TCDD通过AHR和Wnt/β-catenin信号通路交互作用导致腭裂的机制研究

Study on the mechanism of cleft palate caused by TCDD through the interaction of AHR and Wnt/β-catenin signaling pathway

目的探究2,3,7,8-四氯二苯并对二噁英(TCDD)在胚胎腭发育过程中对芳香烃受体(AHR)和Wnt/β-catenin信号通路的影响。方法将36只怀孕的C57BL/6N小鼠随机分为对照组(n=12)、TCDD组(n=12)和TCDD+CH223191组(n=12),并在孕7.5 d(GD7.5)至GD10.5分别对不同组小鼠进行灌胃处理。实验分别在GD13.5、14.5、15.5安乐处死小鼠获取胚胎头部和腭突组织。统计各组腭裂率;HE染色观察胎鼠腭部发育情况;免疫组化染色检测胎鼠腭突组织中蛋白CYP1A1和β-catenin的定位及表达,以及增殖标志物PCNA、Ki67的表达情况;Western blot检测AHR和Wnt/β-catenin信号通路上相关蛋白的表达;TUNEL染色法检测腭突组织中细胞凋亡情况。结果(1)对照组的小鼠胚胎未发生腭裂,而TCDD组的小鼠胚胎腭裂率为96.77%,共同暴露于TCDD和CH223191的小鼠胚胎腭裂率为81.82%。(2)HE染色显示对照组胎鼠腭突在GD13.5未发生接触,GD14.5两侧腭突接触并形成中线上皮缝(MES),GD15.5两侧腭突完全融合;而TCDD组胎鼠腭突在GD13.5~15.5均未发生接触;TCDD可导致胎鼠腭部前、中、后段均发生腭裂。(3)Western blot结果显示TCDD可以激活AHR信号通路;Wnt/β-catenin信号通路受TCDD影响,Wnt 3a和β-catenin蛋白在GD13.5和GD14.5中相对表达水平升高,而在GD15.5中降低(P<0.05);磷酸化GSK-3β/总蛋白的比值在GD13.5,GD14.5中相对升高,而在GD15.5中相对下降(P<0.05);细胞周期蛋白Cyclin D1在GD13.5~15.5中表达水平逐渐降低,并于GD14.4之后低于对照组(P<0.05)。TCDD+CH223191组β-catenin在GD15.5的相对表达高于对照组和TCDD组(P<0.05);CYP1A1在GD15.5的相对表达量低于另外两组(P<0.05)。(4)TUNEL染色结果显示GD15.5的腭间充质区域凋亡细胞比率高于对照组(P<0.05)。结论在胚胎腭发育的关键时期,暴露于TCDD的小鼠胚胎会发生腭裂,这可能与AHR和Wnt/β-catenin信号通路之间存在相互作用有关,这种相互作用会对腭突融合过程细胞的增殖与凋亡产生一定影响,从而导致腭裂的发生。

Objective To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)on the aromatic hydrocarbon receptor(AHR)and Wnt/β-catenin signaling pathways during embryonic palatal development.Methods Thirty-six pregnant C57BL/6N mice were randomly divided into control(n=12),TCDD(n=12),and TCDD+CH223191(n=12)groups and treated with gavage from gestation day 7.5(GD7.5)to GD10.5 for different groups of mice.The mice were euthanized at GD13.5,14.5,and 15.5 to obtain embryonic head and palatal process tissues.The mice of cleft palate in each group was counted;HE staining was used to observe the development of the palatal process;Immunohistochemical staining was used to detect the location and expression of the proteins CYP1A1 andβ-catenin,as well as the expression of the proliferation markers PCNA and Ki67 in the palatal eminence tissues of foetal mice;Western blot was used to detect the expression of related proteins on the signaling pathway of AHR and Wnt/β-catenin;and apoptosis was detected in the palatal tissues by TUNEL staining.Results(1)The mouse embryos in the control group did not develop cleft palate,while the rate of cleft palate in the TCDD group and the TCDD+CH223191 group was 96.77%and 81.82%,respectively.(2)HE staining showed that the palatal process of the control group of fetuses did not make contact at GD13.5,and that the palatal process of the two sides contacted and formed the palatal epithelial suture(MES)at GD14.5.The palatal process was completely fused at GD15.5,while the palatal processes of TCDD group were not in contact at GD13.5~15.5;TCDD can cause cleft palate in the anterior,middle and posterior segments of the palate of foetal mice.(3)Western blot results showed that TCDD could activate the AHR signaling pathway;Wnt/β-catenin signaling pathway was affected by TCDD,the relative expression levels of Wnt 3a andβ-catenin proteins were elevated in GD13.5 and GD14.5,while they were decreased in GD15.5(P<0.05);the ratio of phosphorylated GSK-3β/total protein was relatively elevated in GD13.5 and GD14.5,while it was relatively decreased in GD15.5(P<0.05);the expression level of cell cycle protein Cyclin D1 was gradually decreased in GD13.5~15.5 and was lower than that of control group after GD14.4(P<0.05).The relative expression ofβ-catenin in GD15.5 in the TCDD+CH223191 group was higher than that in the control and TCDD groups(P<0.05);the relative expression of CYP1A1 in GD15.5 was lower than that in the other two groups(P<0.05).(4)TUNEL staining showed that the apoptotic cell ratio in the palatal mesenchymal region of GD15.5 was higher than that in the control group(P<0.05).Conclusion Cleft palate occurs in mouse embryos exposed to TCDD during the critical period of embryonic palatal development,which may be related to the existence of an interaction between the AHR and Wnt/β-catenin signaling pathway,and this interaction will have an effect on apoptosis of the cells in the palatal process of fusion,which will lead to the development of cleft palate.