Western blotting.ResultsAfter administration of high-sugar, PCR results showed that Pax6 was the most serious damaged gene in this model. After overexpression of Pax6, hyperglycemia-induced embryo mortality and eye malformation rates were significantly decreased,Otx2 and Six3 gene and gene expression levels were restored. After giving proanthocyanidins, under high glucose conditions, embryo mortality and eye malformation rate were significantly reduced, body weight and the number of somites were increased. Embryonic growth tended to be normal. However, proanthocyanidins did not affect the level of glucose and GLUT1 gene expression in the embryonic eye. In addition, compared with high glucose group, the intervention of proanthocyanidins increased embryonic eye Pax6 gene and protein expressions. For mechanism research, proanthocyanidins lowered levels of ROS and MDA and rebounded SOD and GSH-Px activity. Compared with high glucose group, proanthocyanidins alleviated the oxidative state.ConclusionThis study proved that Pax6 played an important role in hyperglycemia-induced eye malformation in chick embryo. In addition, we firstly discovered that proanthocyanidins could protect against eye malformation induced by hyperglycemia through anti-oxidant effect, which provided preliminary reference for prenatal medication. |
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