The Mechanism Of Z-ligustilide Underlying Chronic Inflammatory Pain

Objective Z-ligustilide (LIG) is one of the main active compounds of Radix Angelica sinensis essential oil. Several lines of evidence have suggested that LIG can attenuate the pain behavior induced by acetic acid or formalin and also has anti-inflammatory effect. Recent studies have shown that glia-mediated neuroinflammation plays an important role in the development and maintenance of chronic pain. Whether LIG could attenuate inflammatory pain through inhibition of neuroinflammation is not known. In this study, we investigated the preventive and therapeutic effects of LIG on chronic inflammatory pain, explored the effect of LIG on CFA-induced microglia and astrocyte activation in the spinal cord. We also investigated the effect of LIG on the activation of MAPK signaling pathway and NF-κB transcription factor, as well as the cytokine and chemokine expression. Our results partially revealed the analgesia mechanism of LIG in chronic inflammatoy pain.Methods (1) Naive mice were intravenously injected with vehicle, or LIG (30mg/kg,60mg/kg), and the effects of LIG on the baseline of mechanical allodynia and heat hyperalgesia were detected.(2) LIG pretreatment group:mice were intravenously injected with vehicle or LIG (30mg/kg,60mg/kg), once a day for5days, respectively, starting from1day before unilateral intraplantar injection of complete Freund’s adjuvant (CFA). Mechanical allodynia and heat hyperalgesia behavior were measured after injection of LIG.(3) LIG post-treatment group:two days after CFA injection, mice were treated with vehicle or LIG (30mg/kg,60mg/kg), once a day for5days.The mechanical allodynia and heat hyperalgesia were measured after injection of LIG.(4) Twenty-four hours after the last injection of LIG, the expression of microglial marker Iba-1and astroglial marker GFAP in L4.5spinal cord were detected by Real-time PCR, Western Blotting and Immunofluorescence.(5) The expression of inflammatory cytokines/chemokines in the spinal cord with LIG pretreatment was detected by Real-time PCR.(6) The effect of LIG on the LPS-induced meditors’production, MAPK and NF-κB activation in BV2cells and astrocytes were detected by Real-time PCR and Western Blotting.(7) Mechanical allodynia and heat hyperalgesia were measured after intrathecal injection of NF-κB inhibitor BAY11-7082. The expression of pNF-icB in spinal cord was detected by Western Blotting and Immunofluorescence. The distribution of pNF-κB-IR cells was examined by immunofluorescence double-staining.Results (1) Intravenously injection of vehicle,30mg/kg LIG and60mg/kg LIG had no effect on the baseline of mechanical allodynia and heat hyperalgesia. LIG pretreatment reduced mechanical allodynia and heat hyperalgesia induced by intraplantar injection of CFA. LIG post-treatment also reduced mechanical allodynia and heat hyperalgesia induced by intraplantar injection of CFA.(2) LIG pretreatment reduced the expression of spinal microglial marker Iba-1, but not astroglial marker GFAP after CFA injection; LIG pretreatment reduced the expression of inflammatory mediators in the spinal cord, such as IL-1β,TNF-α, MCP-1, MIP-1α.(3) In BV2cells, the inflammatory mediators, such as IL-1β,TNF-α, IL-6, KC, MCP-1, and MIP-1α induced by LPS (6h), were significantly reduced by pretreatment with LIG for30min through inhibition of NF-κB pathway rather than MAPK pathway;(4) In astrocytes, the inflammatory mediators induced by LPS (6h), were also significantly reduced by LIG pretreatment through NF-κB pathway.(5) The NF-κB in the spinal cord was activated by CFA. Intrathecal injection of NF-κB inhibitor BAY11-7082attenuated CFA-induced mechanical allodynia and heat hyperalgesia. Western Blotting and Immunofluorescence showed that LIG pretreatment reduced the CFA-induced expression of pNF-KB in the spinal cord; Immunofluorescence double staining showed that pNF-KB was colocalized with astroglial marker GFAP, not microglial marker Iba-1or neuronal marker NeuN.Conclusions LIG reduced CFA-induced chronic inflammatory pain, indicating the antinociceptive effect in both prevention and treatment. LIG pretreatment reduced CFA-induced activation of spinal microglia, the activation of NF-κB in spinal astrocytes, and the expression of inflammatory mediators in the spinal cord. LIG may be involved in the regulation of chronic inflammatory pain through inhibition of the glia-mediated neuroinflammation.