The Relationship Between Autophagy And Wnt Signaling Pathway In Fetal Rat Cortical Neuron Hypoxic-ischemic Injuries

OBJECTIVE:To study the relationship between Wnt signaling pathway and autophagy in cortical neuron hypoxic-ischemic injuries.METHODS:Fourteen to fifteen days pregnant SD rats were selected and the fetuses were removed.The cortex was isolated and cultured for primary cells.Neurons were identified by immunocytochemistry 6-8 days later.The cells were divided into control and treatment groups,and sugar and oxygen stripping treatments were performed at the corresponding time(1,6,12,24 h).The cells were collected at(1,6,12,24h)and the protein expression of LC3II/LC3 I,P62,Beclin-1,Wnt1,Wnt3 a,and ?-Catenin was measured by Western blot;As an intervention time point,the expression of Wnt1,Wnt3 a,and ?-Catenin proteins was detected by treatment with the autophagy agonist rapamycin and autophagy inhibitor 3-MA.RESULTS:With the prolongation of hypoxia time of sugar-oxygen stripping model,the expression of HIF-1? increased gradually.It showed that with the prolongation of hypoxic time,the degree of hypoxic-ischemic injury in cortical neurons aggravated.When the time of hypoxia reached 24 h,the expression of HIF-1? was the highest.Western blot analysis showed that the expression of LC3 II gradually increased with the time of sugar and oxygen stripping time;the expression of Beclin-1 was positively correlated with the time of sugar and oxygen stripping;the expression of p62 was gradually decreased with the extension of sugar and oxygen stripping time.It was shown that autophagy increased with the prolonged injury time in the hypoxia-ischemic injury induced by glucose-oxygenation.When the time of glucose and oxygen stripping was 24 hours,the expression of LC3 II and Beclin-1 was the highest,and the expression of p62 was the lowest.The expression of promoter proteins Wnt1,Wnt3 a,and downstream protein ?-Catenin were detected.The expression of Wnt1 and Wnt3 a showed a tendency of continuous up-regulation as the sugar and oxygen stripping time was prolonged.Beta-Catenin was up-regulated after the start of sugar-oxygen stripping,but it showed a decreasing trend with the extension of sugar-oxygen stripping time.4.Selecting glucose-oxygen-separating hypoxia for 12 h as the intervention time point,the autophagy inducer rapamycin(rapamycin,Rap)and inhibitor3-Methyladenine(3-MA)were used to detect Wnt signal by up-regulating and down-regulating autophagy.Pathway expression of major proteins,analysis of autophagy on the regulation of Wnt signaling pathway under hypoxia-ischemia conditions.[This is not an experimental result.Please remove.The expression of Wnt1 and Wnt3a was similar to that of LC3 II.The expression of ?-Catenin was significantly higher in 3-MA+sweet oxygen stripping group than in the control group,but was significantly lower in the Rap+sweet oxygen stripping group,which was opposite to the change trend of autophagy..The above results suggest that the initiation of Wnt signaling pathway is consistent with the initiation of autophagy,whereas the expression of Wnt downstream protein is contrary to autophagy activity.CONCLUSION:The activation of Wnt upstream signaling pathway is consistent with autophagy,while the expression of Wnt downstream protein is the opposite of autophagy.