Role And Mechanism Of Achyranthes Bidentata Polypeptide On Promoting Proliferation Of Schwann Cells

Objective:To understand the effect of Achyranthes bidentata polypeptide on the proliferation of Schwann cells in primary culture and in the repair of sciatic nerve defects after different time,we study the overall effect and mechanism analysis of Achyranthes bidentata on Schwann cells,which is helpful for the exploration of peripheral nerve injury therapeutic targets,and provides a more targeted approach for the clinical application of Achyranthes bidentata polypeptide for clinical treatment of peripheral nerve injury.Methods:1.Culture primary Schwann cells in vitro.Complete medium(Control group),complete medium containing Achyranthes polypeptide(ABPPk group),complete medium containing NGF(NGF group)were added at different time points respectively.The proliferation of Schwann cells was determined by CCK8,cell cycle assay,Edu proliferation assay,and the protein expression of PCNA and Ki67.2.Prepare a model of rat sciatic nerve crush.After sciatic nerve crush injury in rats,saline(Control group),physiological saline containing Achyranthes polypeptide(ABPPk group),and normal saline containing NGF(NGF group)were injected under the injured epicardium,respectively.At the time point of 1d,4d and 7d,the injured nerve was taken from the animal.The proliferation and neuromorphological changes of Schwann cells were observed by Western blot and immunohistochemistry.3.Whole transcriptome sequencing and Schwann cell proliferation regulation mechanism analysis.The Schwann cells cultured in vitro were respectively added to complete medium(Control group),complete medium containing Achyranthes polypeptide(ABPPk group)and complete medium containing NGF(NGF group),The cells of each group were obtained at 15min,0.5h,lh,2h,3h,6h,12h and 24h,and the whole transcriptome was sequenced.Through bioinformatics analysis tools,cluster analysis and protein interaction network analysis of Schwann cell proliferation function-related genes were used to compare the similarities and differences between ABPPk and NGF to promote Schwann cell proliferation.The IPA database was used to analyze the gene related to Schwann cell proliferation function,and the miRNA and lncRNA data were further used to analyze the ceRNA network.4.The effect and regulation mechanism of ABPPk on Schwann cells.Further bioinformatics analysis was performed on the-whole transcriptome sequencing results.The data of the ABPPk group and the NGF group were compared and analyzed by the IPA database at various time points of the Canonical Pathway and the disease function,Cluster cluster analysis and GO analysis was performed.Different functions of Schwann cells,which is cells migration,differentiation and myelination,were analyzed,and WGCNA analysis was performed at all time points to compare the mechanism of action between ABPPk and NGF,and real-time quantitative PCR was performed.Results:1.In vitro studies have shown that ABPPk can effectively promote the proliferation of Schwann cells.After ABPPk addition,significant cell cycle changes,increased Edu staining,and increased expression of PCNA and Ki67 protein were observed,2.In vivo studies have shown that ABPPk can significantly increase the proliferation of Schwann cells in the sciatic nerve injury segment of rats,and with the prolongation of time,the proliferation of Schwann cells becomes more obvious.3.The whole transcriptome sequencing and bioinformatics analysis of Schwann cell proliferation-related genes showed that Pmp22,Mmp9,Egr2,Lama2 and Gdnf genes were mainly involved in the proliferation of Schwann cells in the early 15min of the ABPPk group,while there were fewer genes regulated in 15mins such as Pmp22,Egr2,Erbb2 and Cnd4 in the NGF group.The genes highly expressed in the two groups at 0.5h?3h which still had certain similarities.At 12h,the two groups were mainly negatively regulated and at 24h.There are many genes that are positively regulated.The ABPPk group mainly includes Ccnd3,Lama2,Nrgl,Sdc3,and Cdc42,while the NGF group mainly includes Erbb2,Cnd45 Rnf10,Ccnd3,Sdc3,and Cdc42.4.Comparsion analysis analysis showed that the ABPPk group mainly participated in signaling pathways such as Protein Kinase A Signaling,NF-?B Signaling,mTOR Signaling,Integrin Signaling,and related to the functions of molecular transport,axon growth,neurotransmission,cell movement,synaptic transmission,which are closely related and constantly changing over time.The NGF group is mainly involved in IL-8 Signaling,CREB Signaling in Neurons,Integrin