基于SMAD4/PI3K/AKT/GSK-3β通路研究叶酸预防砷诱导胎鼠先天性心脏病的分子机制

Folic acid protects arsenic-induced congenital heart disease by up-regulating SMAD4/PI3K/AKT/GSK-3βpathway

目的探索SMAD4/PI3K/AKT/GSK-3β通路在叶酸预防砷诱导胎鼠先天性心脏病中的可能作用机制。方法选取30只Sprague-Dawley大鼠随机分为:对照组、砷暴露组和叶酸+砷干预组。收集胎鼠心脏,H-E染色观察胎鼠心脏病理改变。TUNEL试剂和ki67免疫组织化学染色检测胎鼠心肌细胞凋亡/增殖状态。RT-PCR及WB测定胎鼠心脏组织中Smad4、Gata4、Nkx2.5及PI3K/AKT/GSK-3β通路的表达水平。CO-IP探索SMAD4和NKX2.5的相互作用关系。结果砷暴露组胎鼠先天性心脏病的发病率为13.3%,对照组和叶酸+砷干预组均为0%。砷暴露组的凋亡心肌细胞较对照组和叶酸+砷干预组显著增加,而增殖心肌细胞明显减少(P<0.05)。砷暴露组胎鼠心脏组织中Smad4、Gata4、Nkx2.5mRNA和蛋白水平相较于对照组和叶酸+砷干预组明显下调(P<0.05)。SMAD4和NKX2.5存在相互作用。砷暴露组胎鼠心脏组织的总PI3K、总AKT、总GSK-3β的蛋白表达水平相较于对照组和叶酸+砷干预组均无显著性差异(P>0.05),但p-PI3K、p-AKT、p-GSK-3β的蛋白表达水平比对照组和叶酸+砷干预组均显著性下调(P<0.05)。结论围孕期补充叶酸可缓解母鼠砷暴露引起的胎鼠心肌细胞增殖减少,凋亡增加,逆转砷暴露引起的Smad4的表达下调,修复砷导致的PI3K/AKT/GSK-3β通路损伤,减少砷诱导先天性心脏病的发生。

Background To explore the possible mechanism of action of folic acid in the prevention of arsenic-induced congenital heart disease in fetal mice about the SMAD4/PI3K/AKT/GSK-3β pathway.Methods Thirty Sprague-Dawley rats were randomly divided into control group,arsenic exposure group and folate+arsenic intervention group.Fetal hearts were collected and cardiac pathological injury were observed by H-E staining.Apoptosis/proliferation status of cardiomyocytes was detected by TUNEL reagent and immunohistochemical staining with ki67.The expression levels of Smad4、Gata4、Nkx2.5 and PI3K/AKT/GSK-3β pathways in heart tissue were determined by RT-PCR and WB.CO-IP explores the interaction relationship between SMAD 4 and NKX 2.5.Results The incidence of congenital heart disease in the arsenic-exposed mice was 13.3%,and 0% in both the control group and the folate+arsenic intervention group.Apoptotic cardiomyocytes were significantly increased in the arsenic-exposed group compared with the control group and the folate+arsenic intervention group,while the proliferating cardiomyocytes were significantly reduced(P<0.05).The levels of Smad4,Gata4,Nkx2.5 mRNA and protein in the heart tissue of the arsenic-exposed group were significantly downregulated compared with the control group and folate+arsenic intervention group(P<0.05).SMAD4 interact with NKX2.5.The protein expression levels of total PI3K,total AKT,and total GSK-3βof the arsenic-exposed group were not significantly different from the control group(P>0.05),but the protein expression levels of p-PI3K,p-AKT,and p-GSK-3β were significantly lower than the control group and folate+arsenic intervention group(P<0.05).Conclusion Perinatal folic acid supplementation can alleviate the decreased proliferation and increased apoptosis of fetal mice caused by arsenic exposure,reverse the downregulation of Smad4expression caused by arsenic exposure,repair the damage of PI3K/AKT/GSK-3β pathway,and reduce the occurrence of arsenic-induced congenital heart disease.