Investigation of the role of DAMP molecules in the pathogenesis of Liver Injury

DAMP分子在肝损伤发病机制中的作用研究

• 批准号: 7547365
• 负责人: Brittany V Murphy
• 金额: $ 2.74万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7547365
• 关键词: Acetaminophen; Alcohol consumption; Allopurinol; Antibodies; Autoimmune Process; Biological Assay; Cell Line; Cell surface; Cells; Condition; Conditioned Culture Media; Data; Dendritic Cells; Diagnosis; Dose; Enzyme-Linked Immunosorbent Assay; Goals; HMGB1 Protein; HMGB1 gene; Heat-Shock Proteins 70; Hepatic; Hepatocyte; Immune; Immune response; Immunohistochemistry; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Investigation; Kupffer Cells; Link; Liver; Liver diseases; Marketing; Measures; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Molecular Target; Mus; Nature; Necrosis; Pathogenesis; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Play; Preclinical Drug Evaluation; Predisposing Factor; Prevention; Proteins; Reaction; Recombinant Proteins; Research; Role; Safety; Severities; Stream; Stress; Surface; Time; Tissues; Urate Oxidase; Uric Acid; Virus Diseases; chemokine; cytokine; drug development; drug mechanism; drug withdrawal; extracellular; in vivo; injured; macrophage; prevent; release factor; research study

DESCRIPTION (provided by applicant): The idiosyncratic nature, severity and poor diagnosis of drug-induced liver injury (DILI) make these reactions a major safety concern during drug development, as well as the most common cause for the withdrawal of drugs from the pharmaceutical market. Evidence suggests that aside from drug-induced direct damage to hepatocytes, an inflammatory innate immune response is triggered, which leads to the exacerbation and progression of liver injury. However, the role of damaged hepatocytes in the induction of inflammatory responses has not been investigated. We hypothesize that drug (more often reactive metabolite)-induced injury to hepatocytes causes the release of damage-associated molecular pattern (DAMP) molecules, which contribute to the progression of liver injury through pro-inflammatory activation of hepatic macrophages (Kupffer cells, KC). Acetaminophen (APAP)-induced liver injury in mice will be used as a model, and the specific aims of the proposed studies will be to determine i) whether APAP-stimulated hepatocytes release DAMP molecules that can activate hepatic KC, ii) - iv) whether HMGB-1, HSP-70, and uric acid represent key DAMP molecules released by APAP-stimulated hepatocytes. Mouse primiary hepatocytes will be isolated and treated with various concentrations of APAP in vitro. After various time points, the conditioned medium will be collected and used to stimulate KC, which will be isolated from mouse liver and purified by FACSorting. Activation of KC by the soluble factors released into the conditioned medium will be determined by measuring mRNA and protein levels of various pro- inflammatory cytokines and chemokines. The release of HMGB-1, HSP-70, and uric acid by APAP- challenged hepatocytes in vitro and in vivo will be determined by ELISA and immunohistochemistry. Pro- inflammatory activation of KC by HMGB-1, HSP-70 and uric acid will be investigated using recombinant proteins and cystal uric acid in vitro. Further, the roles of HMGB-1, HSP-70, and uric acid in causing the progression of APAP-induced liver damage through stimulation of KC will be determined using neutralizing anti-HMGB-1 and anti-HSP-70 antibodies, as well as allopurinol and uricase. The findings of the proposed research will contribute to our long-term objective of understanding the molecular and cellular mechanisms of DILI, and developing strategies to prevent these reactions and screen for drug candidates' potential of causing DILI.

描述(由申请人提供)：药物性肝损伤(DILI)的特殊性质、严重程度和较差的诊断使这些反应成为药物开发过程中的主要安全问题，也是导致药物退出药品市场的最常见原因。有证据表明，除了药物对肝细胞的直接损伤外，还会触发炎性先天免疫反应，从而导致肝损伤的加重和进展。然而，受损的肝细胞在诱导炎症反应中的作用还没有被研究。我们假设药物(更常见的是反应性代谢物)诱导的肝细胞损伤导致损伤相关分子模式(DAMP)的释放，这些分子通过促炎症激活肝巨噬细胞(Kupffer cell，KC)而促进肝损伤的进展。本研究将以对乙酰氨基酚(APAP)诱导的小鼠肝损伤为模型，研究的具体目的将是：1)APAP刺激的肝细胞是否释放可激活肝脏KC的湿润分子；2)-4)HMGB-1、HSP-70和尿酸是否代表APAP刺激的肝细胞释放的关键湿润分子。体外分离小鼠原代肝细胞，用不同浓度的APAP处理。在不同的时间点后，收集条件培养液，用来刺激KC，从小鼠肝脏中分离KC，并用FACSorting进行纯化。释放到条件培养液中的可溶性因子对KC的激活将通过测量各种促炎细胞因子和趋化因子的mRNA和蛋白水平来确定。体外和体内APAP攻击的肝细胞释放HMGB-1、HSP-70和尿酸的能力将通过ELISA法和免疫组织化学法测定。用重组蛋白和半胱氨酸尿酸体外研究HMGB-1、HSP-70和尿酸对KC的促炎活性。此外，将通过中和抗HMGB-1和抗HSP-70抗体以及别嘌醇和尿酸来确定HMGB-1、HSP-70和尿酸在通过刺激KC导致APAP诱导的肝损伤进展中的作用。拟议的研究结果将有助于我们的长期目标，即了解DILI的分子和细胞机制，开发预防这些反应的策略，并筛选候选药物导致DILI的可能性。