肠道菌群参与小鼠甲基苯丙胺成瘾的机制研究

In our previous study, we found that chronic methamphetamine(METH) administration reduced intestinal microbial flora diversity of mice and upregulated expression of TNF-α in the nucleus accumbens (NAc) of mice. However, the mechanism between METH-induced diversification of gut microflora and METH addiction remain unclear. In the present study, C57BL/6 and dopamine D3 receptor knockout (D3R -/-) mice were used as the subjects to experience a chronic METH administration regimen which including an escalating dose of drug use for one month, two weeks’ withdrawal and a challenge dose finally. The changes of gut microbiota and their metabolite of mice in response to METH are analysed via 16S rRNA gene sequencing and NMR technique. qPCR, Western blotting and ELISA are used to measure the expression changes of addiction-associated genes (D3R, GABAR, BDNF) and inflammatory factors in the NAc of mice in response to METH. Base on these studies, we will further clarify the relationship between METH addiction and intestinal microflora. Furthermore, antibiotics and short chain fatty acids(SCFA) are given throughout three phases of METH administration to regulate the intestinal microflora of mice. The effects of regulating intestinal flora on the addictive behaviors and the related genes and inflammatory factors in METH addictive mice are observed, and the regulatory role of D3R involved in it is also determined. This research will reveal the role of regulating intestinal microflora in the treatment of METH addiction, and lay a theoretical foundation for further clarifying the mechanism of METH addiction and opening a new treatment method.

我们前期发现，甲基苯丙胺（METH）在上调伏隔核TNF-α表达同时，还可以降低小鼠肠道菌群多样性，但METH引起的肠道菌群改变是否参与METH成瘾及其作用机制尚不清楚。本项目拟以C57BL/6小鼠及多巴胺D3受体（D3R）基因敲除小鼠为研究对象，采用16srDNA扩增子测序和NMR技术，检测METH对成瘾小鼠肠道菌群和肠道菌群代谢产物的作用；利用qPCR、Western blotting和ELISA等方法，检测成瘾小鼠大脑伏隔核D3R、GABAR、BDNF等基因以及炎症因子表达变化的规律，进一步明确METH成瘾和肠道菌群之间的关系。通过给予抗生素和短链脂肪酸干预小鼠肠道菌群，观察肠道菌群改变对METH成瘾小鼠成瘾行为及伏隔核相关基因与炎症因子的作用，确定D3R在其中参与的调节作用。探讨通过调节肠道菌群干预治疗METH成瘾的作用，为进一步明确METH成瘾的机制及开辟新的治疗方法奠定理论基础。

我们给予慢性递增剂量的甲基苯丙胺（Methamphetamine，METH）处理小鼠模拟人的药品滥用模式、并建立了METH诱导的条件性位置偏好（Conditioned place preference，CPP）形成和METH戒断小鼠模型，发现上述METH给药模式均导致小鼠的肠道菌群发生显著改变，尤其是与短链脂肪酸（Short-chain fatty acids，SCFAs）生成相关菌群的丰度下降。在慢性递增给药模式，抗生素（Antibiotic，ABX）干预影响METH暴露小鼠的运动活性并促进炎症，而SCFAs可能调节METH诱导的行为和肠道菌群变化。METH诱导的CPP形成小鼠盲肠内容物和血清中的SCFAs浓度减少，因此我们推测METH作用与SCFAs密切相关。在探索了不同SCFAs处理方式后，发现经过SCFAs预处理两周并在METH给药期间持续给予SCFAs，可以改善METH诱导的小鼠CPP形成、兴奋性增高以及认知记忆破坏，还可以改善METH戒断引起的认知记忆破坏、社交破坏以及抑郁样行为。同时还发现SCFAs干预可以改善METH导致的菌群变化、盲肠内容物SCFAs浓度减少以及结肠黏膜完整性和连接性的改变，提示SCFAs对METH引起的行为变化有显著的改善作用，且与肠结肠黏膜完整性密切相关。最后，我们构建了SCFAs干预METH诱导CPP形成小鼠伏隔核（Nucleus accumbens，NAc）的单细胞转录组表达谱，发现SCFAs可以改变METH导致的NAc中多种细胞类型、数量及基因表达变化。而且基于SCFAs可以纠正METH作用对小胶质细胞亚群和代谢通路的影响，我们推测SCFAs可能通过改变小胶质细胞亚群的脂质代谢，尤其是花生四烯酸（Arachidonic acid，AA）代谢改善METH的作用。本研究探讨METH作用下肠道菌群的改变，以及口服SCFAs干预对METH作用小鼠的影响及其机制，为发现潜在的治疗靶点提供了理论基础。项目原计划目标基本完成：发表SCI论文4篇；为本学科培养博士研究生1名（来思敏），硕士研究生1名（李国栋）；在研期间邀请1名（张遐）国外本领域的知名专家指导交流，合作发表SCI论文2篇。通过本项目研究工作的实施，课题研究团队之间的协同促进作用得到明显加强，研究能力和水平进一步得到提升，为深入研究药物依赖机制和防治打下了坚实基础。