基于肠道菌群探讨睡眠剥夺大鼠生物节律改变的机制及丹栀逍遥散的干预作用研究

It is well known that staying up late does harm to our health. However, it remains unestablished how it works. It has been reported that sleep deprivation can result in circadian rhythm disorders and changes in the diversity of the gut microbiota, while many cognitive and behavioral diseases are mediated by gut microbiota, which can be ameliorated via restoring the microbiome composition. The major syndrome that Danzhi Xiaoyao powder cures corresponds to common symptoms in patients with insufficient sleep, thus Danzhi Xiaoyao powder has clinically significant efficacy. Therefore, we presented the hypothesis: the disorganized microbiome composition may mediate circadian rhythm disorders induced by sleep deprivation, whose underlying mechanism relates to regulation of circadian clock; circadian rhythm disorders may recover via restoring the microbiome composition, and Danzhi Xiaoyao powder works on circadian rhythms by gut microbiota probably. This issue aims to clarify the key role of gut microbiota in abnormal circadian rhythms caused by sleep deprivation and its impact on regulation of circadian clock using fecal microbiota transplantation, 16S rRNA gene sequencing analysis, Real -time quantitative PCR and Western Blot, as well as to verify how Danzhi Xiaoyao powder regulates circadian rhythm disorders. This will provide preclinical evidence for seeking new target in improving circadian rhythm disorders and experimental proof for Danzhi Xiaoyao powder applied to treatment in dysrhythmia.

熬夜会给人体健康带来诸多问题，但其具体机制尚未完全明确。既往研究发现睡眠剥夺会引起生物节律及肠道菌群结构紊乱，而肠道菌群介导许多中枢认知行为异常的发病机制，改善肠道菌群的结构有助于治疗神经和精神疾病。临床上，丹栀逍遥散的主治证候与长期睡眠不足人群的常见证型症状相符，其临床疗效甚佳。因此，我们提出科学假说：肠道菌群结构异常介导睡眠剥夺引起的生物节律紊乱，其内在机制与生物钟调控机制相关，重建正常肠道菌群结构可改善生物节律紊乱，丹栀逍遥散通过调节肠道菌群改善生物节律。本项目拟采用粪菌移植、16s rRNA测序方法、实时荧光定量PCR法和Western Blot法等技术，旨在阐明肠道菌群在睡眠剥夺引起生物节律紊乱的关键作用和对生物钟调控机制的影响，及丹栀逍遥散干预生物节律紊乱的作用机制，为寻找生物节律紊乱治疗新靶点提供科学依据，及为丹栀逍遥散在睡眠等生物节律相关疾病中的临床应用提供实验依据。

现有的研究表明，睡眠剥夺会引起生物节律及其调控机制紊乱，也会导致肠道菌群结构组成改变，而肠道菌群介导多种中枢认知行为异常的发病机制，改善肠道菌群的结构组成有助于治疗神经和精神疾病，而经典明方丹栀逍遥散亦广泛应用于睡眠障碍且疗效显著。因此本研究尝试探索肠道菌群对生物节律的影响及其内在生物钟机制，以及丹栀逍遥散对改善生物节律及肠道菌群结构的干预作用。. 为此，本研究建立了睡眠剥夺大鼠模型，比较睡眠剥夺大鼠和环境对照大鼠生物节律和肠道菌群结构组成的差异；将睡眠剥夺大鼠粪菌移植到正常大鼠，比较正常对照大鼠、正常粪菌移植大鼠、睡眠剥夺粪菌移植大鼠生物节律和肠道菌群结构组成的差异。采用粪菌移植方法，对睡眠剥夺大鼠重建正常肠道菌群结构，观察其生物节律和生物钟调控机制的改善。予睡眠剥夺大鼠、睡眠剥夺粪菌移植大鼠丹栀逍遥散灌胃干预，观察丹栀逍遥散对大鼠肠道菌群结构组成和生物节律及其调控机制的改变，探讨丹栀逍遥散对睡眠剥夺的干预作用。. 研究结果提示，睡眠剥夺可能引起大鼠生物节律紊乱，推测与视交叉上核Cry2、Rev-erba基因表达升高相关，同时睡眠剥夺会导致大鼠肠道菌群多样性下降及物种组成改变。而肠道菌群紊乱，可能足以引起生物节律的紊乱，与视交叉上核Per2、Cry1、Rev-erba基因表达下降可能相关。重建正常的肠道菌群结构，可能有助于改善生物节律紊乱。丹栀逍遥散可能具有下调视交叉上核Per1、Per2、Cry1、Rev-erba基因表达的作用，并具有增加肠道菌群物种多样性，和改变物种组成的作用。未来将需要更深入的基础研究，进一步明确生物节律的调控机制。