安宫牛黄丸改善出血性中风神经功能障碍的“肠—脑”交互作用机制

Hemorrhagic stroke, i.e. intracerebral hemorrhage (ICH), is very acute and has no effective treatment. Previous study confirmed there exist correlation between brain and intestinal injury after ICH. Angong Niuhuang Pill (AGNH) is a famous traditional Chinese medicine for ICH treatment. The current researches about the effects of AGNH on ICH pay only attention to brain instead of external cerebral pathway including gut. The results from our study showed that AGNH could protect gut barrier in ICH mice. AGNH could reduce the levels of blood LPS and inflammatory mediators. These findings indicated that the protective effects of AGNH on ICH may be related to regulating the gut-brain interaction. The present project will use ICH mice model and membrane barrier model in vitro to investigate the effects of AGNH on the impairments of blood-brain barrier (BBB) and brain-gut barrier. Garcia score and electroencephalogram were used to observe the neurological function after bleeding in different time points, and the changes of membrane barrier, the neurotransmitters and the inflammatory mediators will be evaluated at the same time, so as to elucidate the dose-effect relationship and time-effect relationship of AGNH. Then, the effects of AGNH on gut-brain interaction will be investigated by stimulating or blocking the sympathetic and parasympathetic nerve, regulating the levels of LPS and ammonia in blood, checking bacterial translocation with 16S rRNA sequencing detection. The present study aimed to elucidate the characteristics and mechanisms of AGNH attenuating the ICH injury by regulating gut-brain interaction. This project will provide a theoretical basis for traditional famous prescription (e.g. AGNH) being used to treat ICH. And it also supplies a new idea for researching and developing a strategy of treatment of ICH.

出血性中风（ICH）起病急，无有效治疗药物。研究证实ICH时的脑、肠损伤具有相关性。安宫牛黄丸（AGNH）为治疗ICH及昏迷的要药。现有研究只关注了AGNH治疗效应的“脑”作用，却忽视了其可能具有的“脑”外（如肠）作用途径。我们新近发现AGNH能保护ICH模型小鼠肠粘膜屏障，降低血LPS及脑组织炎症，提示AGNH治疗ICH的机制可能与其调节肠-脑通路作用有关。本课题结合小鼠脑出血和体外膜屏障（血脑与肠粘膜屏障）模型，以Garcia评分和脑电图等观察中风后不同时程神经功能状态，同时检测膜屏障、脑内外神经与炎症介质等的变化，明确AGNH神经保护作用的量效、时效关系，以及与膜屏障的关系和分子机制；进而通过干预交感、副交感神经，调节血LPS、血氨，检测脑、肠介质及肠菌易位等，明确AGNH改善ICH神经功能的肠-脑交互作用机制，为传统名方临床应用提供实验依据，为ICH的治疗提供新思路。

采用右侧基底节注射胶原酶法制备脑出血动物模型，给予不同剂量安宫牛黄丸（AGNH），Garcia test、错步实验、旷场实验评价神经功能；评价脑水肿程度及血脑屏障通透性；应用M受体、N受体阻断剂和AGNH，评价动物神经功能，肠、肺等重要器官病理损伤；GC-MS检测单胺类神经递质水平；FD4方法研究AGNH对肠黏膜屏障通透性的影响；ELISA法检测血液内毒素含量；观察AGNH对炎症因子及相关信号通路的影响；取动物肺、肝、脾、肠内容物、肠系膜淋巴结进行16sRNA测序；体外，培养Caco-2细胞，运用血清药理学方法研究AGNH对肠黏膜的保护作用和机制。结果显示，AGNH能够提高脑出血动物Garcia test评分，降低错步率，增加运动距离，表明AGNH能够明显改善脑出血动物神经功能；AGNH能够明显降低脑组织含水量，降低脑组织伊文思蓝含量，表明AGNH能够改善脑出血动物血脑屏障功能；阿托品、长托宁、甲基牛扁亭可减弱AGNH增高神经功能评分作用，阻断AGNH对脑出血小鼠肠、肺组织的保护作用，减弱AGNH降低血内毒素含量的作用；AGNH可降低脑出血动物血液NE水平、肠组织DA和5-HT水平；AGNH能够降低脑出血动物肠黏膜屏障通透性，减少血液中LPS含量，增加肠组织紧密连接蛋白的表达和血管活性肠肽的含量；AGNH可抑制脑出血动物肠道细菌易位。表明AGNH抗脑出血作用可能与其调节交感神经和副交感神经系统，减轻肠粘膜损伤，从而抑制内毒素及肠道细菌易位有关。AGNH血清能够提高LPS刺激Caco-2细胞的跨膜电阻，降低Caco-2细胞通透性，增加Caco-2细胞紧密连接蛋白表达；AGNH可降低TNF-α、IL-6含量及MEKK1、JNK和c-Jun磷酸化水平，表明AGNH对中风后肠粘膜具有保护作用，抑制肠炎症反应（机制与与炎症相关的MEKK1/JNK/c-Jun信号通路有关）；本课题明确了肠黏膜屏障与血脑屏障在AGNH改善出血性中风所致神经功能障碍中的作用，为其用于出血性中风急性期的治疗提供新的实验依据。通过研究AGNH对肠―脑交互作用的影响，为中医“肠脑相通”的经典理论和临床实践中出血性中风治疗的“肠脑并治”提供了支持。