钩吻素子抗糖尿病性神经病理性疼痛作用与其抑制脊髓小胶质细胞M1极化作用的关联机制

It is urgent to develop new durgs and find new drug targets for therapy of diabetic neuropathic pain (DNP). Recently, more and more attention has been focused on microglia M1 polarization for its role in DNP mechanism. Our pilot research have demonstrated that koumine could significant inhibit DNP with high efficiency and low toxicity. And it is suggested that the analgesic effects of koumine might be related with koumine’s ability to inhibit microglia M1 polarization. So firstly, our project is trying to explore the relationship between anti-DNP effects of koumine and its ability to inhibit microglia M1 polarization in spinal cord, by using microglia M1 polarization marker as index in vivo/in vitro. Secondly, we are going to illustrate the role of Notch-RBP-J signaling pathway in koumine’s anti-DNP effects and IRF8 modulation, which is essential in deep understanding the molecular mechanism ofkoumine’s analgesic function in DNP. At last, by observing protein expression and transcription ability of IRF8, we will systematically analyze IRF8 modulating pattern by which koumine inhibit microglia M1 polarization in vivo/in vitro, through which we could confirm critical pharmacology targets of koumine. This project not only provides new drug targerts and approaches for R&D of DNP potential durgs, but also helps deeply understand DNP mechanism, and establishes theory basis for koumine’s novel anti-DNP function.

防治糖尿病性神经病理性疼痛（DNP）新型药物以及新的药物作用靶标与途径亟待研究。近年来日益重视小胶质细胞M1极化在DNP发生中的作用。我们前期工作表明，钩吻素子具有高效低毒抗DNP作用，并且可能与其抑制小胶质细胞M1极化有关。本项目首先拟以小胶质细胞M1极化标志物表达为指标，在整体水平、细胞水平探索钩吻素子抗DNP作用与其抑制脊髓小胶质细胞M1极化的关联规律； 进而探讨钩吻素子抗DNP 作用以及其影响Notch-RBP-J 信号通路的关系；最后从IRF8 表达与功能方面，在整体与细胞水平深入分析钩吻素子如何通过Notch通路调节IRF8 而抑制小胶质细胞M1 极化，明确其作用的关键效应分子，以期较为系统地阐释钩吻素子抗DNP 作用的分子机制。本研究将为防治DNP新药研发提供新的药物作用靶标与途径，并有助于深化理解DNP的发生机制；也为提出钩吻素子抗DNP新功效奠定理论基础。

糖尿病神经病理性疼痛（diabetic neuropathic pain，DNP）是糖尿病的严重并发症之一，属于慢性疼痛。现有的医疗手段对于DNP不仅很难治愈，还有较大的副作用，亟待开发新型有效副作用少的治疗药物。本研究在链脲佐菌素（streptozocin，STZ）诱导的大鼠DNP模型上，观察钩吻素子抗DNP的作用与其抑制脊髓小胶质细胞M1极化的关联规律；进而阐释钩吻素子抑制小胶质细胞M1极化作用的分子机制，明确其作用的分子通路以及关键效应分子。研究发现，单次腹腔注射STZ溶液（70 mg/kg）建立SD大鼠Ⅰ型DNP模型，灌胃给予钩吻素子可以显著减轻糖尿病大鼠的机械痛敏，提示钩吻素子具有显著的抗DNP的作用，并具有剂量依赖性；运用RT-qPCR和western blot方法来检测大鼠脊髓小胶质细胞IRF8、小胶质细胞活化标志物iba-1及M1极化标志物CD86、CD68、TNF-α和IL-1β等mRNA和蛋白的表达水平，结果发现，钩吻素子可能通过下调IRF8的表达而抑制小胶质细胞M1极化，降低致炎细胞因子CD86、CD68、TNF-α、IL-1β等的表达，从而发挥抗DNP作用。观察脊髓鞘内注射Jagged 1对灌胃给予钩吻素子镇痛作用的可能拮抗作用，结果发现，钩吻素子可以抑制STZ诱导的DNP，但给予Jagged 1激活Notch信号通路后可拮抗钩吻素子的抗DNP效应，使痛敏增加，提示钩吻素子抗DNP的作用可能与其调控Notch信号通路有关。本文优先探索钩吻素子抗DNP作用与其抑制脊髓小胶质细胞M1极化作用的关联机制，明确其作用靶标；阐释其作用的分子机制以及关键效应分子，为防治DNP新药研发提供新的药物作用靶标与途径，并有助于深化理解DNP 的发生机制；也为提出钩吻素子抗DNP 新功效奠定理论基础，研究具有原始创新性及实用价值意义。