The Biochemical Action Mechanism of Resveratrol and α-Tocopherol In Alleviating Valproic Acid-Induced Embryonic Teratogenicity

• Main Authors: Pin-Xiao Lin, 林品孝
• Other Authors: Chiu-Lan Hsieh
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/34tbg2

碩士 === 國立彰化師範大學 === 生物技術研究所 === 101 === Valproic acid (VPA) is a sedative frequently used for treatment of epilepsy and pregnant syptoms. VPA has been widely reported to exhibit teratogenicity with a rather complicate action mechanism. To understand the preventive and protective effect of resveratrol (RV) and vitamin E (Vit E) against the VPA teratogenicity, we used the chicken embryo model (CEM) for study. Chicken embryos at HH stage-10 (day 1.5 embryos) were respectively treated with VPA alone, RV alone, Vit E alon, and combined therapy of VPA+RV, and VPA+Vit E. The dosages were VPA 60 M/egg, RV 0.2 and 2.0 M; Vit E 0.2 and 2.0 M, respectively. VPA (60 M) induced a malformation rate 36.5±2.47% (p<0.05) and at the same time a mortality rate 47.7±0.49% (p<0.05). RV and Vit E at 2.0 M respectively reduced the malformation rate to 8.35±1.51% and 5.0±0.0%; and the mortality rate to 5.5±0.7% and 5.0±0.0%. Results also indicated that VPA initiated 1) neural tube defect (NTD); 2) inflammatory edema and liposis in the cervical muscles; and 3) the severe depletion of serum GSH level. RV and Vit E completely rescued the NTD, as evidenced by HE stain of the complete closure of NT in day 3.5 embryos. Literature indicated that NTD could be caused by over accumulation of serum homocysteine (Hcys). HPLC analysis identified the level of HCys 26±3.25%. On treatment with RV and Vit E, the level was reduced to 13.2±0.61M and 15.1±0.01M, respectively. Concomitantly, the serum GSH was restored. On the other hand, the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) which once elevated to 19±4.0 mol/mg protein was found substantially suppressed by RV and Vit E, respectively. Regarding the liposis in cervical muscles, the TG level was 0.26±0.01, 0.21±0.01 and 0.19±0.01 mM, respectively found for the VPA, RV, and Vit E groups. VPA upregulated gene acetyl-CoA carboxylase (ACC) and at the same time downregulated gene carnitine palmitoyltransferase-1 (CPT1). Thus although VPA stimulated the production and initiated the accumulation of carnitine, the liposis status in cervical muscle could not be rescued thereby. In this respect, RV and Vit E successfully alleviated the status of inflammatory edema and liposis of cervical muscles. Alternatively, the 2D proteomic analysis of the cervical muscles in day-1 (HH stage 46) chicks, we selected those protein spots showing significance with p<0.05 for further LC-MS/MS analysis. The proteins apparently appeared in different amount having statistical significance with p<0.05 were identified to be: phosphatidylethanolamine-binding protein 1 (PEBP), ovalbumin, myosin light chain 1 (MYL1), heat shock protein beta-1 (HSPB1, triose phosphate isomerase (TPI), fructose-bisphosphate aldolase C (aldolase C), betaine-homocysteine S-methyltransferase 1 (BHMT1), beta-enolase, filamin-C, dihydrolipoyl dehydrogenase (PDH) complex, pyruvate kinase muscle isozyme (PKM2), serum albumin precursor, ovotransferrin, and peroxiredoxin-1 (Prxd1). As well known, PEBP is related with cell proliferation and NFB modulation. HSBP1 controls cell apoptosis migration and differentiation. TPI, aldolase C, PDH complex and PKM2 are enzymes associated with glycolysis. BHMT1 is the enzyme responsible for converting HCys into methionine. Ovotransferrin is responsible for transferring iron-proteins and is responsible for modulating chondroites and osteogenesis. Prxd1 is able to suppress the oxidative stress.