AMPKα1抑制β-catenin/TCF-4信号阻碍巨结肠乳鼠肠神经嵴衍生细胞迁移和线粒体裂解

AMPKα1 inhibits β-catenin/TCF-4 signaling and impedes intestinal neural crestderived cell migration and mitochondrial lysis in congenital megacolon model rats

目的研究AMPKα 1在先天性巨结肠症(Hirschsprung’s disease, HD)肠神经嵴衍生细胞线粒体裂解与迁移中调控作用。方法建立HD乳鼠模型,Western blot检测结肠组织中S100、NSE,AMPKα 1的蛋白表达水平。分离培养肠神经嵴细胞,AMPKα 1过表达质粒转染肠神经嵴细胞,RTqPCR检测结肠组织中AMPKα 1的mRNA水平,Western blot检测细胞中AMPKα 1,Drp1、Fis1、Mfn1、Mfn2、β-catenin和TCF-4表达。Transwell法检测细胞的迁移,透射电镜观察线粒体形状变化,荧光素酶报告实验检测β-catenin/TCF-4的激活情况。结果HD组织中S100、NSE,AMPKα 1蛋白表达水平降低,AMPKα 1过表达抑制细胞的迁移、增加线粒体的体积、抑制Drp1和Fis1的表达和β-catenin/TCF-4的活性(P<0.05)。结论 AMPKα 1抑制β-catenin/TCF-4信号并阻碍先天性巨结肠乳鼠模型肠神经嵴衍生细胞迁移和线粒体裂解。

Objective To study the regulation of AMPKα 1 in mitochondrial lysis and migration of enteric neural crest-derived cells in Hirschsprung’s disease(HD). Methods HD mouse model was established and the protein expression of S100, NSE and AMPKα 1 in colon tissue was detected by Western blot. The enteric neural crest cells were isolated and cultured, and the AMPKα 1 overexpression plasmid was transfected into intestinal neural crest cells. RT-qPCR was used to detect the mRNA level of AMPKα 1 in colon tissue. Western blot was used to detect the expression of AMPKα 1, Drp1, Fis1, Mfn1, Mfn2, β-catenin and TCF-4. The migration of cells was detected by Transwell method, the shape of mitochondria was observed by projection electron microscopy, and the activation state of β-catenin/TCF-4 was detected by luciferase reporter assay. Results The protein expression of S100, NSE and AMPKα 1 was decreased in HD tissues(P<0.05). Overexpression of AMPKα 1 inhibited cell migration(P <0.05), increased mitochondrial volume(P<0.05),and inhibited the expression of Drp1 and Fis1 and β-catenin/TCF-4 activity(P<0.05). Conclusion AMPKα 1 inhibits β-catenin/TCF-4 signaling and impedes intestinal neural crest-derived cell migration and mitochondrial lysis in congenital megacolon model rats.