轻型肝性脑病相关的肠道微生物宏基因组深度测序分析

Hepatic encephalopathy (HE), as metabolic disease, is a disorder of mental activity, neuromuscular function and consciousness that occurs as a result of either chronic or acute liver failure. This complex neuropsychiatric syndrome is primarily caused by metabolic abnormalities, possibly associated with the change of gut microbiome. Hence, around current research hotspot "whether there are associations between the changes in human microbiome and health/disease", this project will perform three researches for widely revealing the potential association between gut-microbiome-associated human metabolic disorder (e.g. ammonia metabolism) and HE occurrence. Firstly, by using an unbiased deep metagenomic sequencing of gut microbiome, we will broadly compare the difference of gut microbiome structure (composition and variation at different taxonomic level) and function (the diversity of gene and their associated metabolic network) in ten cirrhosis patients with MHE and their healthy relatives, as well as ten cirrhosis patients without MHE. Secondly, we will identify specific gut bacteria closely related to MHE by co-occurring network analysis of gut microbiota and then analyze the correlation between their changes and human metabolic phenotype (e.g. blood ammonia). Finally, we will confirm our above analyzed results by large-scale molecular analyses of gut bacteria 16S rRNA genes in twenty cirrhosis patients with MHE and their healthy relatives, as well as twenty cirrhosis patients without MHE. In a word, the major task of this project will provide basic information for exploring the mechanism of HE occurrence and further provide some foundations for the prevention, diagnosis, and treatment of HE in the coming day.

肝性脑病（HE）是严重肝病引起的、以代谢紊乱为基础的中枢神经系统功能失调的代谢性疾病；肠道微生物改变与HE发生有关。为此，本项目将围绕"人体共生微生物与疾病的关系"这一国际研究热点，从两个方面全面探讨肠道微生物组相关的人体代谢失调（特别是氨代谢）与HE发生具有潜在相关性。首先，通过肠道微生物宏基因组深度测序分析，系统比较肝硬化患者(伴随MHE)及其健康家属、肝硬化患者（非MHE）各10人的肠道微生物组结构（指细菌不同分类水平的组成和变异）和功能（基因多样性和代谢网络多样性）的差异；其次，通过肠道微生物共变异网络分析，鉴定与MHE发生相关的肠道特定细菌类群，分析特定细菌类群的变化与人体代谢表型（如血氨等）的相关性；最后，在各增加20人基础上，对以上分析结果辅以细菌16S rRNA基因大规模分子分析验证。本项目将为探讨HE发生机制提供最根本的信息，并为今后HE预防和诊治奠定一定的基础。

轻微型肝性脑病（MHE）是一种神经认知功能紊乱的代谢性疾病、是肝性脑病发病的起始阶段。肝性脑病的发生是肝硬化患者死亡的主要原因之一，从肝性脑病发展历程（肝硬化→MHE→症状性肝性脑病）来看，MHE阶段的诊断和治疗对缓解肝硬化患者的死亡率非常关键。如何破译肝性脑病患者肠道微生物组结构和功能，对于我们探讨肝性脑病发生机制将具有重大意义。轻微肝性脑病相关的肠道微生物群研究结果显示：1.肝硬化MHE的发生与肠道产氨表型细菌增加有关，MHE患者中产氨的肠道唾液链球菌较不合并MHE的患者显著增高；2.与肝硬化非MHE患者相比，MHE组肠道微生物群的生物多样性发生了明显改变；与健康对照组比，MHE组与肝硬化组患者的肠道菌群多样性均降低了。3.MHE组患者肠道微生物群的丰度和物种数较健康对照组和肝硬化组降低。4. MHE组优杆菌属和苏特雷氏菌属缺失，而普雷沃氏菌属、肠杆菌属等革兰氏阴性菌增多。轻微型肝性脑病肠道菌群调节治疗前后相关的肠道微生物组结构与功能分析研究结果显示：1、MHE患者的肠型（enterotypes)有三个类型，其中两个肠型不同于正常人的肠型。2、联合使用利福昔明和益生菌可显著性影响肝性脑病患者的肠道菌群的代谢功能。3、单独使用利福昔明或联合使用利福昔明和益生菌均可显著性降低肝性脑病患者外周血血氨浓度。我们的研究第一次全面系统的从肠道微生物组成、结构和独特的相互作用方面解析了肠道微生物与MHE发生的相关性。由宿主自身选择导致的肠道微生物的增加和缺失与肝硬化并发MHE具有密切关系。MHE患者所特有的含尿素酶的唾液链球菌的增加,以及该细菌间的相互作用是导致MHE患者肠道微生态失衡和血氨增高的原因。与MHE发生密切相关的肠道微生物之间的互利共生和竞争关系使我们深刻理解了为什么肠道菌群紊乱会导致肝硬化患者并发MHE，为将来MHE患者的诊断、预防、治疗提供了重要的生物和医学证据。以上研究目前已正式发表相关论文5篇(标注资助)，其中1篇SCI论文（标注资助），主编专著2部，通过该课题研究已培养硕士2人，博士1人，课题组成员参加国内学术会议3人次。