胰岛素受体在背根神经节参与炎性痛的机制研究

Evidence for the presence of insulin receptor (InsulinR) has been demonstrated in nervous system. In rodents, InsulinR is selectively distributed in cerebral cortex, hippocampus and spinal cord. Moreover, rather than glia cells, the majority of the InsulinRs locate on neurons, suggesting a direct role of insulin signaling in neuronal activity and communication. However, there are fewer reports about the specific role of insulin signaling in dorsal root ganglion (DRG). Our previous results indicate that the InsulinR-positive neurons were mainly found to be small- (diameter ≤ 20 μm) and medium-sized (20 μm < diameter ≤ 40 μm) and co-expressed with transient receptor potential vanilloid 1 (TRPV1) channel, which is the key factor in inflammatory pain. Pre-incubation of insulin enhances intracellular calcium in TRPV1-positive neurons. Using a whole-cell patch-clamp method, we applied the TRPV1 agonist capsaicin to DRG small neurons in the presence of insulin. With repeated application, insulin potentiated the second capsaicin-activated current without affecting the first capsaicin-activated current. The insulin effect was extracellular calcium-dependent, and was attenuated by an inhibitor of the PI3K/Akt pathway (Wortmannin). Moreover, inflammatory pain leads to the increased expression of InsulinR in DRG, indicating an underlying relationship between inflammatory pain and InsulinR, but the mechanisms remain poorly understood. Based on these considerations, we sought to detect the role of InsulinR in DRG in inflammatory pain. First, we will determine the distinct distribution pattern of InsulinR and PI3K-Akt pathway in DRG; Second, the changes of InsulinR and PI3K-Akt pathway in DRG in inflammatory pain will be detected; Third, the underlying mechanism of InsulinR in inflammatory pain will be discussed in the project. We assume that the role of InsulinR may be associated with nociceptive neurotransmission in DRG, investigating the underlying mechanism in inflammatory pain in greater detail should be informative and may lead to novel intervention strategies.

在痛信息从外周传入中枢的第一站——背根神经节中明确参与炎性痛的关键分子对于寻找疼痛治疗的有效靶点具有重要意义。本课题组发现：免疫组化结果显示胰岛素受体在背根神经节与介导炎性痛的离子通道TRPV1共存于痛信息传递相关的中、小型神经元；钙成像和膜片钳实验结果提示胰岛素受体可能通过其下游最重要的PI3K-Akt通路影响TRPV1通道的活性；炎性痛动物模型背根神经节中胰岛素受体的表达水平显著增加；故我们提出：“胰岛素受体在背根神经节参与炎性痛的发病机制”的假说。本项目以炎性痛模型和TRPV1敲除小鼠为工具，在背根神经节运用多种方法进行以下研究：①胰岛素受体及PI3K-Akt通路在背根神经节的分布模式和神经化学性质；②炎性痛模型小鼠背根神经节中胰岛素受体及PI3K-Akt通路的变化；③胰岛素受体在背根神经节中参与炎性痛的电生理学机制。通过以上研究力争为炎性痛的诊疗提供新的理论依据。

炎性痛是临床上多见的疼痛性疾病，其主要特征是正常的无害刺激亦引起疼痛，给病人带来了极大的痛苦。背根神经节是痛信息从外周传递进入中枢的第一站，是炎性痛传递和整合的重要枢纽。炎性痛时，痛刺激会迅速激活神经内分泌网络，以应对组织修复和细胞代谢等变化。研究表明，胰岛素受体可通过影响神经元的功能和活性参与神经系统的功能和疾病。因此，本项目以背根神经节为研究靶点，采用多种研究方法，探讨胰岛素受体在背根神经节中参与炎性痛信息整合和传递的调控机制，力争为临床上炎性痛的诊治提供新的理论依据。我们的结果表明：① WT小鼠背根神经节中胰岛素受体mRNA与其蛋白表达类似，主要分布在与伤害性信息传递密切相关的中小型神经元中，且与肽能神经元标记物及无髓鞘神经元的标记物大量共存，且大量TRPV1与胰岛素受体共存，与WT小鼠相比，TRPV1 KO小鼠背神经节内胰岛素受体表达无显著差异；② TRPV1 KO小鼠热痛敏的潜伏期高于WT小鼠，而自发运动无差异。CFA足底注射后，WT小鼠出现机械痛敏和热痛敏，而TRPV1 KO小鼠无热痛敏，WT 小鼠和TRPV1 KO小鼠血清中胰岛素水平明显升高，且WT小鼠糖耐量低于TRPV1 KO小鼠。③ 在注射CFA后，WT小鼠背根神经节内胰岛素受体的蛋白表达水平明显增加，TRPV1表达量也随之增加，PI3K-Akt通路被激活。在急性分离培养的背根神经节神经元中，胰岛素可以显著增强TRPV1的表达，给予胰岛素受体拮抗剂S961或PI3K-Akt通路的抑制剂wortmannin可抑制胰岛素的效应。④ 膜片钳结果显示，胰岛素可以增强第二次给予辣椒素之后诱发的TRPV1电流，这种效应是依赖Ca2+离子内流和胰岛素受体及其下游PI3K-Akt通路的。钙成像也证实了胰岛素可显著增加辣椒素引起的TRPV1阳性神经元的钙离子内流，wortmannin和S961可逆转这一效应。我们的研究结果表明，炎性痛状态下，背根神经节神经元中的胰岛素受体通过下游PI3K-Akt通路或/和胞内的钙离子调控TRPV1离子通道的活性，进而参与炎性痛的产生和发展过程。