TLR8 Is Activated By MiR-21 In The Drg And Contributes To The Pathogenesis Of Neuropathic Pain

Objective Neuropathic pain is a kind of chronic pain with unclear mechanisms.As neuropathic pain is long-lasting and severe,it seriously affects the quality of patients’life.However,it is still lack of effective analgesics in clinic,and new therapeutic targets are urgent to be discovered.Mammalian Toll-like Receptor 8（TLR8）promotes defensive inflammatory responses by recognizing endogenous and exogenous stimuli to participate in the immune response process.In addition,TLR8can also recognize single-stranded and double-stranded RNA.miR-21,a pain-related small RNA,can act on TLR8 in immune cells through the form of exosomes in vitro.Focusing on TLR8 and miR-21,we sudied their expression,role and mechanism in DRG underlying neuropathic pain.Methods Unilateral L5 spinal nerve ligation（SNL）was used to establish neuropathic pain model.The mechanical allodynia and heat hyperalgesia of WT and Tlr8^-/-mice were tested after SNL and intrathecal injection of miR-21 mimic in different time points.The cellular localization of TLR8 in DRG neurons was examined by immunofluorescence double staining.The expression of miR-21 and co-localization with TLR8 in DRG neurons were investigated via immunofluorescence with in situ hybridization.RT-PCR was used to detect the mRNA level of several inflammatory cytokines including CCL2,TNF-a,IL-1b,IL-6in DRG after intrathecal injection of TLR8 agonist VTX-2337 and miR-21 mimic.The expression of pNFkB,pERK,pJNK and p-P38 were detected by Western Blot after intrathecal injection of VTX-2337 and miR-21 mimic.Pain behaviors were tested after L5 spinal nerve injection of miR-21 antagomic.Results（1）Knock-out of Tlr8 gene had no effect on basic pain threshold,motor function,expression of nerve fiber markers and chemical transmitters,but relieved the neuropathic pain induced by SNL.（2）TLR8 was mainly localized in lysosomes and endosomes of DRG neurons,less in the endoplasmic reticulum.（3）Intrathecal injection of TLR8 agonist VTX-2337 induced the expression of pERK,but had no effect on the expression of pNFkB,p-P38 and pJNK.SNL induced the expression of pERK,which was inhibited by knockdown of Tlr8 gene.（4）Intrathecal injection of VTX-2337 increased expression of inflammatory cytokines CCL2,TNF-a,IL-1b,which was inhibited by knockdown of Tlr8 gene.SNL induced expression of inflammatory cytokines CCL2,TNF-a,IL-1b,IL-6,which was inhibited by knockdown of Tlr8 gene.（5）The expression of miR-21 was increased in DRG neurons after SNL,with the peak expression at SNL10d.miR-21 was distributed in large,medium and small neurons of DRG,and was labeled with TLR8.（6）Intrathecal injection of miR-21 mimic induced mechanical hyperalgesia and activated the ERK pathway,leading to increased expression of inflammatory cytokines CCL2,TNF-a,IL-1b,and IL-6,which was reduced in Tlr8^-/-mice.（7）L5spinal nerve injection of miR-21 antagomic relieved mechanical hyperalgesia induced by SNL.Conclusions SNL induced persistent upregulation of TLR8 and miR-21 in the DRG.miR-21 may act as an endogenous ligand of TLR8 to activate ERK signaling pathway through TLR8,and promote the release of inflammatory factors to participate in the generation and maintenance of neuropathic pain.