The Role Of PHLDA1 In Neonatal Rats With Hypoxic-Ischemic Brain Damage

Objective:The purpose of this study is to to investigate the expression and mechanism of Pleckstrin homologous domain A family member 1(PHLDA1)in newborn rats with Hypoxic-Ischemic Brain Damage(HIBD)in neonatal rats,and explore whether PHLDA1 is involved in the occurrence of HIBD by affecting autophagy and mitochondrial damage caused by and oxidative stress.Methods:7-day-old newborn rats were used to establish HIBD model and the PC 12 cells were used for OGD/R model,then the changes of PHLDA1,Beclinl,LC3,P62 and mitochondrial damage between model group and control group were detected.Next,after transfecting PC 12 cells and brain with siRNA-PHLDA1 or PHLDA1 over-expression lentivirus,an OGD/R model or HIBD model was established and the cell proliferation activity,LDH release,PHLDA1,LC3,and some mitochondrial damage indicators-ROS,MitoSOX were tested.In the next experiment,PC 12 cells with PHLDA1 knocked down were treated with Rapamycin,PC 12 cells with PHLDA1 over-expression were treated with 3-MA,then the cell proliferation activity,LDH were detected in these cells after OGD/R.Results1.In the neonatal rat HIBD model,the expression of PHLDA1,LC3,Beclinl in hippocampus increased,and the expression of P62 decreased,and the changes were obvious at 24 hours after HIBD;In the OGD/R model,the expression of PHLDA1,LC3,Beclinl in PC 12 cells increased,the expression level of P62 decreased,and the change was obvious at 24 hours after OGD/R;2.SiRNA-PHLDAl transfected PC 12 cells can significantly reduce the expression of PHLDA1 in PC12 cells.Interfering with the expression of PHLDA1 in PC12 cells can reduce the increase of LC3 and Beclinl,reverse the decrease of P62,in PC 12 cells caused by OGD/R,and reduce the degree of cell activity reduction,the degree of increase of ROS,MitoSOX,LDH and the degree of mitochondrial membrane potential reduction,PHLDA1 silencing lentivirus infection of rat brain tissue can reduce the area of brain injury and the damage of neurons;3.The expression of PHLDA1 in PC 12 cells can be significantly increased after transfection of PHLDA1 overexpression lentivirus.PHLDA1 overexpression lentivirus can significantly increase the expression of PHLDA1 in PC 12 cells;PHLDA1 can aggravate the decrease in cell activity,the increase in ROS,MitoSOX,LDH and the degree of mitochondrial membrane potential reduction caused by OGD/R.Lentivirus infection with over-expressing PHLDA1 in rat brain tissue can aggravate the damaged area of brain tissue and the damage of neurons.4.In PC 12 cells that interfered with PHLDA1,the application of the autophagy activator rapamycin will again aggravate the decrease in cell activity caused by OGD/R and the release of LDH.5.In the PC 12 cells with PHLDA1 overexpressing,the application of autophagy inhibitor 3-MA can reduce the degree of cell activity reduction and LDH release after OGD/R.Conclusion:PHLDA1 has a damaging effect on neonatal rats with hypoxic-ischemic brain damage,and the mechanism may be related to the excessive activation of autophagy and aggravation of mitochondrial damage.