Mechanisms of microcystin-induced hepatocellular carcinoma in nonalcoholic steatohepatitis

非酒精性脂肪性肝炎微囊藻毒素诱发肝细胞癌的机制

• 批准号: 10515346
• 负责人: John Daniel Clarke
• 金额: $ 42.08万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-19 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10515346
• 关键词: 3-Dimensional; Acceleration; Address; Age; Alcohols; Algae; Animals; Attenuated; Automobile Driving; Award; Big Data; Career Mobility; Cell Nucleus; Cells; Certification; Cholesterol; Chronic Disease; Cirrhosis; Coculture Techniques; Complement; Country; Cyanobacterium; Cyanotoxin; DNA Methylation; Data; Development; Diet; Diethylnitrosamine; Differentiation Antigens; Dissection; Dose; Environmental Health; Euthanasia; Exposure to; Fatty acid glycerol esters; Fibrosis; Fresh Water; Genes; Hepatocyte; Histopathology; Human; In Vitro; Individual; Injections; Intake; Liver; Liver diseases; Long-Term Effects; Malignant neoplasm of liver; Measures; Mediating; Microfluidics; Modeling; National Institute of Environmental Health Sciences; Pathogenicity; Pathologist; Pathology; Pathway interactions; Persons; Phenotype; Predisposition; Primary Malignant Neoplasm of Liver; Primary carcinoma of the liver cells; Protein Inhibition; Protein phosphatase; Proteomics; Proto-Oncogene Proteins c-akt; Rattus; Research; Role; Science; Scientist; Sprague-Dawley Rats; Strategic Planning; Testing; Toxin; Unhealthy Diet; biological research; carcinogenesis; carcinogenicity; cell determination; cell type; chronic liver disease; cyanoginosin LR; defined contribution; dietary constituent; dietary control; druggable target; in vivo; inhibitor; innovation; microcystin; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; phosphoproteomics; single nucleus RNA-sequencing; stellate cell; stem cell biomarkers; transcriptomics; treatment group; tumor initiators; uptake

Microcystin-LR (MCLR) is the most potent and abundant cyanotoxin produced by freshwater blue-green algae. MCLR exposure is associated with nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). NAFLD has quickly risen to become the most common chronic liver disease in many countries, and HCC is the most common type of primary liver cancer. My K99/R00 data indicate MCLR drives progression of nonalcoholic steatohepatitis (NASH), an advanced form of NAFLD, to a more severe burnt out phenotype (increased fibrosis and reduced steatosis). The burnt out stage is an important pathogenic step towards irreversible cirrhosis and HCC. Preliminary data in Sprague Dawley rats indicate that MCLR-elicited burnt out NASH persists 4 weeks after MCLR exposure, in contrast to the control diet group that returned to a baseline phenotype. In addition, MCLR persistently dysregulated matrisome genes (related to fibrosis) and carcinogenesis genes (de-differentiation/stem cell markers) in NASH. Based on these data we hypothesize MCLR-mediated fibrotic and carcinogenic reprogramming of the liver is enhanced in NASH and promotes the development of cirrhosis and HCC. This hypothesis will be tested in two aims. Aim 1 will determine the role of the protein phosphatase 2A (PP2A)-associated pathways in MCLR-mediated stellate cell activation and hepatocyte de-differentiation. Considering the liver is the primary target for MCLR, there is surprising paucity of research investigating these pathways and none have examined the cell type specific effects of PP2A. STUDY 1.1 will define the contribution of specific PP2A-associated pathways responsible for MCLR-elicited hepatocyte and stellate effects using MCLR and PP2 modulators in human HepaRG hepatocytes and human LX2 stellate cells co-cultured as 3D spheroids in static culture or microfluidics. STUDY 1.2 will test the hypothesis that hepatocytes are the primary MCLR target and stellate cells are activated by hepatocyte damage using HepaRG and LX2 cells cultured individually or in combination in transwell plates. STUDY 1.3 will define the effects of PP2A- pathway modulators on MCLR- and NASH diet-elicited liver pathology in rats fed either a control or a NASH diet. Aim 2 will determine the mechanisms and long-term effects of MCLR-elicited fibrotic and carcinogenic reprogramming on cirrhosis and HCC development in healthy versus NASH. STUDY 2.1 will mimic important aspects of the original MCLR tolerable daily intake study including the dose (40 µg/kg), interval (daily), and duration (3 months) in the context of a tumor initiator (diethylnitrosamine) and a NASH diet. These mechanistic studies will be complemented with phosphoproteomics and single nuclei transcriptomics analyses, potentially providing druggable targets. This research will have sustained impact because it will be the first to comprehensively assess MCLR-elicited cirrhosis and HCC in pre-existing liver disease. By mimicking the TDI study, this research may indicate a lower TDI is required for people with pre-existing liver disease.

微囊藻毒素-LR（Microcystin-LR，MclA）是由淡水蓝藻产生的最强、最丰富的一种蓝藻毒素。 暴露于二甲双胍与非酒精性脂肪性肝病（NAFLD）和肝细胞癌相关 （HCC）。在许多国家，NAFLD已迅速上升为最常见的慢性肝病， 是最常见的原发性肝癌类型。我的K99/R 00数据表明，MPEG4推动了 非酒精性脂肪性肝炎（NASH），一种晚期形式的NAFLD，更严重的烧毁表型 （增加的纤维化和减少的脂肪变性）。燃尽阶段是重要的致病步骤， 不可逆肝硬化和HCC。Sprague道利大鼠的初步数据表明，MCLR引起的烧坏 与对照饮食组恢复到基线相比，NASH在暴露于二甲双胍后持续4周， 表型此外，MMPs持续失调的基质体基因（与纤维化相关）， NASH中的致癌基因（去分化/干细胞标志物）。根据这些数据，我们假设 MCLR介导的肝脏纤维化和致癌性重编程在NASH中增强，并促进了肝细胞凋亡。 肝硬化和HCC的发展。这一假设将在两个目标中得到检验。目标1将决定 MCLR介导的星状细胞活化中的蛋白磷酸酶2A（PP 2A）相关途径， 肝细胞去分化。考虑到肝脏是MMPs的主要靶点， 研究这些途径的研究中，没有一个研究PP 2A的细胞类型特异性作用。研究 1.1将定义负责MCLR诱导肝细胞的特定PP 2A相关途径的贡献 在人HepaRG肝细胞和人LX 2星状细胞中使用MMP 2和PP 2调节剂的星形细胞效应 在静态培养或微流体中作为3D球状体共培养的细胞。研究1.2将检验以下假设： 肝细胞是MMPs的主要靶点，使用HepaRG时，肝细胞损伤会激活星状细胞 和在transwell板中单独或组合培养的LX 2细胞。研究1.3将定义PP 2A的作用- 在喂食对照或NASH饮食的大鼠中，MSH和NASH饮食的通路调节剂引起肝脏病理学。 目的2将确定MCLR引起的纤维化和致癌的机制和长期影响 在健康与NASH中肝硬化和HCC发展的重编程。研究2.1将模拟重要 原始Moglycan每日耐受摄入量研究的各个方面，包括剂量（40 µg/kg）、间隔（每日）和 持续时间（3个月）在肿瘤引发剂（二乙基亚硝胺）和NASH饮食的背景下。这些机械 研究将补充磷酸蛋白质组学和单核转录组学分析，可能 提供可下药的目标。这项研究将产生持续的影响，因为它将是第一个 全面评估MCLR引起的肝硬化和HCC在既存肝病中的作用。通过模仿TDI 这项研究可能表明，预先存在肝脏疾病的人需要较低的TDI。