Mechanisms of Probiotics in Alcoholic Liver Disease

 描述(申请人提供)：严重酒精性肝病(ALD)具有很高的发病率和死亡率。最近的研究表明，益生菌可以逆转酒精诱导的肝脏脂肪变性和炎症，并在动物模型和患者中改善肝酶。我们的实验室表明，给予益生菌菌株鼠李糖乳杆菌(LGG)，显著改善酒精处理小鼠的肝酶和组织学。然而，无论是以活菌形式还是以培养上清液形式，LGG的有益作用机制尚不清楚。我们的总体假设是，补充LGG可增加肠道排泄素相关抗菌肽(CRMP)、肠三叶因子(ITF)的表达和肠道短链脂肪酸(SCFA)的表达，从而改善肠道微生物区系的动态平衡、肠道屏障完整性和肝脏脂肪代谢，这可能是补充LGG减轻酒精性肝损伤的机制。目的1确定痉挛在酒精引起的肠道微生物区系改变中的作用，以及痉挛在LGG对实验性ALD的有益作用中的作用。我们将使用ALD的抽筋基因敲除小鼠模型，利用元基因组学方法来确定抽筋和LGG在酒精诱导的肠道微生物区系变化中的作用。目的2确定LGG在促进ITF表达中的作用，并评价ITF对实验性ALD肠道紧密连接的积极调节作用作为改善肠屏障完整性的机制。我们将使用一种分泌ITF的杯状细胞株来评估IgG在ITF产生中的作用以及ITF的作用 在调节上皮细胞系中的肠道紧密连接。我们还将研究口服重组人ITF减轻酒精诱导的肠道屏障和肝脏损伤的策略。目的3确定丁酸在LGG改善实验性ALD肠管紧密连接中的作用及其机制。代谢组学方法将被用来分析酒精和LGG干预引起的肠腔内SCFA的变化。我们将探索丁酸在肠组蛋白脱乙酰基酶(HDAC)、miR122a和HIF-介导的体外和体内信号转导中调节occludin表达的潜在机制。目的评价和比较免疫球蛋白上清液(LGG)、活性免疫球蛋白(VLGG)和热灭活免疫球蛋白(HiLGG)对小鼠酒精性肝病的防治作用。这三种免疫球蛋白制剂将在脂肪变性、脂肪性肝炎和纤维化的三种ALD实验模型上进行预防/治疗ALD的有效性测试。这项研究将对以益生菌为基础的预防和治疗酒精性肝病的新治疗策略的开发产生重大影响。

 DESCRIPTION (provided by applicant): Severe alcoholic liver disease (ALD) has a high morbidity and mortality. Recent studies demonstrated that probiotics reversed alcohol-induced hepatic steatosis and inflammation, and improved 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, 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 cathelicidin-related antimicrobial peptide (CRAMP), intestinal trefoil factor (ITF) expression and intestinal short chain fatty acids (SCFAs), leading to improved gut microbiota homeostasis, intestinal barrier integrity and hepatic fat metabolism, and these are likely the mechanisms by which LGG supplementation attenuates alcohol induced liver injury. We evaluate mechanisms of LGG action with 4 specific aims: Aim 1 will determine the role of CRAMP in the alcohol-mediated changes in gut microbiota and the role of CRAMP in the beneficial effects of LGG in experimental ALD. We will use CRAMP knockout mouse model of ALD to determine the role of CRAMP and LGG in alcohol-induced changes in gut microbiota using a metagenomic approach. Aim 2 will determine the role of LGG in promoting ITF expression and evaluate whether ITF positively modulates intestinal tight junctions as a mechanism for improved intestinal barrier integrity in experimental ALD. We will use an ITF-secreting goblet cell line to evaluate the role of LGG in the production of ITF and the role of ITF in the modulation of intestinal tight junctions in an epithelial cell line. We will also examine th strategy that oral administration of recombinant human ITF attenuates alcohol-induced intestinal barrier and liver injury. Aim 3 will determine the role and mechanisms of butyric acid in the beneficial effects of LGG on intestinal tight junctions in experimental ALD. A metabolomics approach will be used to analyze the changes in SCFAs induced by alcohol and LGG intervention in the intestinal lumen. We will explore the potential mechanisms underlying the role of butyric acid in the regulation of occludin expression involving intestinal histone deacetylases (HDACs), miR122a, and HIF--mediated signaling in vitro and in vivo. Aim 4 will evaluate and compare the effectiveness of LGG supernatant (LGGs) with viable LGG (vLGG) and heat-inactivated LGG (hiLGG) in the prevention/therapy of ALD in mouse models. These three LGG preparations will be tested for the effectiveness in the prevention/therapy of ALD in three experimental models of ALD of steatosis, steatohepatitis and fibrosis. This study will have a major impact on the development of probiotic-based new therapeutic strategy for the prevention and treatment of ALD.