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

基本信息

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
项目类别：
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

项目摘要

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）：药物诱导的肝损伤（DILI）的特质性质，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问题，也是从制药市场中撤出药物的最常见原因。有证据表明，除了药物引起对肝细胞的直接损害外，还会触发炎症性的先天免疫反应，从而导致肝损伤的加剧和进展。但是，尚未研究受损的肝细胞在诱导炎症反应中的作用。我们假设该药物（更常见的反应性代谢产物）对肝细胞诱导的损伤导致损伤相关的分子模式（DAMP）分子的释放，这通过肝巨噬细胞的促炎性激活（Kupffer细胞，KC）导致肝损伤的进展。 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.小鼠基本肝细胞将在体外分离并用各种浓度的APAP处理。在各个时间点之后，将收集条件培养基并用于刺激KC，该培养基将从小鼠肝脏中分离出来，并通过FACSORTING纯化。通过测量各种炎性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的激活。在体外和体内通过APAP挑战的肝细胞释放HMGB-1，HSP-70和尿酸将由ELISA和免疫组织化学确定。 HMGB-1，HSP-70和尿酸将使用重组蛋白和囊肿尿酸在体外研究KC的炎症激活。此外，使用中和抗HMGB-1和抗HSP-70抗体，以及异硫醇和尿液酶，将确定HMGB-1，HSP-70和尿酸在引起APAP诱导的肝脏损伤进展中的作用。拟议研究的发现将有助于我们理解DILI的分子和细胞机制，并制定策略以防止这些反应并筛选出候选药物引起DILI的潜力。