| BackgroundSpinal Cord Injury(SCI)is a serious complication after vertebral fracture.It is a serious injury of the central nervous system.Epidemiological studies have shown that SCI incidence rates in China are about 100,000 patients per year.SCI is characterized by disability rate,which brings great pain to patients and heavy medical burden to families.After SCI,patients have pain due to vertebral fracture,spinal cord tissue damage,or spinal nerve damage.The International Association for SCI,the American Association for SCI and the American Pain Society classify pain into injurial pain,neuropathic pain and others according to the pathophysiological characteristics of pain after SCI.The prevalence of Neuropathic Pain After Spinal Crod Injury(SCI-NP)is about 40%.Because neuropathic pain involves a complex central mechanism,the treatment effect of neuropathic pain is not ideal.At present,drug therapy is still the main treatment for neuropathic pain after spinal cord injury,but its side effects are large,and the effect of standard pain treatment is limited.Cell therapy is a promising treatment for spinal cord injury and has been shown to be safe and effective in animal and clinical trials.Mesenchymal Stem Cells(MSCs)are used in experimental treatments for a variety of human diseases.Studies have shown that bone marrow mesenchymal stem cells have therapeutic effects in many disease models by inhibiting inflammation and stimulating host cells to affect tissue repair mainly through paracrine mechanisms.Among them,Exosomes are the main components of MSCs paracrine.Exosomes are tiny vesicles with lipid bilayer approximately 30-150 nm in diameter that can be secreted by most cells.They carry and transmit important signaling molecules,promote cell proliferation and angiogenesis,and play neuronal protection and immune regulation roles.Studies have shown that intrathecal injection of exosomes can inhibit the inflammatory response and relieve neuropathic pain caused by spinal nerve ligation.Therefore,it is the purpose of this study to study the influence of MSCs-derived exosomes on SCI-NP and the molecular mechanism of its action.Activated microglia/macrophages exist in the injured site after spinal cord injury and produce inflammatory mediators to inhibit spinal cord repair and regeneration.Activation of microglia cells is closely related to the occurrence and development of pain.Toll-like receptor4(TLR4)is a pattern recognition receptor mainly expressed on the surface of immune cells,while microglia cells are the main immune cells in the central never system.It was found that TLR4-mediated microglia activation plays a key role in the pathological process of nerve injury in the central nervous system and the peripheral nervous system.The molecule My D88 is required for downstream signal transduction of TLR4.After TLR4 recognition of ligand,My D88 recruits downstream IL-1R-associated kinase(IRAK),whose activation leads to phosphorylation of NF-κB(IκB)-kinase complex(IKK complex),which further causes the release of inflammatory cytokines.A growing body of evidence suggests that TLR4-mediated neuroinflammation is closely related to the mechanisms of peripheral and central sensitization of pain.However,it is not clear whether MSCs-derived exosomes can alleviate neuropathic pain by regulating the expression of TLR4 inflammatory signaling pathway and alleviating microglial activation response after SCI.Therefore,in this study,the effect of MSCs-derived exosomes on SCI-NP was investigated by transplantation,and the mechanism of alleviating SCI-NP pain was studied,so as to provide experimental evidence and theoretical basis for the treatment of SCI-NP.Therefore,in this study,the effect of MSCs-derived exosomes on SCI-NP was studied by transplanting MSCs-exosome,so as to provide experimental evidence and theoretical basis for the treatment of SCI-NP.Objective:1.To investigate the activation of microglia after SCI and whether TLR4/My D88/NF-κB are involved in neuropathic pain.2.To investigate the mechanism of MSCs-derived exosomes on neuropathic pain induced by spinal cord injury.Methods :(1)The spinal cord injury model of rats was established.The changes of mechanical pain behavior after spinal cord injury were observed by injection of TLR4 inhibitor TAK242.SD rats were randomly divided into SHAM,SCI+ veh blank control group and SCI+TAK242 treatment group.Rats in the treatment group were intraperitoneally injected with TLR4 inhibitor TAK242(6mg/kg)for 14 consecutive days after operation.The behavioral results of each group were observed 7-14 days after injury,and the expression changes of microglia at the spinal cord injury site in each group were observed by immunofluorescence on the 7th day after surgery.The TLR4 signaling pathway and the expression changes of downstream inflammatory cytokines TNF-α and IL-6 were observed by WB and ELISA.(2)To establish a mouse model of spinal cord injury and observe the effect of TLR4 gene mutation on mechanical pain behavior changes.C57 mice were randomly divided into SHAM,SCI+WT,SCI+TLR4 KO,and the behavioral results were observed 7-14 days after surgery.Similarly,the expression changes of microglia in the spinal cord injury site,as well as the expression changes of TLR4 signaling pathway and downstream inflammatory factors in each group were observed on the 7th day after surgery.(3)A mouse model of spinal cord injury was established to observe the effects of postoperative nasal feeding of MSCs-derived exosomes on mechanical pain behavior changes.C57 mice were randomly divided into SHAM,SCI+ veh and SCI+ Exosome groups.The behavioral results of each group were observed 7-14 days after injury,and the changes in the expression of microglia,TLR4 signaling pathway and downstream inflammatory cytokines at the spinal cord injury site in each group were observed on the 7th day after surgery.(4)An external pain model was established to observe whether MSCs-EX inhibited the TLR4 signaling pathway and reduced microglia activation in vitro.Glutamate was added to cultured mouse cortical neurons in vitro.Results1.On the 7th day after spinal cord injury,obvious mechanical pain sensitivity appeared in rats.In the spinal cord lesions associated with a large number of microglia activation of TLR4 / My D88 nf-kappa B increased signal path expression,and gives the TAK242 after treatment,obvious reverse the microglia activation,inhibition of the over expression of TLR4 signaling pathway,reduce inflammation factor expression,reduce the phenomenon of mechanical pain sensitivity and prompt inhibiting TLR4 could lighten the microglia mediated inflammatory reaction,rational pain alleviate mental derangement.2.WT mice had significant neuropathic pain after spinal cord injury,while TLR4 KO mice relatively relieved this phenomenon.3.Mechanical pain sensitivity also appears after spinal cord injury,and exosomes can effectively alleviate neuropathic pain after treatment.Similarly,exosome therapy can also inhibit the expression of TLR4 signaling pathway and its downstream inflammatory cytokines,suggesting that exosome therapy can alleviate neuroinflammation and neuropathic pain by inhibiting TLR4 signaling.4.Stimulation with 100 um glutamate caused damage to cultured neurons in vitro,and addition of conditioned medium from injured neurons to the cultured BV2 microglial cell line significantly caused expression of the TLR4 signaling pathway in the cells,whereas addition of conditioned medium from MSCs significantly inhibited the expression of the TLR4 signaling pathway in BV2 cells,therefore MSCs derived exosomes can inhibit the TLR4 signaling pathway on microglia Of expression.ConclusionMSCs-derived exosome transplantation can alleviate the neuroinflammation by inhibiting the TLR4/My D88/NF-κB signaling pathway,thus alleviating SCI-NP. |
