4-HNE抑制TNF-α介导的NF-κB信号通路诱导酒精性肝病的发生机制研究

Alcohol liver disease(ALD) remains a leading cause of death from liver diseases in the world. However, the safe and effective therapies for ALD in humans remain elusive, mainly due to the lack of our understanding on its detailed pathogenesis. Therefore, the research aiming to understand the cellular/molecular mechanisms underlying its pathogenesis, and based on which to find the safe and efficacious drugs, is urgently needed. In the proposed project, we will investigate the cellular/molecular mechanisms involved in liver cell death induced by chronic alcohol exposure. Our research will provide an improved understanding of therapeutic options for ALD. Although the mechanisms involved in ALD are multifactorial, it has been well-established that oxidative stress and tumor necrosis factor-α (TNF-α) play pivotal roles in the onset and progression of ALD. Ethanol metabolism produces reactive oxygen species (ROS) and depletes endogenous antioxidants, leading to oxidative stress. Lipid peroxidates are not only the hallmark of intracellular oxidative stress, but also the ultimate mediators of toxic effects elicited by oxidative stress. Increased productions of lipid peroxidates after chronic alcohol exposure were detected in both ALD patients and experimental animals. Of lipid peroxidates, 4-hydroxynonenal (4-HNE) is the most abundant and reactive aldehydic products. We had preliminary data showing that chronic alcohol consumption increased 4-HNE contents in the liver tissue in mice fed standard Lieber-De Carli diet. In our preliminary studies, we found that exposure to exogenous 4-HNE sensitized hepatocytes to TNF-α induced cytotoxicity, potentially via interfering with TNF-mediated NF-κB signaling pathway.Our preliminary data demonstrated that antioxidants, such as N-acetylcysteine（NAC）, can’t confer protection against 4-HNE (together with TNF) induced cytotoxicity, providing at least partial explanation in terms of therapeutic inefficacy of antioxidants in clinical trials. However, our preliminary studies demonstrated for the first time that bezafibrate, a specific PPAR-α agonist, decreased intracellular 4-HNE accumulation in hepatocytes when exogenous 4-HNE was added and protected against 4-HNE/TNF-α induced hepatocyte cell death. Therefore, we believe that the agent with property of facilitating 4-HNE degradation/metabolism represents an ideal therapeutic choice. In this context, PPAR-α?? activation represents a therapeutic target for the treatment of ALD. In this proposal, we will employ multiple state-of-the-arts biomedical technologies, including immunoprecipitation, epidemics analysis, siRNA transfection, gene overexpression, to name a few, to investigate the mechanisms underlying the adverse effects of 4-HNE in hepatocyte cell death. Our ultimate goal is to translate these results to the clinical setting in terms of therapy via 4-HNE removal.

酒精性肝病（ALD）发病机制复杂，危害严重，且临床尚无有效治疗手段，已成为全球关注的公共卫生问题。研究认为，氧化应激是ALD发病过程中的关键环节，通过调控肝细胞信号转导通路，影响ALD的发生、发展。4-羟基壬烯酸（4-HNE）是肝脏长期暴露于酒精后诱发氧化应激而产生的一种致敏因子。我们前期研究表明4-HNE通过抑制肝细胞内TNF-α介导的NF-κB抗凋亡信号通路诱导ALD的发生，而且清除4-HNE可以逆转肝细胞死亡，但机制尚不清楚。因此本课题拟通过蛋白质互作技术和RNA沉默技术深入研究4-HNE抑制NF-κB信号通路的确切作用机制及靶点，拟从细胞信号调控及表观遗传学水平阐明4-HNE促TNF-α介导的肝细胞死亡的作用机理，并从体内和体外水平揭示ALD的发病机制。为ALD临床治疗提供新的策略，也为以4-HNE为靶点的治疗ALD的新型药物研发提供重要的理论指导和依据。

本研究通过体内动物实验和体外细胞实验研究TNF诱导4-HNE致敏肝细胞发生细胞死亡的作用，以期阐明基于NF-κB/ IκBα信号通路4-HNE/TNF协同诱导酒精性肝病的分子机制。结果：⑴体内动物实验: 和对照组相比，ALD模型组小鼠肝脏内甘油三酯、TNF水平升高 (P <0.01)， 4-HNE蛋白质加合物表达增强，肝细胞脂质变性、凋亡数量增加，血浆中丙氨酸氨基转移酶（ALT）水平升高(P <0.01)；肝细胞核内NF-κB（p65）与DNA的结合活性下降，p65蛋白表达减弱，IκBα蛋白磷酸化表达减弱，4-HNE-IκB加合物表达增强。(2)体外细胞实验:①在HepG2及小鼠原代肝细胞中，与TNF组相比,4-HNE/TNF组肝细胞增殖活性下降,随着4-HNE、TNF浓度的增加，增殖活性呈剂量依赖性降低，细胞凋亡增多，细胞释放的乳酸脱氢酶活性升高(P＜0.05)，4-HNE/TNF组明显诱导PPAR蛋白剪切。②在HepG2细胞和小鼠原代肝细胞中,随着4-HNE浓度升高，细胞内4-HNE与蛋白质形成的加合物增多，与TNF组相比，4-HNE/TNF组肝细胞内4-HNE与IκBα形成的蛋白质加合物增多，IκBα的蛋白磷酸化水平降低。③随着4-HNE浓度升高,HepG2细胞核内NF-κB (p65)与DNA的结合活性下降,NF-κB调控基因(cFLIP、cIAP、Bcl-2)的mRNA表达水平下降。④4-HNE对TNF诱导的IKK无明显作用。⑤苯扎贝特通过清除4-HNE来减轻酒精性肝损伤。⑥NA能逆转 H2O2诱导的酒精性肝损伤，但对4-HNE诱导的酒精性肝损伤无明显作用。本研究结果表明，长期饮酒导致肝细胞内4-HNE和TNF的含量增加，TNF诱导了4-HNE致敏的肝细胞死亡，4-HNE作为一个肝细胞致敏因子，可以通过抑制TNF介导的NF-kB抗凋亡信号通路诱导肝细胞死亡，是ALD发生、发展过程中独立于氧化应激的一个新机制。然而，4-HNE与IκBα结合的靶点和机制有待进一步研究。