The Effects And Mechanism Of MiR-23a-3p And MiR-186-5p On Spinal Cord Ischemia-reperfusion Injury In Rats

Objective: Spinal cord ischemia-reperfusion injury(SCII)is associated with many pathophysiological states,such as thoracoabdominal or thoracic aneurysms repair surgery,intraspinal surgery.It remains an unsolved problem resulting in a poor prognosis.Nonetheless,the underlie mechanism of SCII is not well elaborated.This is probably due to the induction of SCII is multifactorial.TRIL(TLR4 interactor with leucine-rich repeats,TRIL)and CXCL13(C-X-C motif living 13,CXCL13)play an important role in nervous system diseases.In recent years,miR-23a-3p and miR-186-5p in mi RNA have attracted more attention in neurological ischemic diseases.Sevoflurane has a protective effect on spinal cord ischemia-reperfusion injury,but whether sevoflurane participates in the protection of SCII by regulating mi RNAs.The purpose of this study was to explore the regulatory mechanism of miR-23a-3p and miR-186-5p in SCII.In addition,whether sevoflurane pretreatment can protect spinal cord from ischemia-reperfusion injury by regulating miR-23a-3p and miR-186-5p was further explored.Methods: Rat models of SCII were established by clamping the aortic arch for 14 minutes.Altered expression of miRNA after SCII was analyzed by microarray chip,and the expression levels of miR-23a-3p and miR-186-5 were evaluated by qRT-PCR.After TRIL inhibition induced by siRNA-TRIL,the expression of TRIL,TLR4,TLR3 and NF-κ B were measured by Western blot and the expression of IL-1 β were measured by ELISA,respectively.Dual-luciferase reporter assay was applied to detect the interaction of miR-23a-3p and TRIL.Overexpression miR-23a-3p was obtained by intrathecal injection of miR-23a-3p in rats.Tarlov score,Evans blue staining and HE staining were used to evaluate the spinal cord injury induced by SCII.The expression of TLR4,TLR3,NF-κ B was detected by Western blot,IL-1 β was detected by ELISA,apoptosis was detected by TUNEL.And the effects of overexpression miR-23a-3p were observed.The time course of CXCL13 expression was measured by Western blot.The localization of CXCL13 was detected by double immunoassay.CXCL13 and CXCR5 were silenced by intrathecal injection of siRNA-CXCL13 and siRNA-CXCR5,respectively.Tarlov score,Evans blue staining and HE staining were used to evaluate the injury induced by SCII.The expression of CXCL13,CXCR5,ERK,p-ERK and caspase-3 were measured by Western blot,and the expression of IL-1 β,IL-6 and TNF-α were measured by ELISA.At the same time,the target genes of miR-186-5p was analyzed for KEGG(Kyoto Encyclopedia of Genes and Genomes)signal pathway and GO(Gene Ontology)enrichment by bioinformatics analysis.Overexpression of miR-186-5p was obtained by intrathecal injection of miR-186-5p in rats.The spinal cord injury induced by SCII was evaluated by Tarlov score,Evans blue staining and HE staining.The expression of CXCL13,CXCR5,ERK,p-ERK,caspase-3,IL-1 β,IL-6,TNF-α were determined following overexpression of miR-186-5p.After preconditioning with 2.4% sevoflurane for 1 h and then washing out for 30 minutes,the expression of miR-23a-3p and TRIL were detected at 12 h after SCII,and the expression of miR-186-5p and CXCL13 were detected at 24 h after SCII.Results: Compared with sham group,the expression of mir-23a-3p and mir-186-5p in SCII group decreased significantly,and the expression of TRIL and CXCL13 protein increased significantly.TRIL inhibition improved the motor function after SCII,reduced BSCB disruption after SCII,increased the number of intact neurons,and reduced the protein expression of TLR4,TLR3,NF-κ B and IL-1 β in the downstream.There is a targeted binding relationship between miR-23a-3p and TRIL.Overexpression of mi R-23a-3p improved the neurological function,decreased the disruption of BSCB,increased the number of intact neurons,and decreased the protein expression of TRIL,TLR4,TLR3,NF-κ B and IL-1 β.The effect of mi R-23a-3p on spinal cord ischemia-reperfusion injury may be due to the targeted regulation of TRIL by miR-23a-3p.CXCL13 increased significantly after SCII,and CXCL13 expressed in neurons and microglia.Silencing CXCL13 or CXCR5 improved the motor function,improved the disruption of BSCB,increased the number of intact neurons,and reduced the protein expression of ERK,p-ERK,caspase-3,IL-1 β,IL-6 and TNF-α.The target genes of miR-186-5p was enriched in go by bioinformatics analysis.It was found that CXCL13 was enriched in several biological processes of miR-186-5p.Overexpression of miR-186-5p improved the neurological functions,decreased the permeability of BSCB,increased the number of intact neurons,and decreased the protein expression of CXCL13,CXCR5,ERK,p-ERK,caspase-3,IL-1 β,IL-6 and TNF-α.miR-186-5p inhibition aggravated SCII.The effects of miR-186-5p on spinal cord ischemia-reperfusion injury might be due to the regulation of CXCL13.Sevoflurane preconditioning could improve the neurological function at 12 h and 24 h after SCII.Sevoflurane preconditioning increased the expression of miR-23a-3p and miR-186-5p,decreased the expression of TRIL and CXCL13 protein after SCII in rats.miR-23a-3p and its target gene TRIL,mi R-186-5p and its potential target gene CXCL13 might be involved in the protective mechanism of sevoflurane preconditioning.Conclusions: 1.In this study,we found that the expression of miR-23a-3p decreased significantly,and the expression of TRIL,TLR4,TLR3,NF-κ B and IL-1β increased after SCII.Through animal model,TLR4 and TLR3 mediated by TRIL were found to be involved in SCI.There is a targeted binding relationship between miR-23a-3p and TRIL.Furthermore,it was found that overexpression of mi R-23a-3p played a protective role in SCII via targeting TRIL.2.We found that the expression of mir-186-5p decreased,the expression of CXCL13 and CXCR5 increased after SCII.CXCL13 was expressed in neurons and microglia after SCII.It was found that CXCL13 / CXCR5 axis promoted SCII by activating ERK mediated neuroinflammation and apoptosis.Further bioinformatics analysis of the target genes of mir-186-5p showed that CXCL13,as one target gene of mir-186-5p,participated in many biological functions of mir-186-5p.Furthermore,it was found that interfering the expression of mir-186-5p might affect SCII by regulating the CXCL13 / CXCR5 axis mediated inflammatory and apoptosis pathways.3.Sevoflurane preconditioning could improve neurological functions after SCII.The protective effect of sevoflurane preconditioning might be due to the increased expression of mir-23a-3p and mir-186-5p,and the inhibition of TRIL and CXCL13.