CYP2A5 and alcoholic liver disease

描述（由申请人提供）：酒精性肝损伤是一个重要的全球性健康问题，也是全球范围内的主要死亡原因。了解这种损伤的机制对于设计新的治疗方法和改善许多患者的生活质量至关重要。乙醇处理导致细胞死亡的机制仍然不清楚。乙醇的许多肝脏毒性作用与其在肝脏中的代谢有关。CYP 2 E1可被乙醇诱导，并能代谢乙醇，被认为是酒精性肝病（ALD）的危险因素之一。据报道，ALD患者的人肝CYP 2A 6表达增加。在小鼠模型中，我们发现小鼠CYP 2A 5（CYP 2A 6的直系同源物）可以通过慢性乙醇喂养诱导，诱导程度远大于CYP 2 E1，非常有趣的是，小鼠中CYP 2A 5的乙醇诱导是CYP 2 E1依赖性的。CYP 2A 5/6代谢重要的临床药物，如香豆素、尼古丁、亚硝胺，因此乙醇对CYP 2A 5/6的诱导具有重要的毒理学意义。在本申请中，我们进一步了解小鼠CYP 2A 5是否可能参与ALD的发病机制。我们假设CYP 2 E1介导的ROS激活氧化还原敏感性核因子-类红细胞2相关因子2（Nrf 2），Nrf 2上调CYP 2A 5，CYP 2A 5可能代谢乙醇并增加氧化应激，这有助于ALD的发病机制，即，CYP 2 E1至少部分通过CYP 2A 5促进ALD。目的1采用体内乙醇喂养模型和体外报告基因转染模型，研究ROS和Nrf 2在乙醇诱导CYP 2A 5中的可能作用。目的2旨在检查CYP 2A 5是否有助于酒精诱导的肝损伤。本研究将利用Cyp 2a 5-/-小鼠研究CYP 2A 5在ALD发病机制中的可能作用，并应用Cyp 2a 5 SiRNA研究CYP 2A 5在乙醇代谢、ROS产生和CYP 2 E1介导的肝损伤中的作用。从长期乙醇液体饲料喂养的WT和cyp 2a 5-/-小鼠中分离的微粒体将用于确定肝微粒体乙醇代谢和CYP 2A 5产生的ROS。乙醇诱导CYP 2A 5和CYP 2 E1的作用是一个新的新发现，因为大多数关于微粒体乙醇代谢的研究都是用缺乏CYP 2A 3（小鼠CYP 2A 5的直系同源物）的大鼠微粒体进行的。我们认为本申请中提出的实验符合R21机制的范畴，这支持探索性研究。希望本研究能回答上述问题，并为乙醇代谢和ALD研究打开一个新的窗口。

DESCRIPTION (provided by applicant): Alcohol-induced liver injury is a significant global health problem and a leading cause of death worldwide. Understanding the mechanisms of this injury is critical for designing new therapies and improving the quality of life of many patients. The mechanisms by which ethanol treatment causes cell death are still not clear. Many of the hepatic toxic effects of ethanol have been linked to its metabolism in the liver. CYP2E1 can be induced by ethanol and is able to metabolize ethanol and is considered as one of the risk factors of alcoholic liver disease (ALD). It was reported that human hepatic CYP2A6 expression was increased in patients with ALD. In a mouse model, we found that mouse CYP2A5, the ortholog of CYP2A6, can be induced by chronic ethanol feeding to a much greater extent than CYP2E1, and very interestingly, ethanol induction of CYP2A5 in mice is CYP2E1-dependent. CYP2A5/6 metabolizes important clinical drugs such as coumarin, nicotine, nitroamines so its induction by ethanol is of major toxicological significance. In this Application, we move towards understanding whether mouse CYP2A5 may be involved in the pathogenesis of ALD. We hypothesize that CYP2E1-mediated ROS activates redox sensitive nuclear factor-eythroid 2-related factor 2 (Nrf2), and Nrf2 upregulates CYP2A5, which may metabolize ethanol and increase oxidative stress which contributes to pathogenesis of ALD i.e., CYP2E1 promotes ALD at least partially through CYP2A5. AIM 1 is designed to examine the possible role of ROS and Nrf2 in the induction of CYP2A5 by ethanol by using an in vivo ethanol feeding model and in vitro reporter gene transfection model. AIM 2 is designed to examine if CYP2A5 contributes to alcohol-induced liver injury. Cyp2a5-/- mice will be used to examine the possible role of CYP2A5 in pathogenesis of ALD, and cyp2a5 SiRNA will be applied to define the role of CYP2A5 in ethanol metabolism and ROS production and CYP2E1-mediated liver injury. Micosomes isolated from chronic ethanol liquid diet fed WT and cyp2a5-/- mice will be used to define hepatic microsomal ethanol metabolism and ROS production by CYP2A5. Ethanol induction of CYP2A5 and the role of CYP2E1 is a novel new finding, because most studies about microsomal ethanol metabolism were carried with rat microsomes, which lack CYP2A3, the ortholog of mouse CYP2A5. We think the experiments proposed in this Application fit in the category of the R21 mechanism, which supports exploratory studies. Hopefully, this study will answer the above questions and open a new window for ethanol metabolism and ALD study.