An Experimental Study On The Effect Of Src On The Neurite Growth And Growth Cone Formation Of Injured Neurons Through PP2A

How to promote neuronal axon regeneration and restore nerve function after central nervous system injury is a major medical problem in the field of neuroscience.The failure of neurite outgrowth or growth cone formation in injured neurons is the main reason that hinders functional recovery after injury.Previous studies have found that Src is a key molecule regulating neuronal axon regeneration and growth cone formation.In the literature,we found that Src associates with PP2 A.PP2A is a target of Src kinase,which can be phosphorylated directly at Tyr307 catalytic subunit of PP2 A and then inhibit PP2 A activity.In addition,literature has shown that PP2 A inhibition promotes neuronal axonal growth.Based on the above background,we speculate that Src promotes neurite outgrowth and growth cone formation through PP2 A in injured neurons.We use mouse hippocampal neuronal cell HT22 and SD rat cortical neurons as oxidative stress models in vitro neuronal.Src activators?SA?,PP2 A inhibitors?Cantharidin?and PP2 A activators?FTY720?were used to interfere with the expression of Src and PP2 A.Using immunofluorescence,Western blot and other morphological and molecular biological techniques to clarify whether Src can promote the neurite outgrowth and growth cone formation through PP2 A.Methods:Cultured HT22 cell and SD rat cortical neurons.The confirmation for the H₂O₂ concentration by CCK8 to construct model of neuron oxidative stress.Morphological observation and statistical analysis were carried out to analyze the changes of neurite length in each group.Using cell protein phosphorylase 2 activity colorimetry to detect the PP2 A activity in each group.Using western blot to detect the p Src and p PP2A/C protein levels in each group.Using F-actin fluorescent specific marker phalloidin?F-actin?to analyze the growth cone changes of primary neurons in each group.Results:?1?The cell viability of HT22 in 250?M H₂O₂ group was 65% and the neurite shortened significantly.So 250?M H₂O₂ can be used to construct the oxidative stress model of HT22.?2?Compared with Control group,the p Src protein in H₂O₂ group increased?P<0.05?and the neurite length of HT22 significantly reduced?P<0.001?.The neurite length of HT22 in H₂O₂+SA group was significantly higher than that of the H₂O₂ group?P<0.001?,and the p Src protein levels in H₂O₂+SA group increased distinctly?P<0.001?.?3?Compared with Control group,the activity of PP2 A in H2O2 group decreased?P<0.05?and the p PP2A/C protein levels increased?P<0.05?.Compared with H2O2 group,the PP2 A activity of H2O2+SA group reduced clearly?P<0.01?and the p PP2A/C protein levels increased?P<0.01?.?4?The neurite length in H₂O₂+Cantharidin group significantly increased than that of the H₂O₂ group?P<0.001?,while that of the H₂O₂+FTY720 group decreased?P<0.05?.In H₂O₂+Cantharidin group,the number and area of growth cones in primary neurons increased significantly?P<0.001?,while that of the H₂O₂+FTY720 group clearly decreased?P<0.01?.?5?The neurite length in H₂O₂+SA group was significantly higher than that in H₂O₂ group?P <0.001?,while that of H₂O₂+SA+FTY720 group decreased comparing with the H₂O₂+SA group?P <0.01?.Statistical analysis of growth cones and processes with F-actin markers showed that compared with H₂O₂ group,the number and area of growth cones in primary neurons in H₂O₂+SA group increased significantly?P<0.001?,while that of the H₂O₂+SA+FTY720 group decreased than H₂O₂+SA group?P<0.01?.Conclusion:Src promotes the neurite growth and growth cone formation of injured neurons through PP2A...