鼠李糖乳酸杆菌在预防和治疗酒精性肝病中的机理研究

Severe alcoholic liver disease (ALD) has a high morbidity and mortality. Recent studies demonstrated that probiotics reversed alcohol-induced hepatic steatosis and inflammation, and improve liver enzymes in animal models and in patients. Our laboratory showed that administration of a probiotic strain, Lactobacillus rhamnosus Gorbach-Goldin (LGG), significantly improved liver enzymes and histology in alcohol treated mice. However, adverse events have been noted with probiotic use. In addition, probiotics may not be always effective in intestinal disorders with altered epithelium, as the bacteria must colonize intestine to be effective. This colonization may be impaired with illness and medications (especially antibiotics). As an alternative, probiotic culture supernatant and dead probiotics have been used in recent studies for intestinal disorders. Our preliminary data showed that LGG supernatant administration protected the liver against acute-alcohol exposure-induced injury. However, the beneficial mechanisms of action of LGG, either in viable bacteria form or in culture supernatant form, are not clear. Our overall hypothesis is that LGG increases intestinal short chanin fatty acids (SCFAs), intestinal trefoil factor (ITF) and cathelin-related antimicrobial peptide (CRAMP) expression leading to improved gut microbiome homeostasis, intestinal barrier integrity and hepatic fat metabolism, and these are likely mechanisms by which LGG supplementation attenuates alcohol induced liver injury. We evaluate mechanisms of LGG action with 4 specific aims: Specific Aim 1 will evaluate and compare the effectiveness of viable LGG, heat-inactivated LGG and LGG supernatant in the prevention/therapy of ALD in mouse models. We will determine whether secreted factors of LGG are responsible for the beneficial effects of LGG in the prevention/treatment of ALD in two ALD mouse models. Specific Aim 2 will determine the role of SCFAs in the beneficial effect of LGG in ALD. We will perform a targeted metabolomic study to analyze the changes in SCFAs induced by alcohol and LGG in the intestinal lumen. We will then explore the link between altered levels of butyrate and hepatic HDAC3 expression and hepatic fat metabolism regulators. Lastly, we will test our hypothesis that the beneficial effects of LGG is via activation of butyrate by using butyrate receptor knockout mice. Specific Aim 3 will determine the role of CRAMP in alcohol and LGG-mediated changes in gut microbiota using a metagenomic approach and CRAMP knockout mice .Specific Aim 4 will determine the role of LGG in promoting ITF expression and secretion leading to improved intestinal mucus barrier integrity by using intestinal-specific ITF knockout mice. This study will have a major impact on the development of probiotic-based new therapeutic strategy for the prevention and treatment of ALD.

近期的研究表明在动物模型及临床患者中使用益生菌可以逆转酒精引起的脂肪性肝炎，改善肝功能。我们的研究表明鼠李糖乳酸杆菌 GG (LGG)以及培养上清液显著性地改善了酒精引起的小鼠的肝功能损伤及肝脏组织学表象。但是这种保护作用的机制尚不清楚。我们假设LGG增加了肠道的短链脂肪酸的产生，小肠三叶因子（ITF）以及抗菌肽（CRAMP）的表达导致了肠道菌群的改善，肠道屏障的完整性以及肝脏脂肪的代谢，这很可能是LGG对酒精性肝病其保护作用的机制。本项目的主要研究内容：1.研究和比较三种不同的LGG制剂对预防和治疗酒精肝的作用；2.研究短链脂肪酸尤其是丁酸在LGG对酒精引起的肠道代谢紊乱及肝脏脂肪堆积调节中的作用；3.研究CRAMP在酒精和LGG对肠道菌群调节中的作用；4.研究ITF在酒精和LGG对肠道屏障完整性中的作用。本项目的完成将对发展益生菌为基础的对酒精性肝病的预防和治疗提供新的方向。

研究表明益生菌可以逆转酒精引起的脂肪性肝炎，改善肝功能。我们研究表明鼠李糖杆菌GG（LGG）以及培养上清液（LGGs）显著的改善了酒精引起的小鼠肝功能损伤及肝脏组织学表象。因此，我们通过利用正常小鼠、Cathelin相关抗菌肽(CRAMP)全身特异性基因敲除鼠和缺氧诱导因子（HIF）-1α肠道特异性基因敲除鼠建立多种酒精性肝病动物模型并结合体外细胞实验，研究和比较不同的LGG制剂对预防和治疗酒精性肝病的作用机制，研究发现：1、添加LGGs到酒精性饮食中显著降低了酒精引起的肝脏脂肪堆积；2、LGGs处理可以增加肠道短链脂肪酸的含量；3、LGGs可以抵消酒精饲喂引起的肠道CRAMP表达量下降，CRAMP全身特异性基因敲除后可以显著加重酒精引起的肝损伤，并通过体外实验进一步证实CRAMP可以显著降低LPS诱导的炎症反应；4、LGG对酒精性肝病的有益作用是由缺氧诱导因子（HIF）-1α来介导的。本课题系统的探讨了LGG在预防和治疗酒精性肝病中的重要作用，并深入剖析了其作用机制，为临床上预防和治疗酒精性肝病提供更完整的理论依据和潜在的预防、治疗手段。