基于肠道乳糖酶对七味白术散治疗小鼠菌群失调腹泻的机理研究

Diarrhea is a common clinical symptom of many diseases. Qiweibaizhusan has a great effect on curing diarrhea. Intestinal enzymes, which are mainly secreted from animal and microbes, have significant impacts on the transformation functions in the gastrointestinal tract. Insufficiency of the intestinal lactase, which hydrolyzes lactose, is associated with many kinds of diarrhea. Our previous studies showed that lactose metabolism in mice was reduced by mixed antibiotics modeling. And lactose metabolism in mice with dysbacteriosis diarrhea was improved by Qiweibaizhusan. But the adjustment function and mechanisms of Qiweibaizhusan on intestinal lactase are not investigated. In order to investigate the curative mechanisms of Qiweibaizhusan upon dysbacteriosis diarrhea, this project will use enzymological techniques and molecular biological techniques combining with metagenome technology. The mouse model with dysbacteriosis diarrhea will be established by administrating mixed antibiotics, and then treated with Qiweibaizhusan. Intestinal content and mucosa will be collected respectively. The changes of intestinal lactase activity in the tested mice will be detected. Gene polymorphisms of the lactase from mice, bacteria and fungi will be analysed, respectively. The gene expression level of lactase from mice, bacteria and fungi will be measured, respectively. The relevance of intestinal lactase activity, gene polymorphism of lactase, and gene expression of lactase will be determined. The relationship between the curative effects of Qiweibaizhusan upon dysbacteriosis diarrhea and the intestinal lactase including activity, gene diversity and gene expession in the tested mice will be analysed. Outcomes of the project will provide experimental basis by using lactase to elucidate curative mechanisms of Qiweibaizhusan upon dysbacteriosis diarrhea. The research findings and methodologies may have a great significance in using disease related important gastrointestinal enzyme for establishing standardization technology of Chinese medicine diagnosis and treatment based on syndrome differentiation and elucidating curative mechanism of traditional Chinese medicine.

腹泻是许多疾病的常见症状。七味白术散对腹泻疗效显著。肠道酶来自动物及微生物，乳糖酶缺乏与多种腹泻相关。前期研究发现，抗生素造模降低小鼠对乳糖的代谢，七味白术散提高菌群失调腹泻小鼠对乳糖的代谢，其对乳糖酶的影响与机理未见报道。本项目拟运用抗生素制备菌群失调腹泻小鼠，灌胃七味白术散，分别采集肠道内容物及粘膜，将酶学、分子生物学和宏基因组技术结合，分析乳糖酶活性、（小鼠、细菌及真菌）乳糖酶基因多样性、（小鼠、细菌及真菌）乳糖酶基因表达量，从酶活、基因及基因表达等探析七味白术散对乳糖酶的影响机制。结合七味白术散对菌群失调腹泻的疗效，揭示七味白术散治疗菌群失调腹泻的机理。项目结果为利用乳糖酶阐明七味白术散治疗菌群失调腹泻的机理提供实验依据。项目研究成果与技术体系，对利用疾病相关重要肠道酶建立中医药辨证论治标准化诊疗技术及阐明中医药疗效机理意义重大。

七味白术散对多种腹泻疗效显著，乳糖酶缺乏与多种腹泻相关。通过动物实验，复制菌群失调腹泻模型，研究了抗生素及七味白术散对小鼠肠内容物与黏膜乳糖酶及蔗糖酶活性的影响，发现抗生素使小鼠肠道乳糖酶及蔗糖酶活性显著下降，七味白术散能显著提高腹泻小鼠的乳糖酶及蔗糖酶活性；研究了抗生素及七味白术散对腹泻小鼠乳糖酶基因13910位点多态性的影响，发现造模和治疗后，小鼠乳糖酶基因13910位点没有发生变异，说明乳糖酶活性与小鼠乳糖酶基因13910位点多态性无关；建立了细菌乳糖酶基因第二代高通量测序技术，发现造模小鼠肠道内容物细菌乳糖酶基因多样性明显降低，与正常小鼠差异显著，小鼠肠道黏膜细菌乳糖酶基因主要来源于变形菌门、放线菌门和部分未分类单元；发现七味白术散提高了肠道内容物Eggerthella、Acidovoorax、Stenotrophomonas等的丰度，降低了模型小鼠Fretibacterium、Serratia及Micromonospora等的丰度；七味白术散增加了肠道黏膜乳酸杆菌属、溶杆菌属和拟杆菌属等的丰度；发现汉逊德巴利酵母菌减少了肠道内容物细菌乳糖酶基因来源种类，改变了肠道内容物细菌乳糖酶基因来源群落结构，显著提高了Enterobacter、Modestobacter及Fretibacterium等的丰度；从肠道黏膜来看，与正常组比，模型组和酵母菌组嗜酸菌属和寡养单胞菌来源的丰度显著降低，而柠檬酸杆菌属、肠杆菌属和假单胞菌属来源的丰度显著升高，但模型组与酵母菌组差异不显著；研究了抗生素及七味白术散对小鼠及肠道细菌乳糖酶基因表达的影响，发现造模及七味白术散对小鼠及肠道细菌乳糖酶基因在肠道内容物和黏膜中的表达均差异不显著；运用16S rRNA第二代高通量测序技术分别研究了造模、七味白术散及酵母菌治疗对小鼠肠道内容物和黏膜细菌多样性的影响。发表论文40篇，其中SCI 12篇；学术交流16人次，学术报告4次；培养硕士生5名，博士生1名。研究结果利于阐明七味白术散对菌群失调腹泻疗效的机理，形成的技术体系，可用于探明中医药疗效的微生态学机理。