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: 8304355
• 负责人: Brittany V Murphy
• 金额: $ 1.55万
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8304355
• 关键词: Acetaminophen; Alcohol consumption; Allopurinol; Antibodies; Autoimmune Process; Biological Assay; Cell Line; Cell surface; Cells; 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; 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 candidate; 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（由申请方提供）：药物性肝损伤（DILI）的特异质性质、严重程度和诊断不佳使这些反应成为药物开发期间的主要安全性问题，也是药物从药品市场撤回的最常见原因。有证据表明，除了药物诱导的肝细胞直接损伤外，还引发了炎症性先天免疫反应，导致肝损伤的加重和进展。然而，损伤的肝细胞在诱导炎症反应中的作用尚未研究。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）分子的释放，这些分子通过肝巨噬细胞（枯否细胞，KC）的促炎激活促进肝损伤的进展。将使用对乙酰氨基酚（APAP）诱导的小鼠肝损伤作为模型，并且所提出的研究的具体目的将是确定i）APAP刺激的肝细胞是否释放可以激活肝KC的DAMP分子，ii）- iv）HMGB-1、HSP-70和尿酸是否代表由APAP刺激的肝细胞释放的关键DAMP分子。将分离小鼠肝细胞，并在体外用不同浓度的APAP处理。在不同时间点后，收集条件培养基并用于刺激KC，KC将从小鼠肝脏中分离并通过FACSorting纯化。通过测量各种促炎细胞因子和趋化因子的mRNA和蛋白质水平来确定释放到条件培养基中的可溶性因子对KC的活化。将通过ELISA和免疫组织化学测定体外和体内APAP激发的肝细胞释放HMGB-1、HSP-70和尿酸。将在体外使用重组蛋白和半胱氨酸尿酸研究HMGB-1、HSP-70和尿酸对KC的促炎活化。此外，HMGB-1、HSP-70和尿酸在通过刺激KC引起APAP诱导的肝损伤进展中的作用将使用中和抗HMGB-1和抗HSP-70抗体以及别嘌呤醇和尿酸酶来确定。拟议研究的结果将有助于我们理解DILI的分子和细胞机制的长期目标，并制定预防这些反应的策略，并筛选可能导致DILI的候选药物。