ABSTRACT
Cleft palate(CP) is a widely studied congenital malformation. However, its etiology and pathogenesis still remain unclear. Proteins are fundamental molecules that participate in every biological process within cells. In this study, we established CP mouse models induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and retinoic acid (RA), using proteomics technology isobaric tags for relative and absolute quantitation (iTRAQ) to investigate the key proteins in the formation of CP. Pregnant mice were given a gavage of TCDD 28µg/kg or retinoic acid 80mg/kg of body weight or equivalent corn oil at gestational day 10.5(GD10.5) and sacrificed at GD 17.5. Foetal mice were recorded and collected for further detection. Western blot was performed to verify the iTRAQ results. Eventually, we obtained 18 common differentially expressed proteins in TCDD group and RA group compared with normal control, 17 up-regulated and 1 down-regulated. 14-3-3sigma and Annexin A1 were up-regulated in experimental groups at GD17.5, which was consistent with Western blot. We speculated that the common differentially expressed proteins might be one of the molecular mechanisms in the formation of cleft palate.
Subject(s)
Cleft Palate/chemically induced , Cleft Palate/metabolism , Polychlorinated Dibenzodioxins , Tretinoin , 14-3-3 Proteins/metabolism , Animals , Annexin A1/metabolism , Disease Models, Animal , Female , Male , Mice, Inbred C57BL , ProteomicsABSTRACT
2,3,7,8-Tetrachlrodibenzo-p-dioxin (TCDD) has been shown to induce cleft palate through growth factor and receptor expression changes during palatogenesis. DNA methylation is an important epigenetic modification that can regulate gene expressions and may be involved in TCDD-induced cleft palate. In this study, we investigated the effects of TCDD on the global and CpG DNA methylation status and the expression levels of DNA methyltransferases (Dnmts) in palate tissue of fetal mice. Pregnant C57BL/6J mice were administered with corn oil or TCDD 28 µg/kg at gestation day 10.5(GD10.5), and sacrificed at GD13.5, 14.5, 15.5. Fetal palates were collected for molecular analysis. Global DNA methylation status was detected by Methylamp™ Global DNA Methylation Quantification Ultra Kit. The expression of DNA methyltransferases were examined by quantitative real-time PCR(q-PCR). Methylation Specific PCR (MSP) was performed to analyze CpG methylation status of Dnmts. We found that the global DNA methylation level and the expression of Dnmt3a were higher at GD13.5 in the TCDD group. The methylation level of CpG site 2 in the promoter region of Dnmt3a in the control group was higher than that of the TCDD group at GD13.5. The low CpG methylation level of Dnmt3a at GD13.5 which causes the up-expression of Dnmt3a may induce global hypermethylation in fetal palate tissue. The aberrant global methylation status at GD13.5 may be the cause of palate malformation in fetal mice induced by TCDD.
Subject(s)
Cleft Palate/chemically induced , DNA Methylation/drug effects , Palate/embryology , Polychlorinated Dibenzodioxins/toxicity , Animals , DNA Modification Methylases/metabolism , Female , Gene Expression/drug effects , Mice , Mice, Inbred C57BL , Palate/drug effects , Palate/metabolism , Pregnancy , Real-Time Polymerase Chain ReactionABSTRACT
OBJECTIVE: To evaluate whether or not administration of folic acid and resveratrol have preventive effects on cleft palate formation as well as the comparison of the two drugs' s effects. METHODS: Pregnant mice were randomly divided into 9 groups, with 8 mice in each group. The TCDD group mice were dosed with TCDD 28 microg/kg body weight on gestation day 10 (GD 10) animals in folic acid group were respectively dosed with folic acid 15, 10, 5 mg/kg and TCDD 28 microg/kg; resveratrol treated mice were divided into 3 groups: resveratrol 50 mg/kg were orally administered for 6 consecutive days, from gestational day GD 8 to GD13 in resveratrol (GD8-13 ) group; resveratrol 50 mg/kg were orally administered for 6 consecutive days, from gestational day GD 8 to GD13, followed hy an oral administered with TCDD on GD10 in resveratrol (GD8-13) + TCDD group; resveratrol 50mg/kg and TCDD 28 microg/kg were used by gavage administration at GD10 in resveratrol (GD10) + TCDD group. Control mice were treated with the same volume of water for 6 consecutive days from GD8 to GD13 and were given a single dose of corn oil on GD10. The pregnant mice weight and embryos, the number of live, cleft palate, dead and resorption fetal mice were recorded on GD 17.5. The coronal sections of the fetal mice heads were prepared at GD 17.5 and observed by microscopy. RESULTS: Total frequency of clefts was 92.86% in TCDD group, 84.00% (15 mg), 73.08% (10 mg), 84.00% (5 mg) in folic acid + TCDD groups, 0% in resveratrol (GD10) group, 74.51% (GD10), 57.78% (GD8-13) in resveratrol + TCDD groups. The frequency of cleft was 0% in the control group. Compared with the control and the TCDD groups, there were significant differences in the number of live, dead and resorption fetal mice in TCCD + resveratrol (GD8-13) group (P < 0.05). No significant differences in embryonic weight, live fetuses weight, the number of live, dead and resorption fetal mice were found in the other groups (P > 0.05). CONCLUSION: Test dose of folic acid and resveratrol both had certain antagonistic effect on cleft palate in mice induced by TCDD, with folic acid 10 mg/kg, resveratrol 50 mg/kg GD8-13 doses having stronger antagonistic action. Effects of both the two drugs have no significant difference, but resveratrol (50 mg/kg, GD8-13) significantly affects the fetal mice's growth and development under TCDD exposure in utero.
