钠通道位点3调制剂BmKI靶向性致痛的信号整合机制研究

发布时间：2019-03-09 14:15

【摘要】：电压门控钠通道位点3调制剂BmK I可剂量依赖性地诱导大鼠产生长达2小时的自发痛、超过72小时的单侧热痛敏、超过1周的双侧机械痛敏及阵发性痛觉大发作等痛相关行为。本论文采用行为学、药理学、免疫组织化学和电生理学等手段，对上述痛相关行为的外周及中枢信号整合机制进行研究。主要结果如下：一、 mTOR信号途径参与BmK I诱导的外周伤害性信息的传输大鼠足底皮下注射10μg BmK I，（1）可诱导大鼠L4-L5腰椎对应的DRG内mTOR及其下游分子p70S6K和4E-BP1的双侧激活；（2）与大鼠痛行为的时程相对应，该信号途径于2小时达到激活峰值，后逐渐恢复；双标结果显示，（3）与对照组相比，疼痛状态下，激活态mTOR、p70S6K和4E-BP1免疫阳性神经元在各类型DRG神经元中的比率显著提高，同时激活态mTOR、p70S6K和4E-BP1免疫阳性神经元的细胞类型构成亦发生了不同程度的变化。上述结果表明，mTOR信号途径在外周伤害性信息的传输编码中具有重要作用。上述细胞类型比重的变化亦提示了电压门控钠通道调制剂诱导痛觉的独特外周机制。二、脊髓mTOR信号途径参与介导BmK I诱导的大鼠痛相关行为大鼠足底皮下注射10μg BmK I后，（1）大鼠脊髓L4-L5腰椎节段内mTOR及其下游分子p70S6K和4E-BP1均呈双侧激活；（2）1-2小时达到激活高峰并在1天后恢复；（3）免疫双标实验显示，绝大多数激活态mTOR及4E-BP1与神经元标志物NeuN共标，另有少量与星形胶质细胞标志物GFAP共标，鲜与小胶质细胞标志物Iba-1共标；激活态p70S6K几乎全部与NeuN共标；（4）鞘内预注射mTOR特异性抑制剂Rapamycin或CCI-779，均可剂量依赖地缓解由BmK I诱导的大鼠自发痛、单侧热痛敏和双侧机械痛敏等行为；有趣的是，（5）注射BmK I后2小时鞘内注射Rapamycin，仍可显著抑制大鼠痛敏相关行为。上述结果提示，mTOR信号途径的激活对于BmK I诱导的中枢神经元敏化的过程不可或缺。三、 p38信号通路参与BmK I诱导的脊髓小胶质细胞的激活大鼠足底皮下注射10μg BmK I，（1）可诱导大鼠脊髓腰椎L4-L5节段内小胶质细胞的双侧激活；（2）脊髓小胶质细胞的激活略迟于神经元内信号分子，于4-8小时达激活峰值，并于1天时开始恢复；（3）同节段内的p38亦呈现类似的双侧时程性激活；免疫双标实验显示，（4）激活态p38几乎全部与小胶质细胞标记物OX-42重合；（5）BmK I注射前30分钟，鞘内注射p38特异性抑制剂SB203580可剂量依赖地缓解BmK I诱导的大鼠同侧热痛敏及双侧机械痛敏；（6）小胶质细胞的激活同时受到抑制。上述结果提示，小胶质细胞可能通过p38信号通路参与介导了BmK I诱导的大鼠痛行为。选择性地抑制p38依赖的小胶质细胞激活可缓解痛觉的中枢敏化，减轻疼痛。四、 BmK I诱导的丘脑-皮层信息整合与互动课题选取大鼠初级体感皮层（Primary Somatosensory Cortex, SI）和前扣带回皮层（Anterior Cingulate Cortex,ACC），以及丘脑背内侧核（Medial Dorsal, MD）和丘脑腹后核（Ventral Posterior Nucleus, VP）为研究脑区，记录319个神经元，研究了BmK I诱导的丘脑-皮层网络动态反应模式。结果发现，（1）BmK I诱导的神经元反应可分为三类：持续兴奋型、持续抑制型和静息型；（2）BmKI注射导致脑区之间的信息流动发生改变：内侧通路至外侧通路的信息流动量显著增加；皮层至丘脑的信息流动量显著增加。上述结果显示，高级中枢通过增强脑区间的信息互动参与与了BmK I诱导的痛觉编码。BmK I模型较为简单的脑神经元群反应模式，，或许能为痛觉研究提供另一视野的便利。
[Abstract]:The voltage-gated sodium channel site 3 formulation, BmK I, can be used in a dose-dependent manner to induce a spontaneous pain of up to 2 hours, a one-sided thermal pain in excess of 72 hours, bilateral mechanical pain-sensitive and paroxysmal hyperalgesia in excess of 1 week, and the like. The whole mechanism of the peripheral and central signals of the above-mentioned pain-related behavior was studied by means of behavior, pharmacology, immunohistochemistry and electrophysiology. The main results are as follows: I. The mTOR signaling pathway is involved in the peripheral nociceptive information induced by BmK I. Subcutaneous injection of 10. m u.g of BmK I in the sole of the rat, and (1) a double of the DRG and its downstream molecules p70S6K and 4E-BP1 corresponding to the L4-L5 lumbar spine in the rat the side activation; (2) the time course of the pain behavior of the rat corresponds to the time course of the pain behavior of the rat, the signal path reaches the activation peak at 2 hours, and then is gradually recovered; the double-labeled result shows that (3) in the pain state, the active state m The ratio of TOR, p70S6K and 4E-BP1 positive neurons to the type DRG neurons increased significantly, and the cell types of the activated mTOR, p70S6K, and 4E-BP1 immunopositive neurons were also different. The results show that the mTOR signaling pathway has a heavy weight in the transmission coding of the peripheral nociceptive information. The change of the specific gravity of the above cell types also suggests that the voltage-gated sodium channel regulator is unique to the induction of hyperalgesia. peripheral mechanism. The involvement of the spinal mTOR signaling pathway in mediating the induction