The Mechanism On Histone Acetylation And Prenatal Alclhol Exposure Cause Fetal Cardiac Abnormalities In Mice

Part 1:Prenatal alcohol exposure increased the level of acetylated histone H3 to cause over-expression of cardiac specific genes in miceObjectiveTo build the prenatal alcohol exposure model, the pregnant mice are taken alcohol by gastric tube. The study is to detect the effect of alcohol on expression of heart development related genes as GATA4, NKX2.5, DHAND and EHAND, the changes of acetylated histone H3, the expression of acetylated histone H3 which combined by these genes in promoter region in fetal mice. In the present study, we will test the role that prenatal alcohol exposure can increase the level of acetylated histone H3, thereby causing over-expression of cardiac specific genes in fetal mice.Methods1) Model:The pregnant mice were taken 56% alcohol by gastric tube during E7.5 and E14.5, then collected the fetal mice heart on the last day. Specimens were extracted nucleoprotein and RNA, which were numbered and saved for further study.2) Groups:The pregnant mice were divided into three groups, blank group, DMSO group and Alc proup.3) Method:The histone acetylation enzyme (HATs) activity was detected by colorimetric assay. The level of acetylated histone H3 was measured by western-blotting. The expression of cardiac related genes GATA4, NKX2.5, DHAND and EHAND were determined by real-time-quantitive PCR. The acetyled histone H3 which were combined in the promoter region of GATA4, NKX2.5, DHAND and EHAND were detected by ChIP-real-time quantitive PCR.Results1) Prenatal alcohol exposure significantly increased the HATs activity on E14.5 (p<0.05).2) The level of acetylated histone H3 in Alc group was significantly higher compared to the blank group and DMSO group (p< 0.05).3) The mRNA expression level of cardiac related genes GATA4, NKX2.5, DHAND and EHAND were increased in the Alc group compared to the blank group and DMSO group (p< 0.05).4) The acetylated histone H3 which were combined in the promoter region of cardiac related gene GATA4, NKX2.5, DHAND and EHAND were increased in the Alc group compared to the blank group and DMSO group (p<0.05).Conclusions1) Prenatal alcohol exposure increase the HATs activity in fetal mice.2) Prenatal alcohol exposure can increase the level of histone H3 acetylation, lead to over-expression of AcH3 in fetal mice.3) Prenatal alcohol exposure causes the over-expression of heart development related genes GATA4 and NKX2.5, DHAND and EHAND.4) Prenatal alcohol exposure significantly increase the AcH3 which combined in the promoter region of heart development related genes GATA4 and NKX2.5, DHAND and EHAND.Part2:Curcumin reduces the level of acetylated histone H3 against the over-expression of cardiac specific genes induced by prenatal alcohol exposureObjectiveOn the basis of pregnant alcohol exposure, the pregnant mice were injected with the HATs inhibitor, Curcumin. Curcumin reduces the level of histone H3 acetylation in cardiac tissue, alters the over-expression of cardiac specific genes, and to explore whether it can be used to protect the heart development.Methods1) Model:The pregnant mice were taken 56% alcohol and Curcumin during E7.5 and E14.5, then collected the fetal mice heart on the last day. Specimens were extracted nucleoprotein and RNA, which were numbered and saved for further study.2) Groups:The pregnant mice were divided into five groups, blank group, DMSO group, Ale proup, Cur group and Ale plus Cur group.3) Method:The histone acetylation enzyme activity was detected by colorimetric assay. The level of acetylated histone H3 was measured by western-blotting. The expression of cardiac related genes GATA4, NKX2.5, DHAND and EHAND were determined by RT-Q-PCR. The acetyled histone H3 which were combined in the promoter region of GATA4, NKX2.5, DHAND and EHAND were detected by ChIP-RT-Q-PCR.Results1) The HATs activity of Cur group had significant difference compared to the blank group, DMSO group, or Ale group (p<0.05). There is no significant difference between the Ale and Cur group and blank group, or DMSO