广藿香活性成分对 IBS-D 肠道平滑肌神经元递质释放的靶向调节机制

Diarrhea-predominant Irritable Bowel Syndrome(IBS-D) is one of most common functional bowel disorders. Incooperation with Harvard Medical School, we found that patchouli alcohol(PA)could regulates neurotransmission in the fundus of stomach and colon. Moreover, our group had found that Myosin Va could play an important role in the presynaptic release of neurotransmitters in enteric neurons.So we proposed the hypothesis that the mechanism of PA in the treatment of IBS-D is related to its regulation of microcircuit in enteric neural system by acting on neurotransmitter release via myosin Va. In this proposal, we will investigate (1)the changes of function of smooth muscle and related neurotransmissions in small intestine and colon via stress-induced IBS-D animal model; (2)the essential role of myosin Va and the effect of PA on myosin Va in neurotransduction signaling passway in small intestine and colon.To reach the goal,the following techniques including the intracellular microelectrod recording, molecular cloning, in situ PLA, gene transcription and protein expression of Myosin Va, Myosin Va gene knockout mouse, RNAi and CRISPR-Cas9 were applied. The aim of the present study is to elucidate the underlying mechanism of PA for treating IBS-D. If reaching the goal,we will get a new drug target protein and a lead compound which acts on target protein for the treatment of IBS-D.

腹泻型肠易激综合征（IBS-D）是一种常见的功能性肠病。我们与哈佛大学合作研究中发现：广藿香活性成分广藿香醇能减小胃肠平滑肌张力，非肾上腺素能非胆碱能依赖性地抑制肠神经传导。综合前期发现肌球蛋白Myosin Va在胃肠神经元突触递质释放中起重要作用等资料，课题组提出“广藿香醇通过作用于Myosin Va而抑制递质转运/释放过程，调节肠神经微环路，产生对IBS-D的治疗作用”的假说。本项目利用整体观察比较和细胞内记录、基因敲除、RNAi、CRISPR-Cas9等最新基因组编辑技术，从整体、器官、细胞和分子水平系统探讨（1）Myosin Va在IBS-D肠神经递质释放中的作用及其调控胃肠运动神经传导的分子信号通路机制;（2）广藿香醇对模型动物肠神经传导的影响及其对肠神经元Myosin Va的基因转录与蛋白表达的调控作用，目的是阐明广藿香活性成分治疗IBS-D的分子机制，以期发现治疗IBS新药。

在腹泻型肠易激综合征（IBS-D）发病中，肠神经元的递质转运与释放，以及神经可塑性、肠神经稳态等许多机制尚不十分清楚。研究发现肌球蛋白Myosin Va在胃肠神经元突触递质释放中具有的重要作用。本研究建立IBS-D模型动物，考察Myosin Va在IBS-D模型肠神经传导异常和稳态失衡中的作用；研究广藿香及广藿香醇对IBS-D模型动物肠神经元 Myosin Va的靶向调节机制。.结果：.1.建立慢性束缚IBS-D模型，对慢性束缚应激刺激量等参数进行改良，动态观察造模后不同时间点的粪便粒数、粪便面积、AWR评分、病理检查和远端结肠肌间神经丛（LMMP）神经元数目等，建立稳定可靠的IBS-D模型。并在此基础上观察了IBS-D模型神经传导和神经元稳态的变化。.2.广藿香对IBS-D模型的治疗作用：广藿香配方颗粒和广藿香醇（PA）改善慢性束缚应激IBS-D模型大鼠的内脏高敏感性和腹泻情况。.3.PA对IBS-D模型神经传导的作用：PA能显著上调模型组大鼠结肠nNOS mRNA水平，重塑结肠LMMP神经元及亚型数量、形态和功能；PA上调结肠LMMP的Myosin Va蛋白水平，增加Myosin Va与nNOS、VIP的共定位水平，促进结肠神经元NO释放。PA对结肠平滑肌自主收缩有抑制作用，还能抑制KCl、卡巴胆碱（CCh）和电场刺激（EFS）所致平滑肌收缩均可被PA抑制。.4.PA对Myosin Va缺乏的DBA小鼠的作用:研究发现PA对肠平滑肌舒缩的作用部分依赖于对氮能神经传导的调控，而PA对氮能神经传导的调控依赖于Myosin Va。.5.PA对Myosin Va表达的调控：基因功能验证中，PA对神经递质释放相关蛋白LC8、SLC17A9的调控依赖于对Myosin Va的调节；Myosin Va SiRNA能降低Myosin Va蛋白及相关下游蛋白水平，阻断PA的作用。Myosin Va 过表达慢病毒载体转染肠神经元后，PA对Myosin Va的表达影响不显著。.以上研究表明，广藿香醇作为广藿香治疗IBS-D的有效活性成分，能够恢复肠神经传导，其对NANC神经递质释放相关蛋白的调控依赖于对Myosin Va的精细调节。科学诠释中药治疗功能性胃肠病的机制，为指导中医临床有效防治IBS-D和研发创新药物提供实验证据和基础。