“胆汁酸-TGR5-NLRP3”通路调控背根神经节炎症反应缓解吗啡耐受的机制研究

Morphine is an important opioid analgesic for pain management. However, long-term application of morphine will lead to morphine tolerance, which greatly limits its clinical application. Recent studies have shown that peripheral dorsal root ganglion (DRG) play a critical role in the development of morphine tolerance. Gastrointestinal microbiota was shown to modulate neuroinflammation in the DRGs, which contributed to morphine tolerance, but the underlying mechanisms are unclear. Based on our previous metabonomics study, we found that the fecal metabolites secondary bile acids in morphine tolerant mice were significantly decreased, and activation of the bile acid receptor TGR5 attenuated neuroinflammatory responses and morphine tolerance. Therefore, we proposed a hypothesis that secondary bile acid activated TGR5 to modulate macrophage-neuron cross-talk in the DRGs, and suppressed neuroinflammatory responses and morphine tolerance. This project will employ metabonomics, gene knockout mice, neuron–macrophage coculture, calcium imaging, patch clamp and other methods, on multiple levers of gene, molecular, cell to whole animal, 1) to clarify the alteration of bile acid metabolism induced by morphine tolerance and the roles of gut microbiota. 2) to elucidate the role of TGR5 in effects of bile acid on morphine tolerance. 3) to investigate the mechanisms underlying interaction between macrophage and neuron regulated by TGR5 activation in the DRGs. On one hand, our research results will shed light on the mechanisms underlying morphine tolerance at metabolisic perspective; on the other hand, we will provide new basis and potential therapeutic targets for the clinical management of morphine tolerance.

吗啡是重要的镇痛药物，但长期使用易产生吗啡耐受，限制其临床应用。研究发现外周背根神经节（DRG）是产生吗啡耐受的关键部位，且肠道菌群参与DRG神经炎症及吗啡耐受，但机制不清。我们前期代谢组学研究发现，吗啡耐受小鼠肠道菌群代谢产物次级胆汁酸显著降低，激活胆汁酸受体TGR5可缓解吗啡耐受，并抑制DRG巨噬细胞浸润及神经炎症。我们推测：次级胆汁酸通过激活TGR5信号调控DRG巨噬细胞与神经元交互对话，降低神经炎症进而缓解吗啡耐受。本项目采用代谢组学、基因缺陷小鼠、细胞共培养及电生理等多手段，从基因、分子、细胞到整体动物多层次展开研究，拟探讨：1）吗啡耐受小鼠胆汁酸代谢改变及肠道菌群在其中的作用；2）TGR5受体介导胆汁酸缓解吗啡耐受的作用；3）TGR5受体调控DRG巨噬细胞-神经元相互作用的机制。本研究将从代谢调控神经炎症的新视角探讨吗啡耐受的机制，为临床干预吗啡耐受提供新的理论依据和治疗靶点。

慢性疼痛是众多疾病的常见临床表现之一，吗啡是治疗慢性中重度疼痛的常规用药，但反复应用会导致吗啡耐受。研究阐明吗啡耐受的发生机制，对治疗慢性疼痛和应对“阿片类药物危机”具有重要的意义。我们在先前的工作基础发现肠道菌群代谢产物次级胆汁酸可能调控DRG 神经炎症缓解吗啡耐受，但具体机制不清。因此，从代谢调控神经炎症的新视角阐明代谢产物次级胆汁酸调控吗啡耐受的分子机制，具有重要的科学意义和临床价值。本研究内容包括 1）明确吗啡耐受小鼠胆汁酸代谢改变及肠道菌群在其中的作用；2）阐明背根神经节中巨噬细胞-神经元相互作用的机制。我们通过代谢组学、16S rDNA测序，免疫组织荧光、流式细胞术、钙成像、电生理、单细胞测序及RNA测序等技术，发现长期使用吗啡后胆汁酸重塑，胆汁酸重塑参与背根神经节细胞类型及基因表达的变化。同时我们还发现1.TGR5在脊髓和DRG神经元中有表达，且DRG中TGR5阳性神经元主要分布在中小直径神经元上。2.TRPA1参与DCA导致的DRG神经元激活。3.TGR5基因敲除小鼠的焦虑样行为增加，疼痛、吗啡耐受、运动及抑郁样行为不变。4.TGR5介导了胆汁酸的镇痛，致痒及吗啡耐受的缓解，并且脊髓是胆汁酸的镇痛部位之一。本研究成果从代谢调控神经炎症的新视角提出肠道菌群通过胆汁酸调控吗啡耐受的新理论，胆汁酸及TGR5可能是临床干预吗啡耐受及镇痛的新治疗靶点。