group (p> 0.05).2) The level of AcH3 in heart treated with curcumin was significantly reduced compared to the blank group,or DMSO group (P<0.05).The level of AcH3 in heart treated with alcohol was significantly increased compared to the blank group, or DMSO group (P<0.05). Meanwhile, curcumin can reduce the high level of AcH3 induced by alcohol, there was no difference between the blank group, or DMSO group and alcohol plus curcumin (p>0.05).3) The mRNA expression level of cardiac related genes GATA4, NKX2.5, DHAND and EHAND were decreased in the Cur group compared to the blank group and DMSO group (p< 0.05). There was no difference between the blank group, or DMSO group and alcohol plus curcumin (p>0.05).4) The acetylated histone H3 which were combined in the promoter region of cardiac related gene GATA4, NKX2.5, DHAND and EHAND were decreased in the Cur group compared to the blank group and DMSO group (p<0.05). There was no difference between in alcohol plus curcumin group compared to the blank group and DMSO group (p> 0.05).Conclusions1) Curcumin can reverse the hyper-acetylation status by prenatal alcohol exposure in fetal mice heart.2) Curcumin can cause the over-expression of AcH3 by prenatal alcohol exposure back to normal in fetal mice heart.3) Curcumin can reverse the over-expression of genes GATA4, NKX2.5, DHAND and EHAND caused by prenatal alcohol exposure to recover to near normal levels.4) Curcumin significantly reverse the over-expression of the AcH3 which combined in the promoter region of heart development related genes GATA4, NKX2.5, DHAND and EHAND induced by prenatal alcohol exposure.Part 3:SAHA increase the level of acetylated histone H3 to aggravate the over-expression of cardiac specific genes induced by prenatal alcohol exposureObjectiveBased on the pregnant alcohol exposure, the pregnant mice were injected with the HDACs inhibitor, SAHA. SAHA increases the level of histone H3 acetylation in mice, alters the over-expression of cardiac related genes, and to explore whether it can be used to aggrevate the heart development.Methods1) Model:The pregnant mice were taken 56% alcohol and SAHA during E7.5 and E14.5, then collected the fetal mice heart on the last day. Specimens were extracted nucleoprotein and RNA, which were numbered and kept for further study.2) Groups:The pregnant mice were divided into five groups, blank group, DMSO group, Alc proup, SAHA group and Alc plus SAHA group.3) Method:The histone acetylation enzyme activity was detected by colorimetric assay. The level of acetylated histone H3 was measured by western-blotting. The expression of cardiac related genes GATA4, NKX2.5, DHAND and EHAND were determined by RT-Q-PCR. The acetyled histone H3 which were combined in the promoter region of GATA4, NKX2.5, DHAND and EHAND were detected by ChIP-RT-Q-PCR.Results1) The HATs activity of SAHA group had significant difference compared to the blank group and DMSO group(p<0.05). The HATs activity of alcohol plus SAHA group had significant difference compared to the Alc group and SAHA group(p<0.05).There is no significant difference between the Alc group and SAHA group(p>0.05).2) The level of AcH3 in heart treated with SAHA was significantly increased compared to the blank group, or DMSO group (P<0.05). There was no difference between the Alc group and SAHA group (p>0.05). Meanwhile, SAHA can aggrevate the high level of AcH3 induced by alcohol, the level of AcH3 in heart treated with Alc and SAHA was significantly increased compared to the Alc group, or SAHA group (P<0.05).3) The mRNA expression level of cardiac related genes GATA4, NKX2.5, DHAND and EHAND were increased in the SAHA group compared to the blank group, or DMSO group (p< 0.05). There was no difference between the Alc group and SAHA group (p>0.05). The Alc and SAHA group is was significantly increased compared to the Ale group, or SAHA group (P<0.05).4) The acetylated histone H3 which were combined in the promoter region of cardiac related gene GATA4, NKX2.5, DHAND and EHAND were increased in the SAHA group compared to the blank group and DMSO group (p< 0.05). There was no