Study on the mechanism of cleft palate caused by TCDD through the interac-tion of AHR and Wnt/β-catenin signaling pathway
Objective To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)on the aromat-ic 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)toGD10.5 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 expres-sion of the proteins CYP1A1 and β-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 pro-teins 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 and β-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 de-creased 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.
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