The Effect And Mechanism Of Excessive All-trans Retinoic Acid On Mouse Embryonic Palate Mesenchymal Cell Proliferation

Objectives To investigate the effect and mechanism of excessive all-trans retinoic acid on cell proliferation of mouse embryonic palate mesenchymal.Methods C57BL/6J mice about at 10-weeks were mated(female: maler=2: 1) to genenrate embryos. The observation of vaginal plug was considered as gestation day 0(GD0).30 pregnant mice were random Ly devided into two groups. Timed pregnant mice(GD10) in experimental group with maternal administration of 100 mg/kg body weight of retinoic acid(RA) by gastric intubation were cervical dislocation executed to evaluate growth changes of palatal shelves by using hematoxylin and eosin(H&E)staining. The pregnant mice in control group were treated with corn oil. On GD13, to establish a primary mouse embryonic palate mesenchymal(MEPM) cell culture model, the fetuses were dissected out, and then their palate shelves were isolated. We used the third generation cell to perform the next study. MEPM cells were seeded in a96-well-plate, and then treated with at RA( 0.1 ?mol/L, 0.5 ?mol/L, 1 ?mol/L, 5?mol/L and 10 ?mol/L) for 24 h, 48 h and 72 h. The effects of at RA on cell proliferation were assessed by performing MTT cell viability assay and Trypan blue staining assay. According to our results, we chose an optimal concentration for next analysis. At the same time, the m RNA expression levels of Smad2 and Smad7 were also detected by quantitative RT-PCR. Furthermore, we also investigated the protein levels of Smad2, p-Smad2, and Smad7 by western blot. Statistical analysis between groups was performed using one-way ANOVA. P<0.05 was considered statistically significant.Results In vivo, the sizes of the shelves were smaller than palatal shelves in control on gestation day 13. The palatal shelves of RA-treated embryos on gestation day 14 were not elevated. Compared with complete secondary palate formation in controls, a large gap eventually presented as a wider cleft phenotype in RA-treated group on gestation day 16. The results showed MEPM cell proliferation was the main process in shelf outgrowth. We found at RA inhibited MEPM cell proliferation with both increasing concentration and increasing incubation time, especially at 72 h in vitro. The optimal concentration of at RA was 4.76 ?mol/L. Next, we chose 5?mol/L at RA as the experimental concentration. We found 5 ?mol/L at RA significantly increase the m RNA and protein expression levels of Smad7(P<0.05). We also found at RA inhibit phosphorylation of Smad2 compared with untreated group(P<0.05). However, the protein and m RNA levels of Smad2 did not change in both at RA-treated and untreated group( P>0.05).Conclusion(1) Excessive all-trans retinoic acid induces inhibition of MEPM cell proliferation which could cause cleft palate.(2) Excessive retinoic acid could supress mouse embryonic palate mesenchymal cell proliferation by downregulating the TGF-?/Smad pathway.