Study On The Mechanism Of Exendin-4 Promots Nerve Injury Regeneration Via PI3K Signal Pathway

Objective1.To study the effect of Exendin-4 on the proliferation and differentiation of neural stem cells in the subventricular zone of mature brain.2.To study the protective effect of Exendin-4 on Nogo-A-?20 mediated growth inhibition of SH-SY5Y cells.Methods1.The neural stem cells of the subventricular zone of 5-week C57 mice were extracted and identified,and GLP-1R was knocked out using shRNA,and the knock-out effect was verified by Western Blot,using neural stem cells knocked down or not knocked down by GLP-1R.Subsequent experiments were performed to investigate the effects of Exendin-4 on its proliferation and differentiation.Exendin-4 was used at a concentration gradient of 10 nM,50nM,100 nM,and 250 nM,and its value-added effect was examined by CCK8and Western Blot assay.In the differentiation experiment,immunofluorescence and Western Blot were used to identify the phenotype of the differentiated cells,and the differentiation trend was judged by counting the number of positive cells by immunofluorescence.The effect of exendin-4 on neuron differentiation was verified again after knocking out GLP-1R.Neural stem cells were treated with PI3K and MAPK blockers,and the activation of AKT/CREB and the expression of BDNF were detected by Western Blot to identify possible signal pathway.2.The SH-SY5Y cells were cultured and the 24-well plates were plated with Nogo-A-?20 at a concentration of 0,20,40,60,80,100 pmol/cm ~2,then the cells were seeded with Nogo-A-?20-cultured substrate.The spreading area was evaluated by phalloidin staining and Image J to evaluate its inhibition of cell adhesion growth.The expression of GLP-1R was knocked down by siRNA and its knockdown effect was detected by Western Blot.The cells were pretreated with 150 nM concentration of Exendin-4 to block the inhibition of Nogo-A-?20-mediated cell growth,and GLP was further used.-1R knockdown cells were repeatedly verified.In the neurite outage experiment,SH-SY5Y was induced to differentiate with all-trans retinoic acid,and then differentiated SH-SY5Y was treated with 150 nM Nogo-A-?20 to induce neurite collapse.The blocking effect of Nogo-A-?20 on neurite outgrowth was observed by pretreatment of Exendin-4,and repeated experiments were performed using GLP-1R knockdown cells.Western blot was used to detect the expression of RhoA and ROCK1 in the blank group,Exendin-4 group,GLP-1R knockdown+Exendin-4 group.The activation and expression of RhoA was detected after treatment of cells with PI3K pathway inhibitors,MAPK pathway inhibitors,RhoA inhibitors and Rock1 inhibitors to verify the signal pathway.Results1.Exendin-4 Promotes Neural Stem Cells Proliferation and Induces Differentiation to Neurons via Regulating PI3K/AKT/CREB PathwaysImmunofluorescence was used to identify the neural stem cells in the subventricular zone of mature brain and the expression of GLP-1R.The results of CCK8 and Western Blot showed that Exendin-4 significantly promoted the proliferation of neural stem cells through GLP-1R.Western blot analysis of immunofluorescence revealed that Exendin-4 significantly increased the proportion of neuronal phenotype cells during neural stem cell differentiation,and this effect was performed after shRNA was used to knock out its receptor GLP-1R or block the PI3K pathway.disappear.Western blotting detection of Exendin-4 activates the AKT/CREB pathway in neurons,which in turn increases the expression of BDNF in the cell to promote neuronal differentiation,and this signaling cascade relies on intracellular PI3K rather than MAPK.2.Exendin-4 Down-regulates RhoA Protects SH-SY5Y Cells from Nogo-A-?20 Mediated Growth Inhibition via PI3K PathwayNogo-A-?20 can significantly inhibit the spread adhesion of SH-SY5Y and cause the neurite to collapse.Pretreatment with Exendin-4 protects cells from Nogo-A-?20-mediated cell growth inhibition,and knockdown of its receptor GLP-1R blocks this effect.Western blot analysis showed that Exendin-4 could significantly reduce the expression and activation of RhoA in SH-SY5Y cells.Inhibition of PI3 pathway could block the effect of Exendin-4 on RhoA,while blocking MAPK pathway has no significant effect.ConclusionsExendin-4 increase the proliferation of neural stem cells in the brain SVZ region through the PI3K/AKT/CREB pathway and maintain its neuronal phenotype during differentiation.This study also found Exendin-4 could protects cells from Nogo-A-?20 mediated neurosuppression by reducing the expression and activation of RhoA in the cell via PI3K pathway.