基于“微生物-肠-脑”轴研究凉血通瘀方治疗脑出血的神经保护机制

It is the consensus in traditional and modern medicine that the severity of intracerebral hemorrhage is associated with constipation. Tongfu Xiere method is the important principle of treatment. Master of traditional Chinese medical science--Zhou Zhongying proposed a theory of Yure Zuqiao and founded Liangxue Tongyu prescription. The clinical, animal and in vitro experiments found that this prescription could relieve cerebral edema, reduce inflammatory mediator expression, protect vascular endothelial cells, and had nerve protective effect. But the inner link and the specific mechanism between Liangxue Tongyu prescription and nerve protection are unclear. Modern research proves that gut microbes affect the central nervous system through brain-gut axis neural pathways (neurotransmitters), immune pathways (cytokines), neuroendocrine pathways (gastrointestinal hormones) and direct communication (metabolites). Hence, this project proposes hypothesis--“Liangxue Tongyu prescription exerts nerve protective effects through the regulation of intestinal flora, intervening microbiome-gut-brain axis”. With autologous blood injection method of intracerebral hemorrhage model as the research object, by means of Illumina high-throughput sequencing analysis of the structure of intestinal flora, applying the neurobiology method to appraise brain damage, using molecular biology techniques, the purpose of this project is to verify that this prescription exerts nerve protective mechanisms related to microbiome-gut-brain axis. It is helpful for the prevention and treatment of intracerebral hemorrhage with intestinal flora. It is significant to enrich the theory of treating bowel to cure brain, provide experimental data and clinical guidance for this method treating for brain gut related diseases.

脑出血病情严重程度与腑实便秘密切相关，通腑泻热是重要的治疗法则。国医大师周仲瑛在此基础上提出瘀热阻窍学说，创立凉血通瘀方治疗。经临床、动物和体外实验发现可减轻脑水肿，降低炎性介质表达，保护血管内皮细胞，具有神经保护作用。但凉血通瘀方与神经保护之间的内在联系和具体机制尚不明确。现代研究发现肠道微生物可通过脑-肠轴神经途径(神经递质)、免疫途径(细胞因子)、神经内分泌途径(胃肠激素)以及直接交流(代谢产物)影响中枢神经。据此提出“凉血通瘀方通过调节肠道菌群，干预微生物-肠-脑轴，发挥神经保护作用”假说。以自体血注入法脑出血模型为研究对象，借助Illumina高通量测序解析肠道菌群结构组成，运用神经生物学方法评价脑损害，采用分子生物学技术验证本方神经保护作用与调控微生物-肠-脑轴有关，探索以肠道菌群为靶点防治脑出血的新思路，丰富上病下取、脑病治肠理论，为治疗脑肠相关疾病提供实验资料和临床指导。

脑出血病情严重程度与腑实便秘密切相关，通腑泻热是重要的治疗法则。国医大师周仲瑛在此基础上提出瘀热阻窍学说，创立凉血通瘀方治疗。前期经临床、动物和体外实验发现可减轻脑水肿，降低炎性介质表达，保护血管内皮细胞，具有神经保护作用，但凉血通瘀方与神经保护之间的内在联系和具体机制尚不明确。 .本项目以自体血注入法制作脑出血模型，借助Illumina高通量测序解析肠道菌群结构组成，运用神经生物学方法评价脑损害，采用分子生物学技术探索凉血通瘀方神经保护作用与调控微生物-肠-脑轴有关。结果显示本方可恢复肠道微生物菌群丰富度，促使肠道微生物分布均匀，拟杆菌逐渐减少，厚壁菌逐渐增多，恢复肠道菌群正常构成。进一步合成神经递质（去甲肾上腺素、多巴胺、谷氨酸和脑源性神经营养因子），调控宿主神经系统；改变促炎、抗炎细胞因子（Toll样受体-4、白介素-1β和白介素-6）水平，影响中枢神经；作用于肠道内分泌细胞，调节脑肠肽（胃泌素、胃动素和胆囊收缩素）作用于脑；调节肠道菌群代谢产物（一氧化碳、一氧化氮和硫化氢），直接与中枢神经系统进行交流。证实本方可改善脑出血大鼠神经功能缺损、促进血肿吸收、减轻脑水肿和改善血脑屏障通透性。采用“抗生素鸡尾酒法”制作无菌大鼠模型，反证本方通过肠道微生物发挥神经保护作用。.通过研究证实凉血通瘀方调节肠道微生物通过脑-肠轴神经途径（神经递质）、免疫途径（细胞因子）、神经内分泌途径（胃肠激素）以及直接交流途径（代谢产物）影响中枢神经。明确凉血通瘀方可减轻脑损害，发挥神经保护作用，丰富上病下取、脑病治肠理论；初步阐明凉血通瘀方通过调节肠道菌群，影响微生物-肠-脑轴，发挥神经保护作用，有利于以肠道菌群为靶点预防治疗脑出血，为中医学传统治法治疗中风等脑肠相关疾病提供现代科学依据。