Effects And Mechanisms Of Netrin-1 On Apoptosis,Neurite Outgrowth And Synapse Formation In Cortical Neurons After OGD Injury

Object:To study the effect of Netrin-1 on apoptosis,neurite outgrowth and synapse formation in cortical neurons after OGD injury,as well as the activation of ERK1/2pathway,we used overexpression and interference of lentivirus to regulate the amount of Netrin-1 in cortical neurons.It provides a reliable basis for the therapeutic effect of Netrin-1 on cerebral ischemia.Methods:1.Cortical neurons of rats were cultured and identified in vitro.2.The neurons cultured to the 7^thday were treated with OGD for 90 minutes to establish OGD injury model.And OGD injury was observed by light microscope and laser confocal microscope after one day.3.Before and after OGD injury,we added overexpression and interference of lentivirus Ntn1 and U0126.Then they were divided into the following five groups:normal control group（Control group）,OGD model group（OGD group）,OGD+overexpression Ntn1 group（OGD+LV-Ntn1 group）,OGD+Ntn1 interference group（OGD+LV-Ntn1-RNAi group）,OGD+overexpression Ntn1+ERK1/2 inhibitor group（OGD+LV-Ntn1+U0126 group）.4.The cortical neurons of each group were detected as follows 24 hours after OGD injury:CCK-8 kit was used to detect neuron viability;LDH kit was used to detect neuron toxicity.We used q PCR to detect the expression levels of PSD-95 and GAP-43mRNA,and Western Blot to detect the expression levels of p-ERK1/2,Bax,Bcl-2,Cleaved Caspase-3,PSD-95 and GAP-43 protein.Immunofluorescence staining and Zeiss LSM 710 laser confocal microscopy was used to take photos;Image J software was used to measure the average length of the longest neurite and count the number of the first neurite.The Caspase-3 kit was used to detect the Caspase-3 activity of neurons.Results:1.CCK-8 detection of neuronal survival results:Compared with the normal group,the OGD group was significantly reduced;compared with the OGD group,the OGD+LV-Ntn1 group was significantly increased.However,the OGD+LV-Ntn1-RNAi group was significantly reduced.In addition,the OGD+LV-Ntn1+U0126 group was significantly lower than that in the OGD+LV-Ntn1 group.2.LDH detection of neuronal toxicity results:Compared with the normal group,the OGD group was significantly higher;compared with the OGD group,the OGD+LV-Ntn1 group was significantly lower.However,the OGD+LV-Ntn1-RNAi group was significantly higher.In addition,the OGD+LV-Ntn1+U0126 group was significantly higher than that of the OGD+LV-Ntn1 group.3.q PCR results show that:The expression of PSD-95 and GAP-43 mRNA in each group:Compared with the normal group,the OGD group was significantly decreased;compared with the OGD group,the OGD+LV-Ntn1 group was significantly increased.However,the OGD+LV-Ntn1-RNAi group was significantly decreased.In addition,the OGD+LV-Ntn1+U0126 group was significantly lower than that in the OGD+LV-Ntn1 group.4.Western Blot results show that:The expression of p-ERK1/2 protein in each group:Compared with the normal group,the OGD group was significantly increased;compared with the OGD group,the OGD+LV-Ntn1 group was significantly increased.However,the OGD+LV-Ntn1-RNAi group was significantly reduced.In addition,the OGD+LV-Ntn1+U0126 group was significantly lower than that of the OGD+LV-Ntn1 group.The expression of Bax and Cleaved Caspase-3 protein in each group:Compared with the normal group,the OGD group was significantly increased;compared with the OGD group,the OGD+LV-Ntn1 group was decreased.However,the OGD+LV-Ntn1-RNAi group was increased.In addition,the OGD+LV-Ntn1+U0126 group was significantly higher than that of the OGD+LV-Ntn1 group.The expression of Bcl-2,PSD-95 and GAP-43 protein in each group:Compared with the normal group,the OGD group was decreased;compared with the OGD group,the OGD+LV-Ntn1 group was increased.However,the OGD+LV-Ntn1-RNAi group was significantly decreased.In addition,the OGD+LV-Ntn1+U0126 group was significantly lower than that of the OGD+LV-Ntn1 group.5.Immunofluorescence staining results show that:The average length of longest neurites was significantly shorter in the OGD group compared with the normal group;compared with the OGD group,the OGD+LV-Ntn1group was significantly longer;the OGD+LV-Ntn1-RNAi group was significantly shorter than that of the OGD group;compared with the OGD+LV-Ntn1 group,the OGD+LV-Ntn1+U0126 group was significantly shorter.The average number of primary neurites was significantly decreased in the OGD group compared with the normal group;compared with the OGD group,the OGD+LV-Ntn1group was significantly increased;compared with the OGD group,the OGD+LV-Ntn1-RNAi group was significantly decreased;compared with the OGD+LV-Ntn1group,the OGD+LV-Ntn1+U0126 group was significantly decreased.6.Caspase-3 activity results:The OGD group was significantly increased compared with the normal group;compared with the OGD group,the OGD+LV-Ntn1 group was significantly decreased;the OGD+LV-Ntn1-RNAi group was significantly higher than that of the OGD group;compared with the OGD+LV-Ntn1 group,the OGD+LV-Ntn1+U0126 group was significantly increased.Conclusion:Netrin-1 attenuates apoptosis and facilitates neurite outgrowth and synapse formation in primary cortical neurons after OGD injury.The mechanism may be related of the activation of ERK1/2 and regulation of Bax,PSD-95 expression,etc.