Lysosomal Damage, Impaired Autophagy, and Alcoholic Pancreatitis

溶酶体损伤、自噬受损和酒精性胰腺炎

• 批准号: 8044203
• 负责人: ILYA GUKOVSKY
• 金额: $ 26.92万
• 依托单位: BRENTWOOD BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8044203
• 关键词: Acinar Cell; Acute; Affect; Alcohol abuse; Alcoholic Pancreatitis; Alcohols; Autophagocytosis; Autophagosome; Cathepsins; Cholecystokinin; Data; Disease; Dose; Endotoxemia; Ethanol; Ethanol toxicity; Event; Exocrine pancreas; Experimental Models; Functional disorder; Genetic; Goals; Golgi Apparatus; Human; Hydrolase; Impairment; In Vitro; Knock-out; LAMP-2; Lead; Life; Lysosomes; Mediating; Mediator of activation protein; Membrane Proteins; Mitochondria; Modeling; Mus; Organ; Organelles; Pancreas; Pancreatitis; Pathogenesis; Pathologic; Pathway interactions; Process; Proteins; Rattus; Resolution; Risk Factors; Rodent; Role; Sincalide; Stress; Testing; Trypsin; Trypsinogen; Vacuole; acute pancreatitis; alcohol effect; analog; base; feeding; in vivo; insight; late endosome; mouse model; new therapeutic target; non-alcoholic; novel; novel therapeutic intervention; overexpression; response; stressor; trafficking

DESCRIPTION (provided by applicant): Pancreatitis is a potentially fatal disease of exocrine pancreas the pathogenesis of which remains obscure and for which no specific treatment has been developed. Alcohol abuse is a major etiologic factor for pancreatitis; however, the mechanisms by which alcohol predisposes to this disease remain elusive. Here we propose a novel hypothesis for the pathogenesis of alcoholic pancreatitis stating that a combination of two events is critical for pancreatitis: 1) induction of autophagy and 2) impaired lysosomal function which makes autophagy defective. Autophagy (a.k.a. macroautophagy) is the main cellular degradative, lysosome-driven process. Our recent study has revealed a profound impairment of autophagy in experimental models of nonalcoholic acute pancreatitis. We found that autophagy is activated but its progression/resolution is impaired. Autophagy impairment is caused by lysosomal dysfunction a prominent manifestation of which is defective processing/maturation of cathepsins, major lysosomal hydrolases. Further, our findings revealed that impaired autophagy mediates 2 key pathologic responses of pancreatitis: acinar cell vacuolation and intra- acinar trypsinogen activation. Our results indicate that ethanol feeding alone causes lysosomal dysfunction in pancreas similar to that we found in nonalcoholic pancreatitis. However, ethanol per se does not induce pancreatitis because it does not activate autophagy. Thus, in conditions of basal unstimulated autophagy, the consequences of ethanol- induced lysosomal dysfunction are limited. However, a combination of ethanol feeding, which causes lysosomal dysfunction, and stresses that induce autophagy leads to defective autophagy and thus pancreatitis. Our results suggest that this is the mechanism through which ethanol feeding "sensitizes" rats or mice to pancreatitis induced by low-dose cerulein (CR; a CCK-8 analog) that by itself does not elicit pancreatitis. We propose that similar mechanism mediates pancreatitis induced by the combination of ethanol feeding and endotoxemia (i.e., LPS), the other available "in vivo sensitization" model of alcoholic pancreatitis. Our results indicate that ethanol causes dysregulation of endolysosomal trafficking and the Golgi, associated with defective processing and activity of cathepsins and decreased level of LAMP-2, a major lysosomal membrane protein. We further propose that manipulating the level of autophagy (by, respectively, inhibiting autophagy through Atg5 knockout or stimulating it through overexpression of Atg8/LC3, a critical autophagy mediator) ameliorates or worsens alcoholic pancreatitis. The Specific Aims for the project are: 1. Determine the effects of ethanol feeding alone and experimental alcoholic pancreatitis (i.e., ethanol feeding combined with low-dose CR or LPS) on lysosomal dysfunction in pancreas, and the role of LAMP-2 in these effects. 2. Determine the effects of ethanol feeding alone and experimental alcoholic pancreatitis on endolysosomal trafficking and the Golgi. 3. Determine the effects of ethanol feeding alone and experimental alcoholic pancreatitis on autophagy induction and progression, and the role of LAMP-2 in these effects. 4. Determine the effects of genetically inhibiting or stimulating autophagy on alcoholic pancreatitis. The proposed studies elucidate ethanol's effects on lysosomes, the Golgi, and autophagy in pancreas; suggest a new model for alcoholic pancreatitis; and indicate novel targets for therapeutic approaches to treat or mitigate this disease. Further, similar mechanisms involving ethanol-induced lysosomal dysfunction may mediate alcohol toxicity to other organs. PUBLIC HEALTH RELEVANCE: Pancreatitis is a serious and sometimes lethal disease of exocrine pancreas, the pathobiology of which is poorly understood, and specific treatments for which do not exist. Alcohol abuse is a major risk factor for pancreatitis; however, the mechanisms by which ethanol predisposes to pancreatitis remain unknown. In this project, we will test the hypothesis that a key mechanism is ethanol-induced damage to lysosomes, resulting in impaired autophagy in pancreas. Lysosome is the key cellular degradative organelle, and autophagy is the main process through which cellular components are "digested" in lysosomes. We will use mouse models to test our hypothesis. The results can lead to novel therapeutic approaches, targeting impaired lysosomal function in autophagy, to treat or mitigate alcoholic pancreatitis in humans. Further, similar mechanisms involving ethanol-induced lysosomal damage may mediate ethanol's toxicity to other organs.

