CYP2A5 and alcoholic liver disease

描述(由申请人提供)：酒精性肝损伤是一个重大的全球健康问题，也是全球主要的死亡原因。了解这种损伤的机制对于设计新的治疗方法和提高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。乙醇对肝脏的许多毒性作用与其在肝脏中的新陈代谢有关。乙醇可诱导细胞色素P450-2E_1代谢乙醇，被认为是酒精性肝病的危险因素之一。有报道称ALD患者肝脏细胞色素P450 2A6的表达增加。在小鼠模型中，我们发现，慢性酒精喂养可以诱导小鼠CYP2A5，也就是CYP2A6的同源基因，其诱导程度远远大于CYP2E1，而且非常有趣的是，乙醇对小鼠CYP2A5的诱导是依赖于CYP2E1的。CYP2A5/6代谢重要的临床药物，如香豆素、尼古丁、硝胺等，因此乙醇对其诱导具有重要的毒理学意义。在本申请中，我们将进一步了解小鼠的细胞色素P450 2 A5是否可能参与ALD的发病机制。我们推测，CYP2E1介导的ROS激活了氧化还原敏感的核因子-拟除虫菊酯2相关因子2(Nrf2)，Nrf2上调了CYP2A5，后者可能代谢乙醇，增加氧化应激，从而参与ALD的发病，即至少部分地通过CYP2A5促进ALD的发生。目的1通过体内乙醇喂养模型和体外报告基因转染模型，研究ROS和Nrf2在乙醇诱导细胞色素P450 2A5表达中的可能作用。AIM 2旨在研究CYP2A5是否会导致酒精性肝损伤。Cyp2a5-/-小鼠将被用来研究CYP2A5在ALD发病机制中的可能作用，cyp2a5 siRNA将被应用于确定CYP2A5在乙醇代谢和ROS产生以及CYP2E1介导的肝损伤中的作用。从慢性乙醇液体饲料喂养的WT和cyp2a5-/-小鼠中分离出的微粒体将被用来确定肝脏微粒体的乙醇代谢和由CYP2A5产生的ROS。乙醇对细胞色素P450 2 A5的诱导作用及其作用是一个新的发现，因为大多数关于微粒体乙醇代谢的研究都是以大鼠的微生物体进行的，而大鼠的微生物体中缺乏与小鼠的细胞色素P450 2 A5同源的细胞色素P450 2 A3。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，为乙醇代谢和酒精性肝病的研究开辟新的窗口。

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.