Signaling,Ephrin Receptor Signaling and other signaling pathways,and is mainly related to cell movement and migration,molecular transport,synaptic transmission,and neurotransmission.The change is constantly changing.It is found that the ABPPk group and the NGF group are mainly involved in the signaling pathways,and have some similar functions in biological functions,but the same biological functions play different roles at different time points.5.Cluster analysis showed that the early expression of Unc5b,Tecprl,Paqr4,Mrc2 and Sgkl was highly expressed in the ABPPk group,which played an early regulatory role.At 24h,there was a strong expression of a gene in a region,mainly including Pmnl3,Commd2,Erbb2,Prx,Vasp and Nrgl.In the NGF group,the region with high gene expression was very different from the ABPPk group.The early high-expression genes were scattered.At 24 hours,there was also a region with high expression of genes,including Mtfr2,Dock7,Usp1,Vps16 and other genes.6.The results of GO analysis showed that the main functions of the ABPPk group in the early stage were vascular endothelial growth factor production,response to ATP,positive regulation of axon extension.And in the medium-term,ABPPk mainly participate in neuromuscular junction development,regulation of axon extension,blood vein lumenization,negative regulation of neuron death.Biological processes involved in the later stages are regulation of tissue remodeling,regulation of short-term neuronal synaptic plasticity,neuron projection extension,negative regulation of dendritic cell apoptotic process.The main functions involved in the early stage of the NGF group are neurotrophin signaling pathway,response to ATP,positive regulation of axon extension.The main functions involved in the mid-term is the regulation of myelination,neurotransmitter receptor biosynthetic process,neuron projection morphogenesis,negative regulation of neuron death,positive regulation of neuron differentiation.And the main functions involved in the later period are axon ensheathment,negative regulation of dendritic cell apoptotic process,glial cell fate commitment,positive regulation of dendrite morphogenesis.7.The results of different biological processes of Schwann cells showed that among the 46 genes related to Schwann cell migration function,ABPPk group and NGF group were mainly related to Racl,Vegfa,Pmp22,Bdnf,Pxn and other genes;Schwann cells Among the 33 genes related to differentiation function,the early ABPPk group was mediated by Egr2,while the NGF group was mediated by Igfl gene.Both groups were closely related to Dock7,Lamcl,Nrgl and other genes.A total of 266 genes related to myelination function were involved.In the process of ABPPk group and NGF group,many genes were involved,such as Rxrb,Sgkl,Gabbrl and Cntnl gene,which were specifically expressed in multiple time points in the two groups.However,the gene expression at each time point in the two groups is still very different.8.WGCNA results showed that ABPPk group mainly relatively with histone demethylation promoted transcription,response to ischemia,ABC transporter to promote substance exchange,positive regulation of cell proliferation,cell proliferation apparent regulation,defense signals and regulation of WNT pathway.NGF group is mainly coupled with G protein signal transduction,negative regulation of cell proliferation,positive regulation of cell cycle,sugar homeostasis,sphingolipid signaling pathway activation,cell division and alternative splicing and PI3K and other functional modules.9.Real-time PCR and Western blot results showed that the results of key participating genes were similar to those of whole transcriptome sequencing with time,and had good correlation.Immunohistochemistry results showed that the key regulatory genes were translated and expressed in each group and eo-Iocalized with Schwann cells,indicating that they play a role in the process of ABPPk.C onclusions:1.ABPPk has a significant role in promoting Schwann cell proliferation,and is closely related to Erbb2,Pmp22,Nrgl,Foxo3,Egr2 and other genes.At the same time,the regulatory gene types involved in Schwann cell proliferation have a certain degree of similarity with NGF,but the action time have difference.2.ABPPk and NGF act on Schwann cells,which can regulate neuronal apoptosis,promote axonal extension,synaptic remodeling and other biological processes at different stages.However,ABPPk group also participates in the regulation of biological processes in blood vessels lumenalization and tissue remodeling compared with NGF group.