TLR4/NF-κB Pathway Is Involved In Radicular Pain By Promoting Spinal Microglia Activation And Inflammatory Response In A Rat Model Of Lumbar Disc Herniation

Lumbar disc herniation(LDH)is a common disease in clinic and the main cause of radicular pain(RP),which mainly present with hyperalgesia,allodynia and spontaneous pain.Over the years,although a large number of studies have been carried out at home and abroad,the mechanism of RP remains unclear.Toll-like receptors(TLRs)are crucial components of the pattern recognition receptor(PRR).In recent years,immunological literature has reported that TLRs not only play a crucial role in congenital immunity,but also coordinate acquired immunity well.The activated TLR4 can express various inflammatory cell tissues by signal transduction such as MAPK/NF-κB and NF-κB/IRF3,thus causing inflammation.TLR4 is expressed more in neurons,microglia(MG)and other related cells in the central nervous system(CNS),and the most significantly in MG.TLR4,which is involved in congenital immunity response,is a repetitive domain rich in leucine and a cytoplasmic signal domain of transmembrane protein.Combined with endogenous or exogenous ligands,TLR4 may induce the release of proinflammatory cytokines by activating NF-κB or p38.It is reported that TLR4 antagonist can relieve hyperalgesia caused by nerve injury or genetic defect glial cell activation and inflammatory pain caused by its cofactor CD14.However,the role of TLR4 in LDH induced radicular pain remains unclear.This study aims to clarify the role of TLR4/NF-κB pathway in radiculopathic pain,the establishment of rat LDH mouse model,the activation mechanism of spinal microglia and the subsequent inflammatory response.Microglia is an innate immune effector cell in CNS.Although its number accounts for only about 10% in CNS,it has a significant effect in the physiological orpathological regulation of the immune response of CNS.Current analysis proves that TLR4 is more active in various tissues of brain and spinal cord,such as vascular endothelial cells and smooth muscle cells,and is most active in MG in the CNS.TLR4/NF-κB signal pathway contributes to the CNS damage,and MG is an important source of damage medium.The excessive inflammatory response that occurs when tissue is damaged can have a devastating effect on the recovery of the nervous system.Not only pro-inflammatory cell tissues and chemotactic tissues can be generated,but also activated MG can generate various free radicals,including oxygen free radicals,which can adversely affect nerve cells.TLR4 in microglia will bring reactive oxygen species under the influence of endotoxin,making epithelial cells and endothelial cells to die.How is MG activated in the LDH-induced chronic pain model?How does activated MG cause chronic pain?What is the specific mechanism?All these problems need to be further studied.Objectives: 1.To clarify the role of TLR4/NF-κB(p65)pathway in radicular pain induced by lumbar disc herniation in rats;2.To explore the correlation and mechanism between microglia activation and spinal cord inflammatory response in radicular pain.Chapter 1: Activation of TLR4/NF-κB pathway in spinal cord is an important cause of chronic pain in LDH rats.In this study,rat model of LDH was replicated by autologous nucleus pulposus(NP)transplantation.The mechanical Paw withdraw threshold(PWT)and thermal withdrawal latency(PWL)of ipsilateral and contralateral hind limbs were detected3-21 days after operation,respectively.Findings: ⑴ Compared with sham operation group,NP implanted rats showed significantly lower paw withdrawal threshold(PWT)and paw withdrawal latency threshold(PWL),while the PWT and PWL of contralateral hind limbs had no significant difference.This indicates that NP transplantation can cause unilateral mechanical hyperalgesia and thermal allodynia.Expression and cytological localization of TLR4 and p-p65 in ipsilateral and contralateral spinal dorsal horn of rats in sham operation group and NP transplantation group were detected by using Western blotting and immunofluorescence methods.(3d,7d,14 d,21d).(1)The findings of western blotting showed that the expression of TLR4 and p-p65 in ipsilateral spinal dorsal horn increased significantly 3-21 days after NP transplantation compared with sham operation group,but the expression ofTLR4 and p-p65 in contralateral spinal dorsal horn had no significant difference.⑵The findings of immunofluorescence double staining showed that TLR4 mainly existed in microglia,not in astrocytes and neurons in spinal dorsal horn.We tested the effect of TLR4 antagonist TAK242 on the expression of p-p65 in spinal cord,so as to further prove that activation of TLR4/NF-κB pathway is the cause of chronic pain.Thefindings revealed that TLR4 antagonist TAK242 could reduce the expression of p-p65 in spinal cord without affecting the level of TLR4.TLR4 inhibitor TAK242 and NF-κB inhibitor PDTC can effectively reduce pain,inhibit activation of spinal microglia and reduce myelitis reaction,suggesting that activation of TLR4/NF-κB pathway in spinal cord is an important cause of chronic pain in rats of LDH model.Chapter 2: TLR4/NF-κB pathway induces RP by activating spinal microglia and promoting the expression of inflammatory cytokinesSince TLR4 mainly exists in microglial cells in spinal dorsal horn,we hypothesized that TLR4/NF-κB pathway may cause chronic pain by affecting activation and functions of microglial cells,such as release of inflammatory cytokines.The findings showed that:(1)Compared with the sham-operation group,ELISA results showed the expression of IL-1β,IL-6,TNF-α in spinal cord of LDH rats increased,while the expression of IL-10 decreased.Compared with LDH rats,TAK242 and PDTC decreased IL-1β,IL-6,TNF-α,increased IL-10 expression.It has been proved in many literatures that IL-1β,IL-6,TNF-α can cause central sensitization and chronic pain.Therefore,we believe that the mechanism of chronic pain caused by activation of TLR4/NF-κB pathway in spinal dorsal horn after NP transplantation is related to the promotion of microglial activation and release of inflammatory cytokines.Conclusion:1.NP transplantation can cause mechanical hyperalgesia and thermal allodynia in the ipsilateral hind limbs of rats.2.Activation of TLR4/NF-κB pathway in spinal dorsal horn is an important reason for chronic pain.3.TLR4/NF-κB pathway of spinal cord is involved in radicular pain by stimulating the activation and inflammatory response of microglia,and targeting this pathway may contribute to relieving the radicular pain.