A Novel Active Peptide VD11 Promotes The Recovery Of Structure And Function After Spinal Cord Injury And Its Mechanism

Objective:Spinal cord injury(SCI)is a serious CNS injury that has a significant morbidity,disability,and fatality rate.Due to the CNS’s poor regenerating ability,there are currently few viable therapies for SCI.Peptide medicines are gaining popularity in therapeutic research and development because to their excellent selectivity,biological activity,and minimal antigenicity.Amphibians are good natural suppliers of such small molecular peptides,with strong repair and regeneration activity in injured tissues.Although amphibians have a significant potential to regenerate their spinal cords,few research have looked into the impact of amphibian spinal cord derived peptides on spinal cord injuries.The purpose of this study was to investigate the effect and mechanism of amphibian spinal cord polypeptide VD11 on promoting nerve regeneration and functional recovery after spinal cord injury at both cellular and animal levels.Methods:1)Total RNA was extracted from the spinal cord of Rana rana.The mRNA was purified using a purification kit.The first and second strand cDNA was synthesized using SMART technology,and the active peptide VD11 was sequenced by Illumina MiSeq.2)The effects of active peptide VD11 on mRNA transcription and protein expression of NGF and BDNF in BV2 cells were detected by qPCR and ELISA.3)The OGD/R model of PC 12 cells was established,and the effects of active peptide VD11 on proliferation and axon extension of PC 12 cells after OGD/R injury were observed by MTS and microscope.Cells were divided into control group(without special treatment),3 VD11 groups(treated with 1,10 and 100 nM VD11,respectively),Model group(OGD/R Model)and 3 Model+VD11 groups(treated with 1,10 and 100 nM VD11 after OGD/R modeling).OGD/R model:By introducing N2,the concentration of CO2 and O2 in the three-gas incubator was reduced to 5%,and sugar-free medium was used without FBS,so as to form an anoxic environment in cells and simulate the ischemic anoxic injury of neurons in vivo.4)T10 spinal cord transection model of SD rats was established,and rats in Vehicle,MPED and VD11 groups were given 0.9%normal saline,MPED(30mg/kg)and VD11 peptide(0.1mg/kg)intraspinal injection,twice a day,7 days after surgery,respectively.Artificially assisted urination,3 times a day.BBB exercise score was used to evaluate the effect of active peptide VD11 on the functional recovery of hind limbs after spinal cord injury in rats 28 days after operation.5)The injury area and number of neurons were observed under microscope by H&E staining and Niss staining to determine the effect of active peptide VD11 on spinal cord injury repair.6)The effect of active peptide VD11 on astrocyte regeneration and axon extension after spinal cord injury in rats was determined by immunofluorescence staining.7)Total mRNA transcription group of normal group,30 days after total spinal cord transection(model group)and 30 days after total spinal cord transection(VD 11 group)were detected.The obvious up-regulation or down-regulation pathway of KEGG concentration was detected by WB and other methods to obtain the information of pathway activation or inhibition,and clarify the signaling pathway of VD11 promoting spinal cord injury repair.Results:1)Sequence of active peptide VD11:VD11 contains 11 amino acids,and the sequence is "VDELWPPWLPC".2)qPCR and ELISA showed that active peptide VD11 increased the production of NGF and BDNF in BV2 cells in a dose-dependent manner.3)MTS and microscopic observation showed that active peptide VD11 could promote the proliferation and axon extension of PC 12 cells after OGD/R injury.4)BBB exercise scores showed that active peptide VD11 promoted functional recovery of hind limbs after spinal cord injury in rats,and mortality and body weight after SCI showed that VD11 was safer than MPED in rats.5)Active peptide VD11 promoted tissue repair after spinal cord injury in rats:the general picture of spinal cord tissue of rats 30 days after spinal cord injury,H&E staining and Nessie staining showed that scar size of tissue in VD11 group was significantly smaller than that in Vehicle group and MPED group.The number of viable neurons was significantly more than that of Vehicle and MPED groups.6)Immunofluorescence staining showed that active peptide VD11 promoted astrocyte regeneration and axon extension after spinal cord injury in rats.7)To investigate the mechanism of active peptide VD11 on nerve regeneration:the spinal cord tissues of normal group,model group and model+VD11 group were sequenced.KEGG pathway enrichment analysis revealed that DEGs was enriched in AMPK,AKT and other nerve injury related pathways.Western blotting and immunofluorescence confirmed that VD11 may promote the repair of spinal cord injury through the activation of BDNF/PI3K/AKT pathway.Conclusion:VD11 is the first peptide derived from amphibian spinal cord to promote spinal cord injury repair.Furthermore,this study is the first to explore the association between amphibian-derived peptides and axon regeneration,thus laying the foundation for the development of future drugs and therapies.