Alcoholic Hepatitis: Molecular Mechanisms

描述(申请人提供)：酒精导致近10万人死亡，公共卫生成本每年超过1160亿美元。中性粒细胞脂肪性肝炎是慢性酒精性脂肪性肝炎的重要病理表现之一，它是慢性酒精性脂肪性肝炎进展为肝纤维化/肝硬变和临床肝病的限速步骤。尽管在过去的十年中，我们对肝脏中性粒细胞浸润机制的总体认识已经取得了重大进展，但中性粒细胞从肝血管向肝细胞附着和死亡的确切原因目前尚不清楚。显然，了解慢性酒精中毒患者肝脏中性粒细胞浸润的机制基础是当务之急。在这个项目中，我们的具体目标有三个方面，并基于一个中心假设，即在啮齿类酒精性肝炎模型中诱导骨桥蛋白(OPN)是一种基质细胞蛋白，OPN的诱导是嗜神经细胞渗入肝实质的原因。进一步推测，在OPN介导的肝中性粒细胞浸润之前，肝血管内的中性粒细胞被激活。本实验将通过原位杂交、RT-PCR、免疫组织化学和免疫印迹等方法检测OPN在酒精性肝炎Lieber-DeCarli饮食啮齿动物模型中的表达，并将其与肝脏中性粒细胞浸润和肝损伤进行相关性研究。酒精性肝炎的体内啮齿动物模型和体外研究将通过流式细胞术检测L-选择素和Mac-1(？2整合素)整合素来探讨骨桥蛋白在中性粒细胞迁移到肝脏之前的中性粒细胞激活中的作用。最后，OPN的核心作用将在OPN OPN-/--/-小鼠身上得到证实，并在小鼠中使用OPN中和抗体进行干预实验。该项目的长期目标是设计适当的治疗方法，通过减少中性粒细胞渗入肝脏来预防慢性酒精中毒患者的中性粒细胞脂肪性肝炎。

DESCRIPTION (provided by applicant): Alcohol accounts for nearly 100,000 deaths and public health costs are more than $116 billion per year. Neutrophilic steatohepatitis is one of the important pathologic findings in chronic alcoholics which represents a rate-limiting step in the progression to firbrosis/cirrhosis and clinical liver disease. Although significant advances have been made in our general understanding of the mechanisms of hepatic neutrophil infiltration over the last decade, the precise reason for neutrophil migration from hepatic vasculature to hepatocyte attachment and death are currently unknown. Clearly, understanding the mechanistic basis for hepatic neutrophil infiltration in chronic alcoholic patients is of high priority. In this project, our specific aims are three fold, and are based on the central hypothesis that osteopontin (OPN), a matricellular protein is induced in the rodent alcoholic hepatitis model and induction of OPN is the reason behind neurophil infiltration into hepatic parenchyma. It is further postulated that OPN-mediated hepatic neutrophil infiltration is preceded by neutrophil activation within the hepatic vasculature. Our experiments will focus on assessing OPN mRNA and protein expression in the in vivo alcoholic hepatitis Lieber DeCarli diet rodent model by in situ hybridization, RT-PCR, immunohistochemistry and Western blotting which is then correlated with hepatic neutrophil infiltration and liver injury. The in vivo rodent model of alcoholic hepatitis and in vitro studies will then investigate the role of OPN in neutrophil activation prior to neutrophil transmigration into liver by assessing L-selectin and Mac-1 (¿2integrin) integrin) by flow cytometry. Finally, the central role of OPN will be confirmed using OPN OPN-/- -/- mice and interventional experiments using neutralizing antibody against OPN in mice. The long-term goal of this project is to device appropriate therapies that will prevent neutrophilic steatohepatitis in chronic alcoholics by reducing neutrophil infiltration into liver.