Hepatocyte-hepatic stellate cell axis in potentiation of alcohol and HIV-induced liver injury

肝细胞-肝星状细胞轴增强酒精和 HIV 诱导的肝损伤

• 批准号: 10375398
• 负责人: Moses O New-Aaron
• 金额: $ 4.08万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-05 至 2022-12-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10375398
• 关键词: Adopted; Alcohol consumption; Alcoholic Liver Diseases; Alcoholic liver damage; Alcohols; Animals; Apoptosis; Apoptotic; Cell Culture Techniques; Cell Death; Cells; Cessation of life; Chemosensitization; Cirrhosis; DNA; Data; Development; Ethanol Metabolism; Etiology; Exposure to; Fibrosis; Flow Cytometry; Genes; Goals; Grant; HIV; HIV Infections; HIV Receptors; HIV Seropositivity; Hepatic Stellate Cell; Hepatitis B Virus; Hepatitis C virus; Hepatocyte; Hepatotoxicity; Highly Active Antiretroviral Therapy; Hospitalization; Hour; In Vitro; Incidence; Individual; Infection; Laboratories; Lead; Liver; Liver Fibrosis; Liver diseases; Lysosomes; Mediating; Mentors; Messenger RNA; Modality; Molecular; Necrosis; Oxidative Stress Induction; Pathogenesis; Pathway interactions; Pattern; Persons; Phagocytosis; Polymerase Chain Reaction; Proteomics; RNA; Reactive Oxygen Species; Research; Research Personnel; Research Proposals; Resources; STAT3 gene; Scheme; Source; Supervision; System; Techniques; Testing; Therapeutic; Time; Transforming Growth Factor beta; United States; Up-Regulation; Virus; Work; Writing; alcohol effect; alcohol exposure; alcohol prevention; alkalinity; antiretroviral therapy; cell type; co-infection; digital; end stage liver disease; humanized mouse; in vivo; in vivo Model; liver injury; mortality; pathogen; pre-clinical; prevent; receptor; receptor expression; skills; therapy development; treatment strategy

Project Summary In the era of highly active antiretroviral therapy (ART), liver disease has become a common reason for hospitalization and one of the leading causes of mortality among HIV positive individuals. The etiologies of hepatotoxicity in HIV are multifaceted. HIV mono-infection, HIV co-infection with hepatotropic viruses (HBV and HCV), HIV and alcohol-induced hepatotoxicity and ART-induced hepatotoxicity are known causes of liver disease among HIV-infected individuals. Among all causes of hepatotoxicity, the mechanisms of alcohol- induced hepatotoxicity in HIV-infected cells are unknown. Given that all HIV-infected individuals are on ART due to high ART availability and accessibility, it becomes difficult to evaluate the single effects of alcohol on the liver of HIV-infected individuals. That is why I am adopting an invitro system with the help of my mentors (Drs. Osna and Poluektova) to explore alcohol-induced hepatotoxicity in HIV-infected hepatocytes. My preliminary data under the supervision of my mentors has revealed the following findings: Alcohol metabolites enhanced HIV accumulation in hepatocytes which triggers reactive oxygen species (ROS) that lead to hepatocyte death via apoptotic pathway. When Hepatic Stellate Cells (HSC) were exposed to HIV-infected apoptotic hepatocytes, an upregulation of profibrotic genes such as Col 1A1, TIMP 1 and TGFβ was established after 2 hours of exposure. This implies that there might be a cross talk between hepatocytes and HSC in the premises of HIV and alcohol via apoptotic hepatocytes which results in liver fibrosis. These observations were confirmed in the liver of humanized mice exposed to HIV and alcohol. For therapeutic purposes it has become necessary to understand the mechanisms of alcohol-induced hepatotoxicity in HIV- infected cells. To understand this mechanism, we are confronted with research questions which seek to understand the receptors for HIV entry into hepatocytes and the influence of alcohol metabolites to increase HIV entry. Moreover, the impact of alcohol on HIV degradation in hepatocytes needs to be explored. It is not known if hepatocyte death is due to apoptosis or other cell death mechanisms (such as necrosis or necroptosis) are involved. Hence understanding the type of cell death mechanism will help devise strategies to block/prevent the hepatocyte death, which mediates liver fibrosis. Also, we will explore the pathways involved in the activation of profibrotic pathways to understand if blocking these pathways will prevent activation of fibrotic genes. All the research questions will be tested in both in vitro and in vivo models. Skills that will be gained during the 3-year period dedicated to completing the 3 aims of the research proposal are laboratory techniques such as proteomics, flow cytometry, Real Time Polymerase Chain Reaction (RT PCR) for HIV RNA, mRNA, HIV DNA, digital droplet PCR (ddPCR), flow cytometry, cell culture, and animal handling. During this period, I will not only be thoroughly furnished with grant writing and presentation skills, I will be involved in the management of my resources as a trainee, a skill that is needed to become an independent researcher.

