Analgesic Effect Of Koumine On Neuropathic Pain And Its Spinal Molecular Mechanism

Neuropathic pain is a critical challenge for current clinical care according to its high prevalence, complicated pathogenesis, unbearable pain sensation and difficult treatment, which appeals for research and development of novel drugs for its prevention and treatment. Koumine is an alkaloid of Gelsemium elegans Benth., which is a traditional medicinal plant of China. Koumine is the most abundant molecule among the Gelsemium alkaloids and exerts relatively low toxicity. Previous data show that koumine possesses the potential analgesic property on chronic pain while it is still needed to be further confirmed. Mechanism of analgesic action of koumine on neuropathic pain is expected to be clarified.Dorsal horn of spinal cord is an important region for peripheral neuropathic pain modulation. A growing body of evidences indicates that spinal glia is involved in the modulation of neuropathic pain. Activated spinal glia releases multiple proinflammatory cytokines such as TNF-α and IL-1β. Glia activation plays an important role on the initiation and maintenance of neuropathic pain and is responsible for the algesic action of glia.Does there exist any endogenous and compensatory response to antagonize the algesic action of glia activation? In other words, does there exist any endogenous analgesic mechanism? It needs to be investigated. The analgesic effect of spinal neuroactive steroids on neuropathic pain has been paid more and more attention. TSPO [Translocator protein(18 k Da)] is predominantly expressed in glia in central nervous system. The main function of TSPO is to promote the biosynthesis of neurosteroids. Upregulation of TSPO antagonizes the painful statement of neuropathic pain via its possible analgesic pathway TSPO-allopregnanolone-GABAA receptor-analgesia.This study further confirmed the analgesic effect of koumine on peripheral neuropathic pain by three rat neuropathic pain models in order to propose the possible novel efficacy of koumine on neuropathic pain relief. Based on this, the inhibition of koumine on algesic pathogenesis was studied on one hand. The relationship between the analgesic effect of koumine and the level of spinal proinflammatory cytokines is explored, and then further observe and analyze the correlation between the analgesic effect of koumine and the effect of koumine on the activation of spinal astrocyte. Enhancement of koumine on endogenous analgesic mechanism was studied on the other hand. The relationship between the analgesic effect of koumine and the up-regulated level of spinal allopregnanolone is evaluated, and then further elucidate the correlation between the analgesic effect of koumine and the enhancement of koumine on spinal TSPO. The aim of this study is to overall clarify the spinal molecular mechanism of analgesic effect of koumine on neuropathic pain, to reveal the novel drug target and mechanism of drug action for the research and development of novel neuropathic pain therapeutic drugs and to make further understanding of central mechanism of neuropathic pain pathogenesis.The main results are listed here. 1 Analgesic effect of koumine on neuropathic pain in rats 1.1 Analgesic effect of koumine on chronic constriction injury(CCI) model in ratsAnalgesic effect of koumine on CCI neuropathy was measured by mechanical withdrawal threshold on rat CCI model. Experiment animals were randomly assigned into sham group, model group, koumine treated groups(0.28, 1.4 and 7.0 mg/kg) and positive drug gabapentin control group(40 mg/kg). Corresponding drugs or vehicle was administered subcutaneously twice per day for 7 consecutive days beginning from postoperative day 3. The pain threshold was measured before surgery, on postoperative day 3(pre-dosing) and on 1 h after drug administration(post-dosing) on the morning of postoperative days 5, 7 and 9. Data showed that koumine significantly reversed CCI-induced mechanical evoked pain in a time and dose dependent manner. Koumine 7.0 mg/kg didn’t affect threshold on sham group. 