描述（由申请方提供）：胰腺炎是胰腺外分泌的一种潜在致死性疾病，其发病机制尚不清楚，目前尚无特异性治疗方法。酒精滥用是胰腺炎的一个主要病因，然而，酒精易患胰腺炎的机制仍不清楚。在这里，我们提出了一个新的假设，酒精性胰腺炎的发病机制，指出两个事件的组合是胰腺炎的关键：1）诱导自噬和2）受损的溶酶体功能，使自噬缺陷。自噬（又称自噬）巨自噬）是主要的细胞降解、溶酶体驱动的过程。我们最近的研究揭示了非酒精性急性胰腺炎实验模型中自噬的严重损害。我们发现自噬被激活，但其进展/解决受损。自噬损伤是由溶酶体功能障碍引起的，其突出表现是组织蛋白酶（主要溶酶体水解酶）的加工/成熟缺陷。此外，我们的研究结果表明，受损的自噬介导胰腺炎的2个关键病理反应：腺泡细胞空泡化和腺泡内胰蛋白酶原激活。 我们的研究结果表明，乙醇喂养单独导致胰腺溶酶体功能障碍，我们发现在非酒精性胰腺炎。然而，乙醇本身不会诱发胰腺炎，因为它不会激活自噬。因此，在基础未刺激的自噬条件下，乙醇诱导的溶酶体功能障碍的后果是有限的。然而，导致溶酶体功能障碍的乙醇喂养和诱导自噬的应激的组合导致有缺陷的自噬，从而导致胰腺炎。我们的研究结果表明，这是通过乙醇喂养“敏感”大鼠或小鼠胰腺炎诱导的低剂量雨蛙肽（CR; CCK-8类似物），本身并不引起胰腺炎的机制。我们提出类似的机制介导了由乙醇喂养和内毒素血症联合诱导的胰腺炎（即，LPS），另一种可用的酒精性胰腺炎“体内致敏”模型。 我们的研究结果表明，乙醇引起的内溶酶体运输和高尔基体，与有缺陷的加工和活动的组织蛋白酶和LAMP-2，一个主要的溶酶体膜蛋白的水平下降的失调。我们进一步提出，操纵自噬水平（分别通过Atg 5敲除抑制自噬或通过Atg 8/LC 3（一种关键的自噬介质）过表达刺激自噬）可改善或抑制酒精性胰腺炎。该项目的具体目标是：1。确定单独乙醇喂养和实验性酒精性胰腺炎（即，乙醇喂养结合低剂量CR或LPS）对胰腺中溶酶体功能障碍的影响，以及LAMP-2在这些影响中的作用。2.确定单独乙醇喂养和实验性酒精性胰腺炎对内溶酶体运输和高尔基体的影响。3.确定单独乙醇喂养和实验性酒精性胰腺炎对自噬诱导和进展的影响，以及LAMP-2在这些影响中的作用。4.确定遗传抑制或刺激自噬对酒精性胰腺炎的影响。 拟议的研究阐明了乙醇对胰腺中溶酶体、高尔基体和自噬的影响;提出了酒精性胰腺炎的新模型;并指出了治疗或减轻这种疾病的治疗方法的新靶点。此外，涉及乙醇诱导的溶酶体功能障碍的类似机制可能介导酒精对其他器官的毒性。 公共卫生相关性：胰腺炎是胰腺外分泌的一种严重且有时致命的疾病，其病理生物学知之甚少，并且不存在针对其的特定治疗。酒精滥用是胰腺炎的主要危险因素;然而，乙醇诱发胰腺炎的机制尚不清楚。在这个项目中，我们将测试一个假设，即一个关键的机制是乙醇诱导的溶酶体损伤，导致受损的胰腺自噬。溶酶体是细胞内重要的降解细胞器，自噬是细胞成分在溶酶体中被“消化”的主要过程。我们将使用小鼠模型来验证我们的假设。这些结果可能导致新的治疗方法，靶向自噬中受损的溶酶体功能，以治疗或减轻人类酒精性胰腺炎。此外，涉及乙醇诱导的溶酶体损伤的类似机制可能介导乙醇对其他器官的毒性。