Targeting HMGB1-TLR4 signaling by miR-216a-5p elevation alleviates the inflammatory behavioral hypersensitivity.

Neuroinflammation induced by microglial activation has a critical role in inflammatory pain. In this study, we detected the function of miR-216a-5p in the progression of inflammatory behavioral hypersensitivity. Here, decreases of miR-216a-5p and up-regulation of high-mobility group box1 (HMGB1) were observed in complete freund's adjuvant (CFA)-induced inflammatory pain model in mice and LSP-activated BV2 microglia. HMGB1 was identified as a target of miR-216a-5p by luciferase reporter system. Ectopic expression of miR-216a-5p suppressed microglial marker IBA-1 expression and subsequent pro-inflammatory cytokine releases (IL-1β, IL-6 and TNF-α) from LPS-activated microglia. Additionally, LPS exposure enhanced the protein expression levels of HMGB1, TLR4 and p-p65 NF-kB in microglia, which were abrogated following miR-216a-5p overexpression. Intriguingly, transfection of HMGN1 cDNA into BV2 microglial cells reversed the inhibitory effects of miR-216a-5p elevation on microglial activation-triggered inflammatory response. Intrathecal delivery of LV-miR-216a-5-p ameliorated CFA-evoked mechanical and thermal hyperalgesia in mice. Concomitantly, overexpressing miR-216a-5p also restrained the inflammatory response and microglia activation in CFA-induced inflammatory mouse models, concomitant with the decreases in the expression of HMGB1, TLR4 and p-p65 NF-kB in spinal cord. Thus, these findings highlight that miR-216a-5p may alleviate inflammatory behavioral hypersensitivity by blocking microglia-mediated neuroinflammation via targeting the HMGB1-TLR4-NF-kB pathway, supporting miR-216a-5p as a potential therapeutic avenue for inflammatory pain.