The Effect Of Shh Signaling Pathway Changes On Autophagy In Mouse Embryonic Palatal Mesenchymal Cells

Objective:C57BL/6J mouse embryonic palatal mesenchymal cells were cultured in vitro and treated with dexamethasone and SAG.Then,detecting the expression of Shh signaling pathway related proteins and the level of cell autophagy in mouse embryonic palatal mesenchymal cells to explore the effects of the changes in Shh signaling pathway on autophagy during the process of dexamethasone-induced transformation of cleft palate in mouse.Methods:C57BL/6J mouse embryonic palatal tissues of ED14.5 were obtained.The primary mouse embryonic palatal mesenchymal cells were identified by immunocytochemistry.The cells were cultured in vitro to the third generation and divided into 4 groups according to different intervention conditions.When conventional culture as Control group,adding 1×10^-6mol/L dexamethasone as DEX group,adding 1×10^-6mol/L SAG after dexamethasone is treated for 48h as DEX+SAG group,and simply adding 1×10^-6mol/LSAG for 24h as SAG group.The 4 groups of cells would be used in the following experiments.Experiment 1:In Control group,DEX group and DEX+AG group,the cells were be observed the number of autophagosomes/autophagolysosomes by transmission electron microscopy;Experiment 2:The cells of 4 group were collected,then the total protein of each group of cells were extracted to detect the protein expression level of Shh signaling pathway key factors like Shh,Ptch1,Smo,Gli3A/R,CyclinD1 and autophagy markers as LC3Ⅱ/Ⅰ,P62 and Beclin-1 by Western Blot.Results:Experiment 1:A small number of autophagosomes/autophagolysosomes were observed in a part of cells in the Control group and DEX group.The difference between the two groups was not statistically significant（P>0.05）.A large number of autophagosomes/autophagolysosomes were seen in DEX+SAG group,and the difference was statistically significant compared with Control group and DEX group（P<0.05）.Experiment 2:（1）The related proteins of autophagy:Compared with the Control group,in DEX group,there was no significant difference in the expression levels of LC3Ⅱ/Ⅰand Beclin1（P>0.05）,and the expression level of P62 was significantly reduced（P<0.05）;In DEX+SAG group,the expression levels of LC3Ⅱ/Ⅰand Beclin1 was significantly increased（P<0.05）),the expression level of P62 was significantly reduced（P<0.05）;In SAG group,Beclin1’s expression level was significantly increased（P<0.05）,the expression levels of LC3Ⅱ/I and P62 were significantly reduced（P<0.05）.Compared with DEX group,in DEX+SAG group,the expression levels of LC3Ⅱ/Ⅰand Beclin1 were significantly increased（P<0.05）,the expression level of P62 was significantly decreased（P<0.05）;In SAG group,the expression levels of LC3Ⅱ/Ⅰand P62 were significantly decreased（P<0.05）,the expression level of Beclin1 was significantly increased（P<0.05）.Compared with SAG group,the expression levels of LC3Ⅱ/Ⅰand Beclin1 were significantly increased（P<0.05）,and the expression level of P62 was significantly decreased（P<0.05）in DEX+SAG group.（2）The related proteins of Shh signaling pathway:Compared with the Control group,in DEX group,the expression level of Ptch1,Smo,Gli3A/R,CyclinD1were significantly decreased（P<0.05）,and there was no significant difference in Shh expression level（P>0.05）;In DEX+SAG group,Gli3A/R expression level was decreased（P<0.05）,the expression levels of Ptch1 and CyclinD1 were increased（P<0.05）,the expression levels of Shh and Smo showed no significant change and no statistical significance（P>0.05）;In SAG group,the expression levels of Ptch1,CyclinD1,Smo and Gli3A/R were significantly increased（P<0.05）,and there was no significant difference in Shh’s expression level（P>0.05）.Compared with DEX group,in DEX+SAG group,the expression levels of Ptch1,Smo,Gli3A/R and CyclinD1 were increased（P<0.05）,and the expression level of Shh showed no significant change（P>0.05）;In SAG group,The expression levels of Ptch1,Smo,Gli3A/R and CyclinD1 were all significantly increased（P<0.05）,but there was no significant difference in the expression level of Shh（P>0.05）.Compared with SAG group,the expression levels of Ptch1,Smo,Gli3A/R and CyclinD1were all were reduced（P<0.05）in DEX+SAG group,while there was no significant difference in the expression level of Shh（P>0.05）.Conclusion:1.Dexamethasone can inhibit the Shh signaling pathway of mouse embryonic palatal mesenchymal cells;2.Dexamethasone alone cannot induce autophagy in mouse embryonic palatal mesenchymal cells;3.Activating the Shh signaling pathway alone can induce autophagy in the mouse embryonic palatal mesenchymal cells;4.Dexamethasone may interact with the Shh signaling pathway and then further stimulate cell autophagy through other signals.