| Summary: | 碩士 === 國立彰化師範大學 === 生物技術研究所 === 100 === The teratogenicity of antiepilepsy drug valproic acid (VPA) mostly is found in genetic and somatic levels, causing teratogenesis involving neurotubular defects (NTD), lumbosacral meningomyelocele, anencephaly, and leg dysfunction due to spina bifida aperta. A diversity of nutraceutics have been tried to alleviate the risk of VPA-teratogenicity. The effect was varying. In order to promote the prescription, to find out its action mechanism can be rather crucial. We hypothesize that early stage teratogenicity can be prevented by using some nutraceutics. Folic acid (FA), Vitamin C (Vit C) and N-acetyl cysteine (NAC), each 10 mM, were tested for their preventive effects against VPA (30 mM, 100 μL) in day-1.5 of chicken embryo at HH stage-10. Proteomic and biomarker analysis were conducted in day-5.5 embryos at HH stage-28.
VPA at 30 mM showed the higher malformation rate (66.7%) with the least mortality (22.2%). Pathological findings indicated that the cervical muscle was more susceptible to VPA injury than the ankle muscle. VPA downregulated the levels of superoxide dismutase (SOD), glutathione (GSH), histone deacetylase (HDAC), and folate; upregulated the levels of H2O2, homocysteine. FA, Vit C, and NAC significantly upregulated SOD, but only Vit C alone activated GSH. Vit C and NAC downregulated H2O2 while FA was totally ineffective. All three nutraceutics rescued HDAC and simultaneously suppressed homocysteine accumulation and raised folate level comparably, although less effectively by NAC.
At HH stage-28 (day 5.5), differential expression of proteins were identified by image analysis (p<0.05) and LC-MS/MS. Several proteins were found changed significantly including ovotransferrin, retinol-binding protein 4, carbonic anhydrase 2, apolipoprotein A-I, NADH-cytochrome b5 reductase, protein SET and 60S ribosomal protein L22. Ovotransferrin is an iron binding transport protein and regulates cartilage and bone formation. The Retinol-binding proteins are likely to play a role in the regulation of retinoid metabolism and retinol-related gene expression. In addition, the product carbonic acid transformed from carbon dioxide and water by carbonic anhydrase 2 tends to affect the angiogenesis, acid-base balance, bone resorption, urea cycle and fatty acid production. Apolipoprotein A-I, a cholesterin transporer, plays a role in metabolism of steroid hormones. Protein SET with a variety of biological functions can be involved in gene regulation, apoptosis, and posttranslational modification of histone acetylation, etc. In embryonic development, VPA induced malformation by disturbing the metabolism of retinol and lipid, the regulation of calcium or iron ion concentration, the pH homeostasis, the production of hormone production, and the regulation of signal transduction and gene expression. In addition to the above cited, co-treatment with folate, Vit C or NAC decreased oxidative stress, recovered HDAC activity and regulated folate metabolism, revealing an overall effect in amelioration of certain degree of malformation caused by VPA.
Based on these data, we conclude VPA possesses “Multiple Point Action Mechanism”. To prescribe medicine multiple combination has to be taken into consideration.
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