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.

项目摘要 在高效抗逆转录病毒疗法(ART)的时代，肝脏疾病已成为 住院是艾滋病毒感染者死亡的主要原因之一。致病原因 HIV对肝脏的毒性是多方面的。HIV单一感染、HIV与嗜肝病毒(乙肝)混合感染 和丙型肝炎病毒)、艾滋病毒和酒精引起的肝毒性以及ART引起的肝毒性是已知的肝脏原因 在感染艾滋病毒的个人中传播疾病。在肝毒性的所有原因中，酒精的机制是- 在HIV感染细胞中诱导的肝毒性尚不清楚。鉴于所有艾滋病毒感染者都在接受抗逆转录病毒治疗 由于抗逆转录病毒药物的高度可获得性和可获得性，很难评估酒精对 HIV感染者的肝脏。这就是为什么我在导师的帮助下采用体外培养系统的原因 OSNA和Pluektova博士)探讨酒精对艾滋病毒感染的肝细胞的肝毒性。我的 在我导师的监督下，初步数据揭示了以下发现：酒精代谢物 增加HIV在肝细胞中的积聚，从而触发活性氧物种(ROS)，从而导致 肝细胞通过凋亡途径死亡。肝星状细胞(HSC)暴露于HIV感染的时间 肝细胞凋亡，促肝纤维化基因Col 1A1、TIMP 1和转化生长因子β上调 在暴露2小时后确定。这意味着在肝细胞和细胞之间可能存在串扰 HSC在HIV和酒精的存在前提下，通过肝细胞的凋亡而导致肝纤维化。这些 在暴露于艾滋病毒和酒精的人源化小鼠的肝脏中观察到了证实的结果。用于治疗 目的了解酒精对人类免疫缺陷病毒(HIV)肝毒性的作用机制已成为必要。 被感染的细胞。为了理解这一机制，我们面临着一些研究问题，试图 了解HIV进入肝细胞的受体以及酒精代谢产物对增加的影响 艾滋病病毒进入。此外，酒精对肝细胞中艾滋病毒降解的影响还需要探讨。它不是 已知肝细胞死亡是由于细胞凋亡还是其他细胞死亡机制(如坏死或 坏死性上睑下垂)。因此，了解细胞死亡机制的类型将有助于制定策略 阻断/防止肝细胞死亡，而肝细胞死亡是肝纤维化的中介。此外，我们还将探索所涉及的路径 在激活促纤维化通路中了解阻断这些通路是否会阻止活化 纤维化基因。所有的研究问题都将在体外和体内模型中进行测试。这些技能将成为 在致力于完成研究提案的三个目标的三年时间内获得的是实验室 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、信使核糖核酸、艾滋病毒DNA、数字液滴聚合酶链式反应(DdPCR)、流式细胞仪、细胞培养和动物处理。在.期间 在此期间，我不仅将全面掌握助学金的撰写和演示技巧，我还将参与 作为一名实习生，管理我的资源，这是成为一名独立研究人员所必需的技能。