difference between the Ale group and SAHA group (p> 0.05). The Ale and SAHA group is was significantly increased compared to the Ale group, or SAHA group (P<0.05).Conclusions1) SAHA can aggravate the hyper-acetylation status by prenatal alcohol exposure in fetal mice heart.2) SAHA can intensify the over-expression of AcH3 by prenatal alcohol exposure in fetal mice heart.3) SAHA can aggravates the over-expression of related genes GATA4, NKX2.5, DHAND and EHAND caused by prenatal alcohol exposure.4) SAHA significantly aggravates the over-expression of the AcH3 which combined in the promoter region of heart related genes GATA4, NKX2.5, DHAND and EHAND induced by prenatal alcohol exposure.Part 4:Alcohol exposure increase the level of acetylated histone H3 to cause the over-expression of cardiac related genes in H9C2 cells via JNK signaling pathwayObjectiveBased on the intervention of alcohol, SP600125 was used to block the JNK signaling pathway in H9C2 cells, to observe the changes in the level of Histone H3 acetylation and over-expression of downstream cardiac specific genes caused by alcohol exposure, to explore whether JNK mediated the effects of intervention by alcohol in H9C2 cells. In this study, we prove that JNK signaling pathway mediated the hyper-acetylation of histone H3 and over-expression of cardiac specific genes induced by alcohol exposure.Methods1) The optimal dose:After treated the H9C2 cells with alcohol for 36 hour, added different concentrations of SP600125 in the cells to block the JNK pathway. MTT assay was used to detect the cells viabilities to determine the optimal dose of SP600125.2) Groups:The H9C2 cells were divided into five groups, blank group, DMSO group, Alc proup, SP group and Alc plus SP group.3) Method:The histone acetylation enzyme activity was detected by colorimetric assay in H9C2 cells. The level of acetylated histone H3 was measured by western-blotting in H9C2 cells. The expression of cardiac related genes GATA4, TBX5, DHAND and EHAND were determined by RT-Q-PCR H9C2 cells. The acetyled histone H3 which were combined in the promoter region of GATA4, TBX5, DHAND and EHAND were detected by ChIP-RT-Q-PCR in H9C2 cells.Results1) There was no difference between the SP group and DMSO group when treated with 0μM,5μM,7.5μM and 10 μM of SP600126 in H9C2 cells (p> 0.05). There was significant difference between the SP group and DMSO group when treated with 15μM,20 μM and 25 uM of SP600126 in H9C2 cells (P<0.05).2) The HATs activity and level of AcH3 in Ale group had significant difference compared o the blank group and DMSO group(p<0.05). The HATs activity and level of AcH3 in SP group had no difference compared o the blank group and DMSO group(p>0.05). The HATs activity and level of AcH3 in Ale and SP group had no difference compared o the blank group, DMSO group, or SP group(p> 0.05).3) The mRNA expression of cardiac related genes GATA4, DHAND and EHAND were increased in the Ale group compared to the blank group, or DMSO group (p<0.05). The Ale and SP group is was no difference compared to the blank group, DMSO group, or SP group (p>0.05). There was no difference between the SP group and the blank group, or DMSO group (p>0.05). The mRNA expression of cardiac specific genes TBX5 has no difference between the groups (p>0.05).4) The AcH3 which were combined in the promoter region of cardiac related gene GATA4, DHAND and EHAND were increased in the Ale group compared to the blank group, SP group,or DMSO group (p<0.05). The Ale and SP group is was no difference compared to the blank group, DMSO group, or SP group (p>0.05). The AcH3 which were combined in the promoter region of TBX5 has no difference between the groups (p>0.05).Conclusions1) The optimal dose of SP600125 is 15 μM.2) JNK pathway mediate the effect that Ale increase the HATs activity and level of histone H3 acetylation.3) JNK pathway mediate the effect that Ale increase the mRNA of cardiac related genes GATA4, DHAND and EHAND.4) JNK pathway mediate the effect that Ale increase the AcH3 which were combined in the promoter region of cardiac related genes GATA4, DHAND and EHAND.