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: 8212065
• 负责人: ILYA GUKOVSKY
• 金额: $ 26.31万
• 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/8212065
• 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; public health relevance; 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（由申请人提供）：胰腺炎是一种潜在致命的外分泌胰腺疾病，其发病机制仍不清楚，并且尚未开发出具体的治疗方法。酗酒是胰腺炎的主要病因；然而，酒精诱发这种疾病的机制仍然难以捉摸。在这里，我们对酒精性胰腺炎的发病机制提出了一个新的假设，指出两个事件的组合对于胰腺炎至关重要：1）诱导自噬；2）溶酶体功能受损，导致自噬缺陷。自噬（又称巨自噬）是溶酶体驱动的主要细胞降解过程。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受损。我们发现自噬被激活，但其进展/解决受到损害。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（主要的溶酶体水解酶）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两种关键病理反应：腺泡细胞空泡化和腺泡内胰蛋白酶原激活。 我们的结果表明，单独喂食乙醇会导致胰腺溶酶体功能障碍，类似于我们在非酒精性胰腺炎中发现的情况。然而，乙醇本身不会诱发胰腺炎，因为它不会激活自噬。因此，在基础未刺激自噬的条件下，乙醇诱导的溶酶体功能障碍的后果是有限的。然而，乙醇喂养会导致溶酶体功能障碍，再加上诱导自噬的应激，会导致自噬缺陷，从而导致胰腺炎。我们的结果表明，这是乙醇喂养使大鼠或小鼠对低剂量雨蛙素（CR；CCK-8类似物）诱导的胰腺炎“敏感”的机制，而低剂量雨蛙素本身不会引起胰腺炎。我们提出类似的机制介导由乙醇喂养和内毒素血症（即LPS）联合诱导的胰腺炎，这是酒精性胰腺炎的另一种可用的“体内致敏”模型。 我们的结果表明，乙醇会导致内溶酶体运输和高尔基体失调，与组织蛋白酶的加工和活性缺陷以及主要溶酶体膜蛋白 LAMP-2 水平降低有关。我们进一步提出，操纵自噬水平（分别通过 Atg5 敲除抑制自噬或通过 Atg8/LC3（一种关键的自噬介质）过度表达刺激自噬）可改善或恶化酒精性胰腺炎。该项目的具体目标是： 1. 确定单独的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. 确定单独喂食乙醇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. 确定单独喂食乙醇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. 确定基因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溶酶体、高尔基体和胰腺自噬的影响；提出酒精性胰腺炎的新模型；并指出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：胰腺炎是一种严重的、有时是致命的胰腺外分泌疾病，其病理生物学知之甚少，并且尚无具体治疗方法。酗酒是胰腺炎的主要危险因素；然而，乙醇诱发胰腺炎的机制仍不清楚。在这个项目中，我们将测试一个假设，即一个关键机制是乙醇诱导的溶酶体损伤，导致胰腺自噬受损。溶酶体是关键的细胞降解细胞器，自噬是细胞成分在溶酶体中“消化”的主要过程。我们将使用小鼠模型来检验我们的假设。这些结果可以带来新的治疗方法，针对自噬中受损的溶酶体功能，来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。