Alcoholic Hepatitis: Molecular Mechanisms

酒精导致近10万人死亡，每年的公共卫生费用超过1160亿美元。嗜中性脂肪性肝炎是慢性酒精中毒患者的重要病理学表现之一，它代表了向纤维化/肝硬化和临床肝病进展的限速步骤。虽然在过去的十年中，我们对肝脏中性粒细胞浸润机制的总体理解已经取得了重大进展，但中性粒细胞从肝血管系统迁移到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非常重要的。在这个项目中，我们的具体目标有三个方面，并且基于中心假设，即骨桥蛋白（OPN），一种基质细胞蛋白在啮齿动物酒精性肝炎模型中被诱导，并且OPN的诱导是嗜神经细胞浸润到肝实质中的原因。进一步假设OPN介导的肝中性粒细胞浸润之前是肝血管系统内的中性粒细胞活化。本实验通过原位杂交、RT-PCR、免疫组化和Western blotting等方法，观察大鼠酒精性肝炎模型中OPN mRNA和蛋白的表达，并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评估L-选择素和Mac-1（β 2整合素）来研究OPN在中性粒细胞迁移到肝脏之前中性粒细胞活化中的作用。最后，将使用OPN OPN-/- -/-小鼠和在小鼠中使用针对OPN的中和抗体的干预实验来证实OPN的中心作用。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浸润到肝脏中来预防慢性酒精中毒者的嗜中性脂肪性肝炎。

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.