Analgesic Effect Of Koumine On Diabetic Neuropathic Pain And Its Possible Brain Mechanism

Objective: Analgesic effect of koumine(KM)on the diabetic neuropathic pain(DNP)and its characteristics were observed on rat streptozocin-induced DNP model,to provide the experimental proofs for the potential clinical application of koumine as a hopeful drug in the prevention and treatment of DNP.And then explore the relationship between the therapeutic effect of koumine on DNP and the activation of astrocyte、the level of proinflammatory cytokines and allopregnanolone in prefrontal cortex and amygdala.Preliminary analysis of the possible brain mechanisms to analgesic effect of koumine on DNP provide the theoretical basis for the clinical application of the koumine and make further understanding of central regulatory mechanism of DNP.Methods:(1)An intraperitoneal injection of 70 mg/kg streptozocin induces the rat DNP model,experimental animals were randomly assigned into control group,model group,koumine treated groups(0.28,1.4 and 7.0 mg/kg)and gabapentin control group(100 mg/kg).Corresponding drugs or vehicle was administered intragastricly once a day for 8 consecutive days beginning from day 22 after streptozocin administration.Analgesic effect of koumine and its characteristics were observed by mechanical withdrawal threshold on rat streptozocin-induced DNP model.(2)After behavioral test was finished,the brain was harvested after perfusion fixation,and the effect of koumine on astrocyte marker glial fibrillary acidic protein reactivation was evaluated by fluorescence immunohistochemistry.After behavioral test was finished,the brain was harvested,separate the prefrontal cortex and amygdale,and the effect of koumine on astrocyte marker glial fibrillary acidic protein reactivation was evaluated by western blot.(3)The level of proinflammatory cytokine in prefrontal cortex and amygdala was determined by enzyme linked immunosorbent assay(ELISA).The level of allopregnanolone in prefrontal cortex and amygdala was determined by ELISA.Results:(1)Data of the behavioral experiment showed that mechanical withdrawal threshold(MWT)of rats administered koumine was notably enhanced in contrast to the model group,and the effect of koumine is dose-dependent and time-dependent.Moreover,the effect can be maintained for more than 80 h after stopping the drug,which suggested koumine has significant anti-pain effect in streptozocin-induced DNP rats.(2)Immunofluorescence histochemical assay showed that astrocytes activation,the fluorescence of GFAP and the average optical density were notably enhanced,the number of positive astrocyte was increased and the body is bigger,astrocyte was reactive in the DNP model rats in contrast to the control group;the fluorescence of GFAP and the average optical density were decreased,the number of positive astrocyte was decreased,the status of astrocyte return to active type after koumine treated.Western blot assay showed that the expression of GFAP in prefrontal cortex and amygdala was enhanced,;koumine can significantly against the prefrontal cortex and amygdala of the rat model GFAP levels rise.(3)The results of ELISA showed that levels of TNF-? and IL-1β in prefrontal cortex and amygdala were notably enhanced in the DNP model rats in contrast to the control group,koumine could significantly inhibit the increasing expression.(4)ELISA test showed that the level of allopregnanolone has no significant change compared with the model group,koumine could significantly improve the level of allopregnanolone.Conclusion: 1.Intragastric administration koumine has a significant analgesic action in DNP.2.The analgesic affection of koumine on DNP might be associated with the inhibition of astrocytes activation and down-regulation of proinflammatory cytokines TNF-? and IL-1β,and the up-regulation of allopregnanolone in prefrontal cortex and amygdala.