肠道菌群-肠-脑轴功能稳态失衡介导吗啡镇痛耐受以及痛觉过敏的机制研究

Morphine is the most effective opioid for relieving moderate and severe pain at present, but long-term application of morphine can cause tolerance and addiction, which limits its clinical application. The dysregulation of microbiota-gut-brain axis plays a key role in many kinds of neurological diseases, but its role in morphine tolerance and hyperalgesia is still unclear. In our previous study, we found that the gut microbiota in morphine tolerance mice was disturbed, the intestinal permeability was significantly increased, and translocation of the intestinal bacteria happened. Morphine tolerance and neuroinflammatory responses in dorsal root ganglia and spinal cord were significantly reduced in mice treated with antibiotics cocktail or in toll-like receptor 2 knockout mice. Therefore, we proposed a hypothesis that dysregulation of microbiota-gut-brain axis plays a key role in morphine tolerance and hyperalgesia in mice. This project will employ high-throughput sequencing, molecular biology, flow cytometry, electrophysiology, animal behavioral testing, and other methods, 1) to clarify the mechanism underlying morphine-altered gut microbiota community and enhanced intestinal permeability and its association with morphine tolerance and hyperalgesia; 2) to clarify the mechanisms for neuroinflammation induced by morphine via gut microbiota products (e.g. LTA and short-chain fatty acids); 3) to elucidate the mechanisms underlying the hyperexcitability of dorsal root ganglia neurons induced by long-term morphine treatment. On one hand, our research results will shed light on the mechanisms underlying morphine tolerance and hyperalgesia at systemic level; on the other hand, we will provide new clues for the clinical management of morphine tolerance and hyperalgesia.

阿片类药物吗啡是目前缓解中重度疼痛的最有效药物，但长期应用会导致吗啡耐受和成瘾，而限制了其临床应用。肠道菌群-肠-脑轴功能稳态失衡在多种神经系统疾病中发挥关键作用，但是在吗啡耐受和痛觉过敏中的作用仍不清楚。我们前期研究发现，吗啡耐受小鼠的肠道菌群紊乱，肠道渗透性明显增加。吗啡耐受和痛觉过敏及神经炎症反应在抗生素处理或TLR2敲除小鼠均明显减轻。因此，我们的研究假设是：肠道菌群-肠-脑轴功能稳态失衡在吗啡耐受及痛觉过敏中起关键作用。本项目拟采用高通量测序、分子生物学、流式细胞技术、电生理学、动物行为学等方法，1）明确吗啡诱导肠道菌群紊乱及肠道屏障受损的机制及其与吗啡耐受的关联性；2）阐明肠道菌群产物LTA及短链脂肪酸参与吗啡诱导神经炎症的机制；3）初步阐明吗啡耐受导致痛觉神经元超兴奋的机制。本研究将在系统水平上阐明吗啡耐受和痛觉过敏的机制，有望为临床缓解吗啡耐受和痛觉过敏提供新的思路。

吗啡作为一种常用的阿片类镇痛药物，其主要毒副作用包括吗啡耐受、痛觉过敏、呼吸抑制、便秘和成瘾等。近年来随着吗啡的使用量的上升，应对吗啡副作用的药物也亟待开发。本项目围绕吗啡耐受引起的肠道菌群-肠-脑轴的稳态失衡进行深入研究，取得的主要成果如下：（1）吗啡耐受小鼠存在明显的肠道微生态失衡，肠道屏障被破坏。此外，移植吗啡耐受小鼠的肠道菌群可以加速正常小鼠吗啡耐受发展，而清除肠道菌群能缓解吗啡耐受进程；（2）采用抗生素来清除肠道菌群能够缓解吗啡耐受引起的背根神经节（DRG）的神经炎症，从而降低吗啡耐受小鼠DRG神经元的超兴奋性；（3）吗啡耐受小鼠DRG内发生TLR2依赖的巨噬细胞浸润及神经炎症。敲除TLR2能够改善吗啡耐受诱导的肠道微生态失衡，同时降低吗啡耐受小鼠DRG内巨噬细胞浸润和神经炎症；（4）吗啡耐受小鼠肠道菌群中次级胆汁酸合成相关菌群丰度发生改变，导致胆汁酸代谢稳态失衡，血清中脱氧胆酸（DCA）含量显著下降；（5）牛黄熊去氧胆酸（TUDCA）通过巨噬细胞上的TGR5受体缓解DRG神经炎症，进而缓解吗啡耐受的进展。然而，脱氧胆酸（DCA）通过巨噬细胞上的FXR受体释放内啡肽缓解吗啡耐受的进展；（6）脊髓小胶质细胞内未折叠蛋白反应（UPR）中的IRE1α通路参与小鼠吗啡耐受、吗啡痛敏和吗啡戒断引起的神经炎症，靶向抑制IRE1α通路能够缓解这些吗啡的副作用。综上所述，本研究发现吗啡耐受导致小鼠肠道菌群微生态失调，尤其是胆汁酸合成相关的肠道菌群改变导致胆汁酸稳态失衡，血清中DCA水平显著降低。补充次级胆汁酸明显抑制了DRG巨噬细胞浸润以及神经炎症，最终延缓了吗啡耐受的症状。最后，我们还阐明了脊髓小胶质细胞中的IRE1α参与小鼠吗啡耐受、痛敏和戒断的作用及机制。因此，本项目验明了肠道菌群-肠-脑轴稳态失衡在吗啡耐受中发挥重要作用，并且有望提供缓解吗啡耐受等副作用的新的治疗策略和新药研发的靶点。