摘要

糖尿病（Diabetes）是一种以高血糖为主要特征的代谢类疾病，由其引发的一系列并发症对人体健康产生极大危害。目前临床上已有多种降血糖药物，但其控制血糖及并发症症状的效果尚有待提高，而且有的还伴有一定副作用。肠道菌群作为人体的“隐形器官”，与代谢性疾病的发生发展密切相关，补充益生菌与益生元能够通过对肠道菌群的正向调节改善糖脂代谢的紊乱。因此，深入挖掘肠道菌的功能并实现其精准干预对于改善代谢性疾病意义重大。    

由于肠道菌群种类众多，且绝大多数难以人工纯培养。此外，当前的测序技术对于低丰度的群体很难实现精准检测，导致目前的菌群研究多数只能从整体微生态的层面进行探究，很难深入到菌种/菌株水平来阐释其功能。因此，本研究将对课题组前期建立的人类肠道细菌库进行大规模的体外筛选实验，旨在多角度多层次的考察常见肠道细菌的降糖能力，并挑选出功效优良的菌株开展体内功效确认及降糖机制研究，同时考察具有益生元功效的中药苦瓜改善糖脂代谢紊乱的能力以及对肠道菌的调控作用。主要研究结果如下：

1. 大规模体外降糖活性筛选发现多种肠道细菌具有良好的降糖潜力

我们考察了1919株肠道细菌的培养物上清液对HepG2肝癌细胞糖消耗水平的影响。结果显示，有146株菌株具有较好促进糖消耗的效果。同时，利用糖摄取实验和α-糖苷酶抑制剂筛选实验进一步检测了146株菌促进HepG2细胞糖摄取和抑制α-葡萄糖苷酶和α-淀粉酶活性的能力，最终发现有22株菌表现出良好的综合降糖功效，主要分布在双歧杆菌属和乳杆菌属。对22株菌的降糖功效进行综合排序，发现植物乳杆菌、戊糖乳杆菌、干酪乳杆菌、唾液乳杆菌、青春双歧杆菌表现出显著且稳定的降糖作用。

2. 动物实验证明戊糖乳杆菌和干酪乳杆菌具有优良的降糖功效

我们挑选出体外降糖功效最强的7株菌进行体内药效学评价。分别灌胃给予正常小鼠和链脲佐菌素诱导的糖尿病小鼠活菌菌液2周和7周，检测糖脂相关指标，戊糖乳杆菌（B和C菌）和干酪乳杆菌（D菌）能够显著抑制正常小鼠餐后血糖水平和降低糖尿病小鼠空腹血糖与餐后血糖水平，促进胰岛素分泌，改善胰岛素抵抗。对于脂代谢情况，B、C、D菌显著降低糖尿病小鼠血清中甘油三酯水平和肝脏总胆固醇水平。进一步利用苏木精-伊红染色法对糖尿病小鼠胰腺和皮下脂肪组织进行观察，发现B、C、D菌能够改善胰岛形态，增加胰岛细胞数量，减小皮下脂肪细胞体积。

3. 分子药理实验揭示乳杆菌通过抑制肝脏糖异生和促进肠源性GLP-1的合成发挥降糖功效的机制

利用qRT-PCR实验检测糖尿病小鼠肝脏糖异生通路和结肠中胰高血糖素样肽-1（GLP-1）合成通路关键基因的mRNA转录水平，初步确定了B、C、D菌一方面可抑制肝脏糖异生Akt-Foxo1-Pepck / G6pase通路，另一方面可能通过激活Gpr41和Gpr43与短链脂肪酸（SCFAs）的结合，进而促进GLP-1的分泌，最终起到调节糖脂代谢的作用。ELISA法检测小肠内分泌STC-1细胞上清和糖尿病小鼠血清中GLP-1的含量也证明了B、C、D菌能显著提高GLP-1的分泌及入血能力。

4. 具益生元功效的苦瓜通过促进肠道SCFAs产生菌生长发挥降糖和降脂功效

灌胃给予高脂饲料喂养小鼠苦瓜粗提物（Momordica charantia L.，MC）4周，对苦瓜改善糖脂代谢的功效进行体内验证。并通过抗生素处理和粪便移植实验证明了肠道菌群在苦瓜调节血糖血脂过程中的关键作用。进一步对小鼠粪便菌群进行16S rDNA测序和SCFAs含量测定。结果表明，苦瓜可通过促进SCFAs产生菌生产有益于降糖降脂的SCFAs来发挥其改善糖脂代谢的功效。

综上所述，本研究首次在菌株水平上对肠道细菌的降糖功效进行全面扫描，并通过体内药效学评价，发现了2株戊糖乳杆菌和1株干酪乳杆菌具有明确的降糖功效，最后利用分子生物学方法对这3株菌改善糖尿病的作用机制进行探究，为肠菌来源的降糖药物开发奠定了基础。此外，本研究也以苦瓜为例证明了补充中药益生元可通过定向调节肠道菌群及其代谢物，从而改善糖脂代谢紊乱。

Abstract

Diabetes is a metabolic disease characterized by hyperglycemia. The efficiency of current hypoglycemic drugs is suboptimal and often accompanied by side effect. Gut microbiota is closely related to the occurrence and development of metabolic diseases, and microorganism is an important resources for drug discovery, so function of gut microbiome is worthy of in-depth exploration.