of BmK I After subcutaneous injection of 10. m u.g of BmK I in the rat's foot, the mTOR and its downstream molecules p70S6K and 4E-BP1 in the L4-L5 lumbar segment of the spinal cord of the rat were both activated bilaterally; (2)1-2 hours reached the activation peak. The results showed that most of the activated mTOR and 4E-BP1 were co-labeled with the neuron marker NeuN, and a small amount of the GFAP co-labeled with the astrocyte marker, Iba-1, and the activated state p70S6K almost all. and (4) intraoral pre-injection of mTOR-specific inhibitor Rapamycin or CCI-779, both of which can be used to relieve spontaneous pain, unilateral thermal pain and bilateral mechanical pain induced by BmK I; it is interesting to (5) inject Ra in 2 hours after injection of BmK I pamycin, which can still be significantly inhibited The results suggest that the activation of mTOR signaling pathway is sensitive to the central nervous element induced by BmK I. The process is essential. The p38 signal path is involved in the BmK I induction. Subcutaneous injection of 10. m u.g of BmK I, (1), the activation of microglia in the spinal cord, can induce the L4-L5 segment of the spinal cord of the spinal cord of the rat. two-side activation of the microglia; (2) the activation of the microglia in the spinal cord is slightly later than that of the signal in the neuron, reaching the activation peak at 4-8 hours, and recovering at 1 day; and (3) the p38 in the same segment also exhibits similar two-sided timing Activation; immune double-label experiments show that (4) the activated state p38 is almost all coincident with the microglial cell marker OX-42; (5)30 minutes before the BmK I injection, the intraoral injection of p38-specific inhibitor SB203580 can be used to relieve the BmK I-induced rat in a dose-dependent manner. side-heat pain-sensitive and bilateral mechanical pain-sensitive; (6) microglia The activation of the cells is also inhibited. The results suggest that microglia may be involved in the mediation of B by the p38 signaling pathway. mK I-induced rat pain behavior. Selective inhibition of p38-dependent microglia activation can alleviate pain central sensitization of sensation, pain relief,4, BmK I induced The primary Somatosensory Cortex (SI) and the anterior cingulate cortex (ACC) of the rat primary somatosensory cortex (SI) and the anterior cingulate cortex (ACC), as well as the medial dorsal (MD) and the posterior nucleus of the thalamus (Ventral Posterior N), were selected for thalamic-cortical information integration and interaction. In this study,319 neurons were recorded, and BmK I induced by BmK I was studied. The results show that (1) BmK I-induced neuronal responses can be divided into three categories: continuous excitation, continuous type and resting type; (2) BmKI injection results in a change in the flow of information between the brain regions: the medial pathway the amount of information flow to the outer passage is significantly increased; A significant increase in the flow of information from the cortex to the thalamus. The results show that the high-level hub interacts with the information in the brain region Participation in the hyperalgesia induced by BmK I. The BmK I model is a simple model of the neuronal population reaction, perhaps
【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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