RIP3-mediated necroptosis and ethanol-induced liver injury

描述（由申请人提供）：虽然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，在bid缺陷小鼠中，抑制细胞凋亡实际上并没有减轻乙醇诱导的肝细胞损伤、促炎细胞因子/趋化因子的表达或氧化应激。最近，一种新描述的细胞死亡模式，称为坏死下垂，与多种细胞类型的caspase非依赖性细胞损伤有关。坏死下垂以类似于细胞凋亡的方式被激活，但在形态学上，这个过程类似于坏死。涉及受体相互作用蛋白激酶（RIP）的信号机制，包括RIP1和RIP3，介导由TNFa或Fas等死亡配体激活诱导的坏死坏死。在初步研究中，我们首次发现，慢性乙醇喂养后小鼠肝脏中坏死坏死的中枢介质RIP3的表达与肝细胞损伤标志物平行增加。在其他肝损伤模型中，包括四氯化碳(CCl4)-和缺血/再灌注诱导的肝损伤，小鼠肝脏也会诱导RIP3。此外，在试点实验中，我们现在表明rip3缺陷小鼠免受乙醇诱导的肝损伤和炎症。在这里，我们假设慢性乙醇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RIP3缺乏的小鼠，并在乙醇喂养期间使用坏死他汀-1（一种坏死性凋亡抑制剂）治疗。我们还将使用CYP2E1、TNFR1和TLR4缺失的小鼠来确定乙醇喂养时rip3信号通路的上游激活因子。本研究将探索乙醇喂养小鼠肝脏细胞死亡的新途径。这项工作将有助于我们确定新的分子靶点，以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).