1.2 Analgesic effect of koumine on spared nerve injury(SNI) model in ratsAnalgesic effect of koumine on SNI neuropathy was measured by mechanical withdrawal threshold on rat SNI model. Experiment animals were randomly assigned into sham group, model group, koumine- treated groups(0.6 and 3.0 mg/kg) and gabapentin control group(800 mg/kg). Corresponding drugs or vehicle was administered intragastricly twice per day for 7 consecutive days beginning from postoperative day 3. The pain threshold was measured before surgery, on postoperative day 3(pre-dosing) and on 1 h after drug administration(post-dosing) on the morning of postoperative days 5, 7 and 9. Data showed that koumine significantly reversed SNI-induced mechanical evoked pain in a time and dose dependent manner. 1.3 Analgesic effect of koumine on streptozocin(STZ) induced diabetic neuropathic pain(DNP) model in ratsAnalgesic effect of koumine on DNP was measured by mechanical withdrawal threshold on rat STZ-induced DNP model. Experiment 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 per day for 8 consecutive days beginning from day 22 after STZ administration. The pain threshold was measured before STZ administration and on 1 h after drug administration(post-dosing) on the morning of day 21, 22, 24, 26 and 28. Data showed that koumine significantly reversed mechanical evoked pain in DNP rat in a time and dose dependent manner. 2 Effect of koumine on the levels of proinflammatory cytokines in rat lumbar spinal cord with neuropathy 2.1 Effect of koumine on the levels of TNF-α and IL-1β in rat lumbar spinal cord with SNI neuropathyAfter behavioral test was finished, the spinal cord of lumber segment 4 to 6 of sham groups, model groups and koumine treated groups was harvested. The level of TNF-α and IL-1β was determined by enzyme linked immunosorbent assay(ELISA). Data showed that the level of TNF-α and IL-1β was increased in rat lumbar spinal cord with SNI neuropathy. Koumine dose-dependently decreased the level of spinal TNF-α and IL-1β, which negatively correlate the analgesic effect of koumine on SNI rats. 2.2 Effect of koumine on the levels of TNF-α and IL-1β in rat lumbar spinal cord with STZ-induced DNPAfter behavioral test was finished, the spinal cord of lumber segment 4 to 6 of sham groups, model groups and koumine-treated groups was harvested. The level of TNF-α and IL-1β was determined by ELISA. Data showed that the level of TNF-α was increased without significance in rat lumbar spinal cord with DNP. Koumine decreased the level of spinal TNF-α without significance, which coefficient correlation between the effect of koumine on the level of spinal TNF-α and the analgesic effect of koumine on DNP rats is-0.339(P=0.114). The level of IL-1β was increased without significance in rat lumbar spinal cord with DNP. Koumine dose-dependently decreased the level of spinal IL-1β, which negatively correlates the analgesic effect of koumine on DNP rats. 3 Effect of koumine on astrocyte reactivation in rat lumbar spinal cord with neuropathyAfter behavioral test was finished, the spinal cord of lumber segment of sham groups, model groups and koumine-treated groups was harvested after perfusion fixation. The effect of koumine on astrocyte reactivation was evaluated by fluorescence immunohistochemistry and western blot. Data showed that in superficial lamina of ipsilateral spinal cord in SNI rats, the fluorescence intensity of GFAP was increased, astrcyte was reactive, the number of positive astrocyte was increased and the expression of GFAP was elevated. The contralateral spinal cord in SNI rats remained unchanged. After 3.0 mg/kg koumine treatment, the immunofluorescence intensity was decreased, the status of astrocyte return to active type, the number of positive astrocyte and the expression of GFAP was decreased. 4 Effect of koumine on allopregnanolone in rat lumbar spinal cord with neuropathy 4.1 Effect of koumine on the levels of allopregnanolone in rat lumbar spinal cord with CCI neuropathyAfter behavioral test was finished, the spinal cord of lumber segment 4 to 6 of sham groups, model groups and koumine-treated groups was harvested. The level of allopregnanolone was determined by high performance liquid chromatography-tandem mass spectrometry. Data showed that the level of allopregnanolone was increased in rat lumbar spinal cord with CCI neuropathy. Koumine further increased the level of spinal allopregnanolone in a dose-dependent manner, which positively correlates the analgesic effect of koumine on CCI rats. 4.2 Effect of koumine on the levels of allopregnanolone in rat lumbar spinal cord with SNI neuropathyAfter behavioral test was finished, the spinal cord of lumber segment 4 to 6 of sham groups, model groups and