Subject(s)
Abnormalities, Drug-Induced/prevention & control , Cleft Palate/prevention & control , Folic Acid/pharmacology , Polychlorinated Dibenzodioxins/antagonists & inhibitors , Stilbenes/pharmacology , Teratogens , Animals , Cleft Palate/chemically induced , Female , Fetus , Folic Acid/administration & dosage , Humans , Mice , Mice, Inbred C57BL , Pregnancy , Random Allocation , Resveratrol , Stilbenes/administration & dosageABSTRACT
PURPOSE: Kidney ischemia-reperfusion injury (IRI) after kidney transplant remains a major problem, separate from immune rejection that can lead to kidney transplant failure and graft function loss. Free radicals, disturbance of microcirculation and the inflammatory cascade appear to be the main contributors. Radix Codonopsis, a traditional Chinese drug used in vascular diseases, is an antioxidant and free radical scavenger. This study investigates the protective effect and underlying mechanisms of Radix Codonopsis extract saponins on kidney transplantation. METHODS: Renal transplantation was performed after rat kidneys had been stored for 1 h at 4°C. Blood urea nitrogen (BUN), serum creatinine (Scr), superoxide dismutase (SOD) and malondialdehyde (MDA) were assayed; bcl-2 and bax mRNA expression was detected using RT-PCR; bcl-2 and bax protein expression was detected by immunohistochemistry (IHC). Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was used to detect apoptotic cells and determine the apoptosis index (AI). Analysis of variance (ANOVA) followed by Dunnett's test was used when more than two groups were compared. RESULTS: Saponin-treated animals showed increased SOD levels accompanied by decreased MDA, Scr and BUN levels (p < 0.05 vs. untreated controls); bcl-2 mRNA and protein levels were increased in transplanted kidney from treated animals, while bax mRNA and protein levels were decreased (p < 0.05 vs. untreated controls). AI was significantly decreased in transplanted kidneys from treated animals relative to untreated controls (p < 0.05 vs. untreated controls). CONCLUSION: This study clearly demonstrates the protective effects on IRI after kidney transplantation, which may be explained by decreased lipid peroxidation and inhibition of apoptosis.
Subject(s)
Codonopsis , Graft Rejection/prevention & control , Kidney Transplantation/adverse effects , Oxidative Stress/drug effects , Plant Extracts/therapeutic use , Reperfusion Injury/prevention & control , Animals , Apoptosis/drug effects , Disease Models, Animal , Free Radical Scavengers/metabolism , Graft Rejection/metabolism , Graft Rejection/pathology , Immunohistochemistry , In Situ Nick-End Labeling , Male , Malondialdehyde/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/metabolism , Treatment Outcome , bcl-2-Associated X Protein/biosynthesis , bcl-2-Associated X Protein/geneticsABSTRACT
OBJECTIVE: To investigate the effect of the exposure to di- (2-ethylhexyl) phthalate (DEHP) during pregnancy on the DNA methylation level of genomes in the testis of the offspring in mice. METHODS: Pregnant KM mice were randomly divided into three groups, normal control, corn oil and DEHP-exposed. Corn oil and DEHP (500 mg/[kg x d]) were administrated respectively from gestation day 12.5 (GD 12.5) to postnatal day 3 (PND 3). The testes of the offspring were excised on PND 7, and their genomic DNA was treated with EcoR I /Msp I and EcoR I /Hpa II. The genome-wide DNA methylation patterns of the CCGG sites were detected by methylation-sensitive amplification polymorphism (MSAP). The samples were electrophoresed in the ABI 3730 DNA sequencer and the results analyzed by the Genescan3.1. RESULTS: The average incidence of DNA methylation was (34.03 +/- 3.05)% in the DEHP-exposed mice, obviously higher than (28.37 +/- 2.37)% in the normal control and (28.58 2.45)% in the corn oil group, with statistically significant differences (P < 0.05). CONCLUSION: Exposure to DEHP during pregnancy increases the DNA methylation level of the genome in the testis of the offspring and affects the apparent genetic modification of the genome, which may be one of the important toxicological causes of the lesion in the reproductive system.