Study On The Role And Mechanism Of Lappaconitine In The Verification Of Pain

Objective:Lappa contine(LA)is one of the main active constituents of Aconitum.It has been reported that lappaconitine has the characteristics of analgesia,anti-inflammatory,local anesthesia and low temperature.Studies have shown that spinal glial cell-mediated neuroinflammatory responses play an important role in the development of chronic pain.Whether lappaconitine can alleviate chronic inflammatory pain by inhibiting neuroinflammatory response has not been reported.This study explored the prevention and treatment of chronic inflammatory pain by tail vein injection of lappaconitine.The effects of lappaconitine on CFA-induced glial cell activation in rat spinal cord were studied in vitro and in vivo.Activation of pathways and transcription factors as well as the effects of cytokine and chemokine expression to explore mechanisms involved in chronic inflammatory pain.Methods:(1)Normal rats were injected with lappaconitine(2 mg/kg,10 mg/kg)or solvent in the intraperitoneal.Thermal hyperalgesia and mechanical inducing pain behavior in mice were detected by thermal radiation stimulation and mechanical stimulation,and the effect on the basic value of pain-sensitive behavior in normal rats was observed.(2)Lappaconitine pretreatment group: one day before the unilateral plantar injection of complete Freund's adjuvant(CFA),intraperitoneal was injected with lappaconitine(2mg/kg,10mg/kg)or solvent,1 per day.The mechanical tactile pain and thermal hyperalgesia behavior of the mice were measured every day for 5 consecutive days.(3)Post-treatment group: 2 days after CFA unilateral plantar injection,injection of lappaconitine(2mg/kg,10mg/kg)or solvent,once a day for 5 consecutive days,test the rats for thermal hyperalgesia and small daily.Rat mechanical contact induced pain behavior.(4)Lappaconine pretreatment experiment After the last injection,the mouse spinal cord lumbar enlargement L4-5 segment was taken,and the rat spinal cord astrocytes were detected by real-time fluorescent quantitative PCR,Western Blotting and immunofluorescence.The effect of the marker GFAP and microglia marker OX-42 expression.(5)Real-time quantitative PCR was used to detect the effects of lappaconitine preconditioning on the expression of chemokines and inflammatory related cytokines in rat spinal cord.(6)Real-time quantitative PCR,Western Blotting,immunofluorescence method was used to detect the effect of lappaconitine on the expression of inflammatory factors,chemokines and activation of NF-?B in LPS-induced primary microglia.Results:(1)Intravenous injection of lappaconitine(2mg/kg,10mg/kg)or solvent had no effect on the thermal hyperalgesia of rats and the basic value of mechanical tactile pain behavior in mice;Thermal hyperalgesia induced by plantar injection and mechanical tactile allodynia in mice;post-treatment with lappaconitine reduced thermal hyperalgesia induced by CFA foot injection and mechanical tactile allodynia in mice.(2)Pretreatment with lappaconitine reduced CFA-induced expression of OX-42 in rat spinal cord microglia marker and could not reduce CFA-induced expression of astrocyte marker GFAP in rat spinal cord;Gaowu A pretreatment reduces CFA-induced inflammation-related factors TNF-?(tumor necrosis factor),IL-1?(interleukin-1?),IL-6(interleukin-6),MCP-1(mononuclear)Expression of cell chemokine 1),MIP-1?(macrophage inflammatory protein 1?)and the like.(3)Pre-incubation of primary microglia by Lappaconitine significantly inhibited the expression of LPS-induced inflammation-related factors IL-1?,TNF-?,IL-6,KC,MCP-1,MIP-1?;A reduces the expression of inflammatory and chemotactic-associated factors in LPS by inhibiting NF-?B pathway in cells.Conclusions:(1)Low-dose group(2mg/kg)and high-dose(10mg/kg)group could effectively alleviate CFA-induced inflammatory pain response,and the effect of Lappaconitine pretreatment group Better than the post-processing group.(2)Lappaconitine could significantly reduce CFA-induced inflammatory factors(TNF-?,IL-1?,IL-6)and chemokines(KC,MCP-1,MIP-1?)in rat spinal cord and serum.The expression of high-dose group lappaconin showed a more significant difference.(3)Both animal and cell experiments showed that lappaconitine could effectively inhibit the activation of microglia(p<0.05),but there was no significant inhibition on microglia.(4)Lappaconitine might reduce inflammatory factors(TNF-?,IL-1?,IL-6)and chemokines(KC,MCP-1,MIP-1?)by inhibiting the NF-?B pathway.Expression,which has a palliative effect on inflammatory pain.