Mir-26a-5p Alleviates CFA-induced Chronic Inflammatory Hyperalgesia Through Wnt5a/CaMKⅡ/NFAT Signaling In Mice

Background:Inflammatory pain has become one of the major public health problems worldwide,seriously affecting the quality of life of patients and causing a huge economic burden.Most of the drugs currently used for the treatment of inflammatory pain have many side effects and unsatisfactory curative effects.There is an urgent need to redefine and improve management strategies to treat inflammatory pain.Previous studies have shown that miRNAs play an important role in the pathophysiological process of inflammatory pain and have the potential to become a new therapeutic strategy for inflammatory pain.Previous studies by our group have identified the therapeutic effect of miR-26a-5p from human placental mesenchymal stem cell exosomes on chronic pain induced by nerve injury,and another previous study also reported that miR-26a-5p alleviated neuropathic pain in rats and reduced the release of inflammatory cytokines by targeting MAPK6.Therefore,we speculate that miR-26a-5p could play an important role in inflammatory pain through its antiinflammatory effect,and as a new therapeutic strategy for inflammatory pain,it has great prospects in clinical transformation.Objectives:1.To explore the role of miR-26a-5p in inflammatory pain.2.Optimize the dosing regimen of miR-26a-5p in the treatment of inflammatory pain.3.To clarify the specific mechanism of miR-26a-5p in alleviating inflammatory pain.Methods:1.In Vivo StudiesA mouse model of inflammatory pain induced by complete Freund’s adjuvant was constructed,and miR-26a-5p agomir was injected intrathecally or subcutaneously to study its anti-nociceptive effect.Bioinformatic analysis of miRNAs was performed to study the downstream mechanism of miR-26a-5p.HE staining,RT-qPCR,Western blot and immunofluorescence were used for further validation.2.In Vitro StudiesThe BV2 cell line was used to construct an in vitro inflammation model,and the downstream mechanism of miR-26a-5p in alleviating inflammatory pain was supplemented by RT-qPCR,Western blot,immunofluorescence and Elisa experiments.Results:1.Single intrathecal and local subcutaneous injection of miR-26a-5p reverses hyperalgesia in mice with inflammatory pain.2.Both administration methods of miR-26a-5p reduced the inflammation in the peripheral and spinal cord.3.Wnt5a is the direct downstream target gene of miR-26a-5p.4.Wnt5a co-localizes with neuronal and microglial marker proteins in the dorsal horn of the mouse spinal cord.5.miR-26a-5p inhibits the release of inflammatory cytokines by inhibiting the non-canonical Wnt signaling pathway.6.miR-26a-5p alleviates the activation of microglia in vivo and inhibits LPSstimulated BV2 cell inflammation in vitro.Conclusions:1.miR-26a-5p can be used to treat inflammatory pain.2.Both intrathecal injection and subcutaneous injection of miR-26a-5p can relieve inflammatory pain and reduce inflammation of subcutaneous tissue and spinal cord,but intrathecal injection has a longer pain relief period and more significant analgesic effect than subcutaneous injection.3.miR-26a-5p inhibits the non-canonical Wnt signaling pathway of spinal cord microglia and produces analgesic effect through anti-neuroinflammation.