Study On The Relationship Between Autophagy And Apoptosis Of Mouse Embryonic Palatal Mesenchymal Cells Induced By Dexamethasone

Objective:In this study,C57BL/6J Mouse embryonic palatal mesenchymal cells were treated with DEX and RAPA,By detecting the changes of autophagy and apoptosis level in mouse Embryonic Palatal Mesenchymal Cells,the purpose of this study was to explore the effect of DEX on autophagy and apoptosis of mouse Embryonic Palatal Mesenchymal Cells during the development of mouse embryonic palatal process.Methods:The palatine process tissue of C57BL/6J mouse embryos was obtained from14.5d embryos.The primary cells of mouse Embryonic Palatal Mesenchymal Cells were obtained by enzyme digestion and identified by immunocytochemistry.mouse Embryonic Palatal Mesenchymal Cells were cultured until the third generation.According to different intervention conditions,mouse Embryonic Palatal Mesenchymal Cells were randomly divided into control group（routine culture）,DEX group(intervention with 1×10^-6mol/L Dexamethasone),RAPA group（intervention with3μmol/L Rapamycin）and DEX+RAPA group(intervention with 1×10^-6mol/L Dexamethasone+3μmol/L Rapamycin).The above groups of cells would be used in the following experiments.Experiment 1:the numbers of autophagy lysosomes and/or autophagosomes in control group,DEX group,RAPA group and DEX+RAPA group were observed by transmission electron microscope.Experiment 2:the total cellular proteins of control group,DEX group,RAPA group and DEX+RAPA group were extracted to detecte the protein expression levels of autophagy markers LC3Ⅱ/Ⅰ,Beclin-1,P62 and apoptosis markers Caspase-3,Bcl-2 and Bax by Western blot technique.Results:Experiment 1:A certain number of autophagosomes/autophagy lysosomes could be seen in Control group,and very few autophagosomes/autophagy lysosomes could be seen in some cells in DEX group,and the difference between the two groups was statistically significant（P<0.05）.A large number of autophagosomes/autophagy lysosomes could be seen in RAPA group,which were significantly different from those in control group and DEX group（P<0.05）.The number of autophagosomes/autophagy lysosomes in DEX+RAPA group was significantly higher than that in DEX group,and the difference between the two groups was statistically significant（P<0.05）.Compared with the RAPA group,the number of autophagosomes/autophagy lysosomes in DEX+RAPA group decreased and the difference was statistically significant（P<0.05）,but it was no significant difference from those in control group and DEX+RAPA group（P>0.05）.Experiment 2:（1）autophagy-related protein:compared with the control group,the expression of LC3Ⅱ/Ⅰand Beclin1 was significantly decreased（P<0.05）and the expression of P62 was significantly increased in DEX group（P<0.05）,In RAPA group,the expression of LC3Ⅱ/Ⅰ,Beclin1 was significantly increased（P<0.05）,and the expression level of P62 was significantly decreased（P<0.05）;In DEX+RAPA group,the expression of LC3Ⅱ/Ⅰwas significantly increased（P<0.05）,While the expression of Beclin1 and P62was not significantly different（P>0.05）.Compared with DEX group,the expression of LC3Ⅱ/Ⅰand Beclin1 in RAPA group was significantly increased（P<0.05）,while the expression of P62 was significantly decreased（P<0.05）;in DEX+RAPA group,the expression of LC3Ⅱ/Ⅰand Beclin1 was significantly increased（P<0.05）,while the expression of P62 was significantly decreased（P<0.05）.Compared with RAPA,the expression of LC3Ⅱ/Ⅰand Beclin1 in DEX+RAPA group decreased significantly（P<0.05）,but the expression of P62 had no significant difference（P>0.05）.（2）Apoptosis-related protein:In comparison with that control group,the expression of Bax and Caspase-3 was significantly increased and the expression of Bcl-2 was significantly decreased in DEX group（P<0.05）,but it was no significant difference in the expression of Bax,Caspase-3 and Bcl-2 between RAPA group and DEX+RAPA group（P>0.05）.Compared with DEX group,the expression of Bax and Caspase-3 was significantly decreased and the expression of Bcl-2 was significantly increased in RAPA group（P<0.05）;In DEX+RAPA group,the expression of Bax and Caspase-3 was significantly decreased and the expression of Bcl-2 was significantly increased（P<0.05）.Compared with RAPA group,the expression of Caspase-3 was increased（P<0.05）and the expression of Bcl-2（P<0.05）was decreased in DEX+RAPA group,but there was no significant difference in the expression of Bax（P>0.05）.Conclusion:Dexamethasone may induce apoptosis of mouse Embryonic Palatal Mesenchymal Cells by inhibiting autophagy.