The Effects And Mechanisms Of Velvet Antler Polypeptides On Proliferation And Migration Of Schwann Cells And Nerve Regeneration

BackgroundPeripheral nerve injury is one of the common clinical diseases,mainly manifested as motor and sensory dysfunction.However,the function recovery after peripheral nerve injury is still not ideal,and even leads to permanent disability,which seriously threatens the quality of life.Therefore,it is urgent to explore a new effective repair scheme for peripheral nerve injury.Traditional Chinese medicine has been proved to have a good effect on nerve regeneration.Velvet antler peptide(VAP),as the main polypeptide active substance in aelvet antler,has been widely studied for its anti-aging,scarring reduction and regulation of angiogenesis.In the peripheral nervous system,studies have shown that VAP can promote nerve repair after injury,but its mechanism remains unclear."Wallerian degeneration" appeared at the site and distal end,and the myelin sheath of axon disintegrated after peripheral nerve injury.Schwann cells play an important role in the repair of peripheral nerve injury through migration and formation of Büngner bands to provide regeneration channels for axons.After peripheral nerve injury,the level of reactive oxygen species(ROS)is significantly increased in the injured site and its distal stump,due to ischemia and inflammation.Excessive ROS results in oxidative stress damage of nerve cells,impaired cell function and blocked peripheral nerve regeneration.This study will investigate the effect of VAP on the migration function,the protection under oxidative stress,and unveil the underlying mechanisms.This study will explore the effect of VAP on the migration function of Schwann cells,and the regulatory effect of VAP on the function of Schwann cells under oxidative stress.Thus,this project will provide a promising therapeutic strategy for peripheral nerve injury.Objectives:1.The effect of VAP on the proliferation and migration of SCs and the protective effect of oxidative stress were investigated in vitro.2.In vitro experiments were conducted to explore the mechanism of VAP regulating SCs migration.3.Transcriptome sequencing was used to analyze the mechanism of VAP regulation of SCs function.4.Animal experiments further verified the effect and mechanism of VAP on peripheral nerve regeneration.Methods:1.The effects of different concentrations of VAP on the proliferation function of Schwann cells were detected by CCK-8 assay and live/dead cell staining.2.Hydrogen peroxide(H2O2)was used to construct the oxidative stress model.The capacities of VAP in modulating oxidative stress,cell protection and proliferation were verified by CCK8,reactive oxygen species(ROS)assay and live/dead cell staining.3.Transwell assay was used to evaluate the effect of VAP on schwann cells migration function.4.In order to study the molecular mechanism of VAP regulation of SCs,the SCs were firstly interfered with SB203580,a p38 MAPK signaling pathway inhibitor,and LY294002,a PI3K-Akt signaling pathway inhibitor,respectively,to detect the migration of SCs in the presence of inhibitors.5.The effect of VAP on P38 MAPK and PI3K-Akt signaling pathway was evaluated by detecting the activation levels of different concentrations of VAP on these two pathways.SCs were then treated with P38 MAPK signaling pathway inhibitor SB203580 and PI3K-Akt signaling pathway inhibitor LY294002.To study the changes of P38 MAPK and PI3K-Akt signaling pathways under the intervention of pathway inhibitors,and the effects of different concentrations of VAP on p38 MAPK and PI3K-Akt signaling pathways.6.Western blot and RT-PCR were used to detect the effects of VAP on surface receptors(UNC5A,UNC5B,UNC5C and UNC5D)of Schwann cells,and to search for possible targets of VAP.7.RNA was extracted from Schwann cells after VAP treatment for transcriptome sequencing.Transcriptome sequencing analysis was performed for GO and KEGG analysis and pathway analysis to further explore the mechanism of VAP regulation of Schwann cell function8.We assessed the therapeutic effect of VAP in a sciatic nerve injury model.The measurement of sciatic nerve index and nerve