The Mechanism Of AS-IV Regulating Rho A Pathway To Promote Neurite Regeneration

Objective:To investigate the mechanism of Astragaloside IV（AS-IV）regulating Rho A pathway on neurite extension,and to provide a theoretical basis for the treatment of neurite outgrowth after ischemic stroke-induced excito-amino acid toxicity.Methods:（1）CCK-8 method was used to detect the survival rate of retinal ganglion cells（RGCs）with different concentrations of AS-IV and different concentrations of glutamate（Glu）for 24 h.（2）Pull-down and WB were used to detect the relationship between the activation of Rho A pathway proteins and the time of glutamate damage.（3）RGCs were pretreated with AS-IV for 24 h,and Glu was added to co-culture for 1 h.The intracellular Ca²⁺concentration of RGCs was detected by flow cytometry,the activity of each protein was detected by Pull-down and WB methods,and the neurites and growth cones were observed by cytoskeleton staining.The change.（4）RGCs were treated with Glu,the total protein of RGCs at different time points was extracted,and the expression of Rho A pathway and its downstream proteins was detected by Western blotting.（5）RGCs were pretreated with AS-IV for 24 h,and Glu was added to co-culture for 12 h and 48 h,respectively.The expressions of Rho A,Rac1 and Cdc42 in RGCs were detected by Real-time PCR,immunofluorescence and Western blotting（6）TUJ1 and RGCs were stained with F-actin,and the changes of RGCs neurites were detected by confocal imaging.Results:（1）AS-IV toxicology test:when the concentration was between 40μg/m L and 160μg/m L,the cell viability gradually decreased,and the cell viability was the lowest at 160μg/m L（****P<0.01）,80μg/m L was chosen as the subsequent experimental condition.Glu toxicology test:With the increase of Glu concentration,the cell activity gradually decreased.At 10 m M,the cell viability was56.71%（****P<0.01）,and this concentration was selected as the subsequent modeling condition.（2）With the prolongation of Glu injury time,the expression of Rho A-GTP gradually increased,and the expression was the highest at 3 h;the expression of Ccd42-GTP,P-PAK1,N-WASP,phosphorylated LIMK and phosphorylated cofilin gradually increased.decreased,so 3h was chosen as the follow-up experiment time.（3）After AS-IV intervention,the fluorescence intensity of Ca²⁺decreased by 14%（***P<0.01）.After 3h of Glu injury to RGCs,Rho A-GTP increased,and under the intervention of AS-IV,compared with the glutamate group,it decreased by 24.15%（*P<0.05）.Rac1-GTP,Cdc42-GTP,N-WASP,P-PAK1 and P-cofilin all decreased after glutamate injury;after AS-IV intervention,compared with the glutamate group,they increased by 64.55%,respectively,31.08%,2%,51.06%and 49.47%（*P<0.05,***P<0.01）.In the glutamate group,the neurites were shortened,the growth cones shrunk into a spherical shape,and no obvious filopodia were found.（4）Dynamic changes of Rho A protein and its downstream molecules:Rho A,Cdc42,and Rac1 were all highly expressed at 12 h of Glu injury,and then gradually decreased with time,and the expression was the lowest at 48 h,while the downstream effector molecules of Rho A protein The phosphorylation expression of ROCK gradually increased with time,and the expression level was the highest at 48h of injury.（5）After 12 h of Glu injury in RGCs,Rho A protein and gene expression increased significantly,while AS-IV intervention decreased;Cdc42protein and gene expression were increased,but there was no significant difference between AS-IV intervention;Rac1 gene expression was damaged after injury decreased,and the expression level of AS-IV intervention increased.After 48 h of Glu induction,the protein expressions of Rac1 and Cdc42 in the Glu group were decreased（****P<0.01）,and both were up-regulated after AS-IV intervention（**P<0.01）.（6）After 48 h of Glu induction,the cell bodies of RGCs swelled and became round,while the number of cells swelled and rounded in AS-IV group was less,and the neurites were accompanied by many filaments.Conclusion:This study clarified the understanding of Glu on different stages of axonal excitatory injury in RGCs through Glu activation of Rho A signaling pathway in RGCs cells.It was found that Glu-induced axonal damage in RGCs was time-dependent with the activation of Rho A signaling pathway.Using AS-IV to inhibit the Rho A pathway,the results show that inhibiting Glu can significantly reduce the activity and protein expression of Rho A and increase the activity and protein expression of Rac1 and Cdc42 proteins,that is,AS-IV antagonizes Rho A-induced growth cone collapse,promotes lamellipodia and Formation of filopodia.To provide theoretical guidance for ischemic stroke neuroprotection and neurite outgrowth therapy.