koumine-treated groups was harvested. The level of allopregnanolone was determined by ELISA. Data showed that the level of allopregnanolone was increased without significance in rat lumbar spinal cord with SNI neuropathy. Koumine further increased the level of spinal allopregnanolone in a dose-dependent manner, which positively correlates the analgesic effect of koumine on SNI rats. 4.3 Effect of koumine on the levels of allopregnanolone in rat lumbar spinal cord with DNPAfter behavioral test was finished, the spinal cord of lumber segment 4 to 6 of sham groups, model groups and koumine-treated groups was harvested. The level of allopregnanolone was determined by ELISA. Data showed that the level of allopregnanolone was decreased without significance in rat lumbar spinal cord with DNP neuropathy. Koumine increased the level of spinal allopregnanolone in a dose-dependent manner, which positively correlates the analgesic effect of koumine on DNP rats. 5 The relationship between analgesic effect of koumine on neuropathic pain and TSPO and TSPO mediating molecular pathway 5.1 Effect of spinal intrathecal PK11195 on the therapeutic action of koumine on neuropathic painEffect of spinal intrathecal PK111P5 on the therapeutic action of koumine on CCI neuropathy was measured by mechanical withdrawal threshold and TSPO antagonist PK11195 on rat CCI model. Rats were conducted intrathecal catheter implantation and CCI surgery. Experiment animals were randomly assigned into vehicle control group, koumine treated group and PK11195 treated groups(0.04, 0.2 and 1.0 μg) and PK11195 control group(1.0 μg). PK11195 or vehicle was administered intrathecally, koumine 7.0 mg/kg or vehicle was administered subcutaneously. The pain threshold was measured 1 h after drug administration. Data showed that koumine significantly increased the pain threshold. PK11195(1.0 μg) reversed the therapeutic effect of koumine while itself didn’t affect threshold on CCI rats. 5.2 Effect of spinal intrathecal provera on the therapeutic action of koumine on neuropathic painEffect of spinal intrathecal provera on the therapeutic action of koumine on CCI neuropathy was measured by mechanical withdrawal threshold and neurosteroid synthetic enzyme inhibitor provera on rat CCI model. Rats were conducted intrathecal catheter implantation and CCI surgery. Experiment animals were randomly assigned into vehicle control group, koumine treated group and provera treated groups(2.5, 5.0 and 12.5 μg) and provera control group(12.5 μg). Provera or vehicle was administered intrathecally, koumine 7.0 mg/kg or vehicle was administered subcutaneously 30 min later. The pain threshold was measured 1 h after drug administration. Data showed that koumine significantly increased the pain threshold. Provera(5.0 and 12.5 μg) reversed the therapeutic effect of koumine while provera 12.5 μg didn’t affect threshold on CCI rats. 5.3 Effect of spinal intrathecal 17 PA on the therapeutic action of koumine on neuropathic painEffect of spinal intrathecal 3α,5α-17-phenylandrost-16-en-3-ol(17PA) on the therapeutic action of koumine on CCI neuropathy was measured by mechanical withdrawal threshold and allopregnanolone antagonist and GABA receptor blocker 17 PA on rat CCI model. Rats were conducted intrathecal catheter implantation and CCI surgery. Experiment animals were randomly assigned into vehicle control group, koumine treated group and 17 PA treated groups(0.5, 1.0, 2.5 and 12.5 μg) and 17 PA control group(12.5 μg). 17 PA or vehicle was administered intrathecally, koumine 7.0 mg/kg or vehicle was administered subcutaneously 30 min later. The pain threshold was measured 1 h after drug administration. Data showed that koumine significantly increased the pain threshold. 17PA(2.5 and 12.5 μg) reversed the therapeutic effect of koumine while 17 PA 12.5 μg didn’t affect threshold on CCI rats.In summary, koumine has a significant analgesic action in neuropathic pain. To inhibit spinal astrocyte reactivation and to down regulate the level of spinal proinflammatory cytokines might be one of mechanisms of analgesic action of koumine on neuropathic pain. The analgesic action of koumine on neuropatic pain might be associated with the upregulation of spinal allopregnanolone. To enhance TSPO and TSPO mediating molecular pathway(TSPO-allopregnanolone-GABAA receptor) might be another one of mechanisms of analgesic action of koumine on neuropathic pain.