RIP3-mediated necroptosis and ethanol-induced liver injury

描述(由申请人提供)：尽管细胞凋亡与酒精性肝损伤的进展有关，但没有直接证据表明抑制细胞凋亡实际上可以预防酒精性肝损伤。我们发现，事实上，抑制细胞凋亡并不能减轻乙醇诱导的肝细胞损伤、促炎细胞因子/趋化因子的表达或Bid缺陷小鼠的氧化应激。最近，一种新的细胞死亡模式，称为坏死性下垂，已被认为与多种细胞类型的caspase非依赖性细胞损伤有关。坏死性下垂以类似于细胞凋亡的方式被激活，但在形态上，这一过程类似于坏死。涉及受体相互作用蛋白激酶(RIP)的信号机制，包括RIP1和RIP3，介导了包括TNFa或Fas在内的死亡配体的激活而导致的坏死性下垂。在初步研究中，我们首次发现，作为坏死性下垂的中枢介质，RIP3在慢性乙醇喂养后的小鼠肝脏中的表达增加，与肝细胞损伤的标志平行。在其他肝损伤模型中，小鼠肝脏中也有RIP3的表达，包括四氯化碳(CCl4)和缺血/再灌注所致的肝损伤。此外，在试点实验中，我们现在表明，RIP3缺陷小鼠可以免受乙醇诱导的肝损伤和炎症的保护。在这里，我们假设慢性酒精喂养后的肝细胞损伤是由RIP3驱动的caspase非依赖性细胞死亡调节的。为了验证我们的假设，我们将使用缺乏RIP3的小鼠，并在酒精喂养期间使用Necrostatin-1，一种坏死下垂抑制剂。我们还将使用缺乏CYP2E1、TNFR1和TLR4的小鼠来确定RIP3信号通路的上游激活物对乙醇喂养的反应。本研究将探索酒精喂养后小鼠肝脏细胞死亡的新途径。拟议的工作将帮助我们确定新的分子靶点，以便更好地治疗酒精性肝病(ALD)。

DESCRIPTION (provided by applicant): Although apoptosis has been associated with the progression of alcohol-induced liver injury, no direct evidence demonstrates that inhibition of apoptosis actually prevents alcoholic liver damage. We found that inhibition of apoptosis did not, in fact, attenuate ethanol-induced hepatocyte injury, expression of pro- inflammatory cytokines/chemokines or oxidative stress in Bid-deficient mice. Recently, a newly described mode of cell death, called necroptosis, has been implicated in caspase-independent cell injury in a variety of cell types. Necroptosis is activated in a fashion similar to apoptosis, but morphologically, the process resembles necrosis. Signaling mechanisms involving receptor-interacting protein kinases (RIP), including RIP1 and RIP3, mediate necroptosis induced by the activation of death ligands, including TNFa or Fas. In preliminary studies, we find, for the first time, that expression of RIP3, a central mediator of necroptosis, is increased in mouse livers following chronic ethanol feeding in parallel to the markers of hepatocyte injury. RIP3 is also induced in mouse liver in other models of hepatic injury including carbon tetrachloride (CCl4)- and ischemia/reperfusion-induced liver damage. Moreover, in pilot experiments we now show that RIP3-deficient mouse are protected from ethanol-induced liver injury and inflammation. Here we hypothesize that hepatocyte injury following chronic ethanol feeding is regulated by RIP3-driven caspase-independent cell death. To test our hypothesis, we will use mice deficient in RIP3 as well as treatment with necrostatin-1, a necroptosis inhibitor, during ethanol feeding. We will also use mice deficient in CYP2E1, TNFR1, and TLR4 to determine upstream activators of the RIP3-signaling pathway in response to ethanol feeding. This study will explore new pathways of cell death in mouse liver following ethanol feeding. The proposed work will help us to determine new molecular targets for better therapeutic management of alcoholic liver disease (ALD).