利用三代测序技术对酒精性肝病肠道菌群精准检测探索缺氧诱导因子与益生菌调控肠道屏障的研究

Previous studies indicated that probiotics prevented the liver injury from alcohol drinking, however, the mechanism is still unclear. The increased intestinal permeability, high level LPS in the blood and dysbiosis of intestinal flora are the three key features of ALD. Our previous studies demonstrated that LGG increased the level of HIF-2ɑ as well as its target genes and the current researches also pointed out that the stability and expression of HIF played an important role in maintaining the intestinal barrier function and the balance of bacterial flora. Thus, the present study, for the first time, will use the third generation of sequencing technology (16S rRNA) to evaluate the microbiota change in ALD. Moreover, it is also the first time to use intestinal specific HIF-1ɑ/2ɑ/1β knock out mouse model to unveil: 1) Whether alcohol drinking will induce intestinal barrier dysfunction, enhance Endotoxemia and liver injury via HIF pathway modulation; 2) The role of HIF in maintaining intestinal barrier integrity; 3) LGG will prevent the intestine and liver damage from chronic alcohol drinking via HIF pathway. We believe the current study will shed light on developing probiotics-based prevention and treatment of drug for ALD.

以往的研究表明，益生菌可预防及逆转酒精引起的脂肪性肝炎，但其保护机制尚不清楚。肠道通透性的增加，内毒素水平的升高和肠道微生态紊乱是酒精性肝病（ALD）的三个重要特征。我们前期研究表明，鼠李糖乳酸杆菌（LGG）在缓减ALD的过程中增加小鼠肠道HIF-2及其靶基因的表达水平，HIF的稳定表达对维持肠道屏障功能完整性和抑制细菌过度生长起着关键作用。本研究将首次使用第三代基因测序技术（全长16S rRNA扩增子测序）准确评估ALD模型中肠道菌群改变，也首次利用肠道特异性HIF-1ɑ/2ɑ/1β和VHL基因敲除小鼠研究:1、酒精是否通过干扰HIF通路导致肠道屏障的功能失调，增加内毒素血症和肝脏损伤；2、研究HIF通路在维持肠道屏障完整性的作用；3、益生菌LGG是否通过肠道HIF通路来保护慢性酒精诱导的肠道和肝脏损伤。本项目的完成对开发益生菌为基础的ALD防治药物提供了新的方向。

以往的研究表明，益生菌可预防及逆转酒精引起的脂肪性肝炎，但其保护机制尚不清楚。肠道通透性的增加，内毒素水平的升高和肠道微生态紊乱是酒精性肝病（ALD）的三个重要特征。我们前期研究表明，鼠李糖乳酸杆菌（LGG）在缓减ALD的过程中增加小鼠肠道HIF-2α及其靶基因的表达水平，HIF的稳定表达对维持肠道屏障功能完整性和抑制细菌过度生长起着关键作用。通过本次研究我们发现，肠道上皮细胞HIF-1α特敲小鼠在受酒精影响更严重，肝脏内脂肪变性和肝内大量炎性细胞浸润，促炎因子表达升高，肝功能明显下降，说明肠道中的HIF能够保护酒精作用下肝脏，抑制肝内脂质合成和炎症；当肠道上皮细胞缺乏HIF表达可导致肠道内菌群紊乱，益生菌乳酸杆菌减少，乳酸杆菌与阿克曼氏菌比例出现失调；肠道内菌群失调是由于上皮细胞的分泌的抗菌肽—β-防御素异常，肠道屏障受损，TJ蛋白、occludin和Claudin-1蛋白表达减少导致的；HIF能够改善抗菌肽的分泌和受损的肠道屏障，调节肠道菌群；恢复肠道菌群能够抑制酒精所致的肝内脂肪变性和炎症；LGG能够通过促进肠道上皮细胞表达HIF-α改善酒精对肝脏的影响；鼠李糖杆菌培养上清液联合骨髓间充质干细胞治疗能够调节NKB和TFH细胞之间的平衡减轻酒精性肝损伤，比单独治疗效果更好；通过服用铜可以通过调节肠道细胞的HIF表达和氧化应激，改善肠道屏障，减少酒精带来的肝损伤。本项目的完成对开发益生菌为基础的ALD防治药物提供了新的方向。