conduction velocity were measured to evaluate the recovery of sciatic nerve function.Toluidine blue staining and transmission electron microscopy were used to evaluate the histology and morphology of the regenerated sciatic nerve.The expression of neurotrophic factors in regenerated nerves was detected by RT-PCR and immunofluorescence assay.Western blot was used to detect the expression of UNC5 family,P38 MAPK signaling pathway and PI3K-AKT signaling pathway in regenerated nerves.Results:1.Through CCK-8 assay and live/dead cell staining,we found that VAP had no significant effect on schwann cell proliferation in the absence of oxidative stress.2.After hydrogen peroxide(H2O2)was used to construct the oxidative stress model on SCs,we added different concentrations VAP in SCs’culture medium.The CCK-8,reactive oxygen species(ROS)assay and live/dead cell staining showed that with the increase of concentration,VAP could stop H2O2 induced oxidative stress kill the SCs,and also promote cell proliferation under oxidative stress.3.It was found that VAP promoted SCs migration in vitro,and 40 μg/mL VAP promoted SCs migration most obviously,while 80μg/mL VAP inhibited SCs migration.4.Both SB203580,a p38-MAPK signaling pathway inhibitor,and LY294002,a PI3K-Akt inhibitor,could significantly inhibit the migration of Schwann cells and inhibit the promoting effect of VAP on SCs migration.The promoting effect of VAP on SCs migration depends on the activation of P38 MAPK and PI3K-Akt signaling pathways.5.VAP can activate the phosphorylation of P38 MAPK and PI3K-Akt,and promote and regulate SCs migration and survival and proliferation under oxidative stress by activating the p38 MAPK and PI3K-Akt signaling pathways,and the degree of effect is related to the concentration of VAP.P38 MAPK pathway inhibitor SB203580 and PI3K-Akt pathway inhibitor LY294002 can significantly inhibit the phosphorylation activation of the pathway,and weaken the function and cell protection of VAP on SCs.VAP can resist the inhibition of p38 MAPK and PI3K-Akt signaling by pathway inhibitors to a certain extent.6.By detecting the expression of Netrin1 receptors(UNC5A,UNC5B,UNC5C and UNC5D)on the surface of Schwann cells,VAP may bind to the receptor UNC5B.7.Transcriptome analysis showed that 2044 differently expressed genes in the VAP group compared with the control group,among which 970 genes were up-regulated and 1074 genes were down-regulated.Differential genes were analyzed by GO analysis and KEGG pathway enrichment analysis.The down-regulated differential genes were enriched in the intermediate fiber cytoskeleton signaling pathway,suggesting that VAP may regulate Schwann cell function through this pathwayThe up-regulated differential genes were enriched in the neuronal activation ligand-receptor interaction signaling pathway,suggesting that VAP may regulate Schwann cell function through ligand-receptor binding,and UNC5B is the most differentially expressed gene in this pathway.Further evidence suggests that UNC5B may be the VAP binding receptor on schwann cells.8.Animal experiments found that VAP can after sciatic nerve injury in rats in functional behavior to improve motor function(SFI)and nerve conduction ability(NCV),on the histomorphology promote rat sciatic nerve axon regeneration,myelination,at the molecular level to promote nerve injury GDNF and the expression of NGF plays and neurotrophic factor secretion.Further studies showed that VAP specifically up regulated the expression of the receptor UNC5B in nerves and activated the P38 MAPK and PI3K-AKT signaling pathways,thereby promoting nerve repair and regeneration.Conclusions:Our datas suggest that VAP therapy can significantly promote peripheral nerve regeneration through modulating Schwann cell function under oxidative stress,via binding to the UNC5B receptor and activating p38 MAPK and PI3K-AKT signaling pathways.Therefore,VAP is expected to be a new therapeutic agent for the repair of peripheral nerve injury.This research provides a new drug target for the treatment of peripheral nerve injury and provides a new theoretical basis for studying the mechanism of nerve regeneration promoted by the drug.