Most of gut microbiome are difficult to be cultured artificially, and metagenomic sequencing technology cannot detect low-abundance members. So the studies can only be explored from the level of overall microecology, and the specific functional bacterial species/strain against diseases are not clear. To solve this problem, our team has built up a large -scale human gut bacteria library. In this study, we assessed the hypoglycemic effect of human gut microbiota on a high-throughput screening platform, and then explored the anti-diabetic mechanism of hypoglycemic strains. At the same time, we also investigated the ability of Momordica charantia L., a traditional Chinese medicine with prebiotic effect, to improve glucose and lipid metabolism and its regulation on gut microbiota. The main results are as follows:

1. Large-scale screening of hypoglycemic gut bacteria showed that many kinds of intestinal bacteria have good hypoglycemic potential.

We investigated the effect of culture supernatant of 1919 gut bacterial/strains on glucose consumption in HepG2 cells. The results showed that 146 intestinal bacteria have potential hypoglycemic effect. Then glucose uptake test and α-glucosidase inhibit assay were used to further verify the ability of 146 strains to promote glucose uptake of HepG2 cells and to inhibit the activities of α-glucosidase and α-amylase. We found that 22 strains showed comprehensive hypoglycemic effect, mainly distributed in Lactobacillus pentosus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus saliva and Bifidobacterium adolescence.

2. Animal experiments showed that Lactobacillus pentosus and Lactobacillus casei have excellent hypoglycemic effect.

Seven strains of bacteria with the strongest hypoglycemic effect in vitro were selected for pharmacodynamic evaluation in vivo. Normal mice and streptozotocin-induced diabetic mice were given live bacterial solution by oral administration for 2 weeks and 7 weeks, respectively. The results showed that Lactobacillus pentosus (B and C) and Lactobacillus casei (D) could significantly decrease postprandial blood glucose in normal mice and reduce fasting blood glucose and postprandial blood glucose in diabetic mice. For lipid metabolism, B, C and D markedly decreased the level of serum triglyceride and liver total cholesterol in diabetic mice. Furthermore, B, C and D could improve the morphology of islets, increase the number of islet cells and reduce the volume of subcutaneous adipocytes.

3. Molecular pharmacological experiments revealed that the anti-diabetes effect of Lactobacillus attribute to inhibiting hepatic gluconeogenesis and promoting intestinal GLP-1 synthesis.

We detected the effects of B, C and D on the expression of critical genes in gluconeogenesis and glucagon-like peptide-1 (GLP-1) synthesis by Quantitative Real-time PCR. Bacteria B, C and D could inhibit AKT-FOXO1-PEPCK/G6Pase signalling pathway to regulate liver gluconeogenesis and promote GLP-1 expression via upregulation of short-shain fatty acid receptor GPR41/43 to regulate glucose and lipid metabolism. Finally, quantification of GLP-1 in STC-1 cell and serum shared that B, C and D could promote the synthesis and secretion of GLP-1 in vivo and in vitro.

4. Momordica charantia L., a traditional Chinese medicine with prebiotic effect, can improve glucose and lipid metabolism by promoting SCFAs- producing bacteria.

We fed C57BL/6J mice with high-fat diet to construct the hyperlipemic animal model, followed by oral administration of Momordica charantia L.(MC) for 4 weeks.  Results showed that MC has a good effect on improving blood lipid and glucose level. Antibiotic treatment and fecal transplantation experiments showed gut microbiome plays a key role in the anti-hyperlipidaemia and anti- hyperglycemia of MC. Furthermore, 16S rDNA sequencing and the content of SCFAs in faeces showed that MC can improve glucose and lipid metabolism by promoting the production of SCFAs.

Overall, this study scanned the hypoglycemic effect of intestinal bacteria at the strain level for the first time. Three functional strains with definite hypoglycemic effect were found and their mechanism to improve diabetes was explored, which laid a foundation for the development of hypoglycemic drugs derived from intestinal bacteria. In addition, we also took Momordica charantia L. as an example to prove that the supplement of traditional Chinese medicine with prebiotic effects can improve the disorder of glucose and lipid metabolism by directionally regulating gut microbiome and their metabolites.