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目的:探讨p300/CBP是否参与介导了孕期酒精暴露所致的心脏核心转录因子GATA4和NKX2.5的过表达,为防治酒精所致的先天性心脏病提供新的切入点。方法:选取C57BL/6小鼠作为研究对象,随机分为对照组、酒精组、DMSO+酒精组和姜黄素+酒精组。收集胚胎16.5d胎鼠心脏,采用染色质免疫共沉淀技术和Western blot检测小鼠心脏组织中GATA4、NKX2.5启动子区域p300/CBP的结合量及组蛋白ac-H3的乙酰化水平。运用RT-PCR检测GATA4、NKX2.5基因mRNA表达量。结果:ChIP-RT-PCR结果显示,酒精能够显著提高GATA4、NKX2.5启动子区域p300/CBP的结合量及组蛋白ac-H3的乙酰化水平(均P<0.01)。Western blot结果表明,酒精能够显著提高胎鼠心肌组蛋白ac-H3表达水平(P<0.01)。RT-PCR结果显示,酒精能够显著上调GATA4、NKX2.5基因mRNA表达量(均P<0.01)。姜黄素(p300/CBP抑制剂)能够显著降低GATA4和NKX2.5基因启动子区域p300/CBP的结合量,纠正酒精所致的组蛋白ac-H3的高乙酰化,并下调GATA4和NKX2.5基因mRNA过表达(均P<0.05)。结论:p300/CBP介导的组蛋白ac-H3高乙酰化可能是孕期酒精暴露致GATA4、NKX2.5基因过表达的关键调控因素。姜黄素通过抑制p300/CBP在启动子区域的结合能显著降低酒精所致的组蛋白H3高乙酰化,下调GATA4、NKX2.5基因过表达,可能成为防治孕期酒精暴露致子代心脏发育畸形的干预新靶点。
OBJECTIVE: To investigate whether p300 / CBP is involved in the over-expression of cardiac core transcription factors GATA4 and NKX2.5 induced by alcohol exposure during pregnancy, and provide a new entry point for the prevention and treatment of congenital heart disease caused by alcohol. Methods: C57BL / 6 mice were randomly divided into control group, alcohol group, DMSO + alcohol group and curcumin + alcohol group. The hearts of embryonic mice were collected for 16.5 days. The binding of p300 / CBP and the acetylation of histone ac-H3 in GATA4 and NKX2.5 promoter regions were detected by chromatin immunoprecipitation and Western blot. The mRNA expression of GATA4 and NKX2.5 was detected by RT-PCR. Results: The results of ChIP-RT-PCR showed that alcohol could significantly increase the binding of p300 / CBP and acetylation of histone ac-H3 in GATA4 and NKX2.5 promoter regions (all P <0.01). Western blot results showed that alcohol significantly increased the expression of histone ac-H3 in fetal rat myocardium (P <0.01). RT-PCR results showed that alcohol could up-regulate the mRNA expression of GATA4 and NKX2.5 (all P <0.01). Curcumin (p300 / CBP inhibitor) significantly reduced the binding of p300 / CBP in the promoter region of GATA4 and NKX2.5 genes, corrected the hyperacetylation of histone ac-H3 by alcohol and down-regulated GATA4 and NKX2.5 Gene mRNA overexpression (all P <0.05). CONCLUSION: Hyperacetylation of histone ac-H3 by p300 / CBP may be the key regulatory factor of overexpression of GATA4 and NKX2.5 during alcohol exposure during pregnancy. Curcumin can significantly reduce the histone H3 acetylation and down-regulate GATA4 and NKX2.5 gene overexpression by inhibiting the binding of p300 / CBP in the promoter region, which may be the prevention and treatment of gestational alcohol exposure induced offspring cardiac malformation Intervention new target.