项目概要 在高度活跃的抗逆转录病毒治疗（ART）时代，肝病已成为导致患者死亡的常见原因。 住院治疗是艾滋病毒阳性者死亡的主要原因之一。 HIV的肝毒性是多方面的，HIV单一感染、HIV与嗜肝病毒（HBV）合并感染。 和 HCV）、HIV 和酒精引起的肝毒性以及 ART 引起的肝毒性是肝脏的已知原因 在所有导致肝毒性的原因中，酒精- 鉴于所有 HIV 感染者都在接受 ART，HIV 感染细胞中诱导的肝毒性尚不清楚。 由于抗逆转录病毒治疗的可用性和可及性较高，因此很难评估酒精对患者的单一影响。 这就是为什么我在导师的帮助下采用体外系统。 （Osna 和 Poluektova 博士）探索酒精诱导的 HIV 感染肝细胞的肝毒性。 在我的导师的监督下，初步数据揭示了以下发现： 酒精代谢物 增强 HIV 在肝细胞中的积累，从而触发活性氧 (ROS)，从而导致 当肝星状细胞（HSC）暴露于 HIV 感染时，肝细胞通过凋亡途径死亡。 肝细胞凋亡，促纤维化基因如 Col 1A1、TIMP 1 和 TGFβ 上调 暴露 2 小时后建立，这意味着肝细胞和肝细胞之间可能存在串扰。 HSC 存在于 HIV 和酒精的场所，通过凋亡的肝细胞导致肝纤维化。 在暴露于艾滋病毒和酒精的人源化小鼠的肝脏中证实了这一观察结果。 出于目的，有必要了解酒精引起的 HIV 肝毒性机制。 为了了解这种机制，我们面临着一些研究问题，这些问题试图 了解 HIV 进入肝细胞的受体以及酒精代谢物增加的影响 此外，酒精对肝细胞内艾滋病毒降解的影响还需要探索。 已知肝细胞死亡是否是由于细胞凋亡或其他细胞死亡机制（例如坏死或 因此，了解细胞死亡机制的类型将有助于制定策略。 阻止/预防介导肝纤维化的肝细胞死亡。此外，我们还将探讨相关途径。 促纤维化途径的激活，以了解阻断这些途径是否会阻止促纤维化途径的激活 所有研究问题都将在体外和体内模型中进行测试。 在致力于完成研究计划的 3 个目标的 3 年期间所获得的成果是实验室 HIV 的蛋白质组学、流式细胞术、实时聚合酶链式反应 (RT PCR) 等技术 RNA、mRNA、HIV DNA、数字液滴 PCR (ddPCR)、流式细胞术、细胞培养和动物处理。 在此期间，我不仅将彻底掌握拨款写作和演讲技巧，我还将参与 作为实习生管理我的资源，这是成为独立研究员所需的技能。