TUG1 Affected The Inflammatory Mechanism Of Spinal Cord Ischemia Reperfusion Injury In Rats Via Regulating MTDH

Objective: Spinal cord ischemia-reperfusion injury(SCIRI)often occurs in thoracic and abdominal aortic surgery,leading to sensory and motor deficits below the injury level,as well as physiological dysfunction such as irreversible bladder,intestinal and sexual functions even life-threatening.Although surgical techniques and management are constantly improving,SCIRI cannot be completely prevented.The molecular mechanism of SCIRI has not been thoroughly studied.The TLR4-mediated NF-?B inflammatory signaling pathway is an important pathway in SCIRI,and the inflammation-related proteins TLR4 interactor with leucine-rich repeats(TRIL)and metadherin(MTDH)play a vital role in the NF-?B inflammatory pathway.The miR-29 b family and LncRNA TUG1 of non-coding RNA involved in nerve system and ischemia diseases begin to get wide attention in recent years.We aim to explore the regulatory mechanism through TUG1 and miR-29b-1-5p based on the existing inflammatory protein in SCIRI.Methods: IR rat model was induced by 14-minute occlusion of aortic arch between the left common carotid artery and left subclavian artery.The rats were randomly divided into Sham group(rats completed the same surgery with no occlusion of aortic arch)and IR group(occlusion of aortic arch).Microarray analysis was performed to investigate the changed miRNAs after IR.RT-qPCR was used to evaluate the expression of miR-29b-1-5p.Overexpression of miR-29b-1-5p was induced by intrathecal injection of mimics plasmid vector,and the hind-limb motor function was evaluated by Tarlov scores and blood-spinal cord barrier(BSCB)permeability was examined using Evans blue(EB)dye.Western blot was performed to assess the protein level of TRIL and MTDH.Knockdown of TRIL was induced by siRNAs plasmid vector to assess the protein level of TLR4/NF-?B/IL-1? signaling pathway.Knockdown of MTDH was induced by siRNAs plasmid vector to assess the protein level of NF-?B and IL-1?.Overexpression of miR-29b-1-5p was induced to assess the protein level of TRIL and MTDH/NF-?B/IL-1? signaling pathway.The interaction between TRIL and miR-29b-1-5p,as well as MTDH and miR-29b-1-5p were tested by dual-luciferase reporter assay.RT-qPCR was used to evaluate the expression of TUG1 after IR.Knockdown of TUG1 was induced by siRNAs plasmid vector to assess hind-limb motor function and BSCB permeability as well as expression of miR-29b-1-5p.The interaction between TUG1 and miR-29b-1-5p was tested by dual-luciferase reporter assay.Knockdown of TUG1+inhibition of miR-29b-1-5p was induced to assess hind-limb motor function and BSCB permeability,as well as protein level of MTDH/NF-?B/IL-1?signaling pathway and astrocyte activation.Knockdown of TUG1+overexpression of TRIL(pcDNA3.3-TRIL plasmid vector)was induced to assess protein level of TLR4/NF-?B/IL-1? signaling pathway and microglia activation.Results: Compared with the Sham group,miR-29b-1-5p expression was significantly decreased in the IR group after IR,and the protein expression of TRIL and MTDH was significantly increased after IR.Overexpression of miR-29b-1-5p exhibited improved hind-limb motor function and reduced BSCB leakage.Knockdown of TRIL decreased protein level of TLR4/NF-?B/IL-1? signaling pathway.Knockdown of MTDH decreased protein level of NF-?B and IL-1?.Overexpression of miR-29b-1-5p decreased protein level of MTDH/NF-?B/IL-1? signaling pathway while exhibited no affect on TRIL.There is a targeted binding relationship between miR-29b-1-5p and MTDH.The level of TUG1 was highly expressed after IR.Knockdown of TUG1 reduced BSCB leakage and improved hind-limb motor function as well as increased expression of miR-29b-1-5p.TUG1 could directly interact with miR-29b-1-5p by acting as an endogenous sponge.IR induced activation of astrocytes.Inhibition of miR-29b-1-5p efficiently reversed the neuroprotection effect,which also reversed the decreased protein level of MTDH/NF-?B/IL-1? signaling pathway and attenuated activation of astrocytes induced by TUG1 knockdown.IR induced activation of microglia.TRIL overexpression reversed the decreased protein level of TLR4/NF-?B/IL-1? signaling pathway and attenuated activation of microglia induced by TUG1 knockdown.Conclusions: IR induced downregulation of miR-29b-1-5p and upregulation of TUG1 and protein expression of MTDH/NF-?B/IL-1? signaling pathway,as well as TRIL mediated TLR4/NF-?B/IL-1? signaling pathway.IR also induced astrocytes and microglia activation.TUG1 regulated inflammatory damage of the MTDH/NF-?B/IL-1?signaling pathway after IR via miR-29b-1-5p.TUG1 regulated inflammatory damage of the TLR4/ NF-?B/IL-1? signaling pathway after IR via TRIL.