■乙型肝炎病毒(HBV)感染发展为急性或慢性肝病,从脂肪变性发展而来的,肝炎,和纤维化到终末期肝病,如肝硬化和肝细胞癌(HCC)。在慢性肝病中,基质硬度增加伴随纤维化,并且被认为是疾病进展的强预测因子。这项研究的目的是建立增强的肝硬度调节纤维化肝组织中HBV感染性的机制。
■对于体外研究,HBV转染的HepG2.2.15细胞在由2kPa(软)或24kPa(硬)刚性的聚电解质多层膜包被的聚二甲基硅氧烷凝胶上培养,模仿健康或纤维化肝脏的硬度。对于体内研究,通过每周两次注射CCl4,持续6周,在C57Bl/6亲本和HBV+转基因(HBVTg)小鼠中诱导肝纤维化。
■我们发现,在坚硬的凝胶附着肝细胞中,HBV标志物水平较高,伴随着细胞上清液中OPN含量的上调以及抗病毒干扰素刺激基因(ISGs)的抑制。这表明必要的“纤维化僵硬度”增加了骨桥蛋白(OPN)的含量,并释放和抑制了抗病毒的先天免疫,导致肝细胞中HBV标志物表达随后升高。体外结果由来自施用CCl4(HBVTgCCl4)的HBVTg小鼠的数据证实。这些小鼠表现出更高的HBVRNA,DNA,HBV核心抗原(HBcAg),和HBV表面抗原(HBsAg)肝纤维化诱导后的水平由Col1a1,SMA上升判断,MMPs,和TIMPsmRNA,并通过增加肝脏硬度。重要的是,CCl4诱导肝细胞的促纤维化激活,与对照小鼠相比,HBVTg小鼠的肝脏硬度更高。HBV标志物和OPN水平的升高对应于HBVTgCCl4小鼠与HBVTg对照小鼠中ISG活化的降低。
■根据我们的数据,我们得出的结论是,肝脏硬度增强OPN水平,以限制肝细胞中的抗病毒ISG激活,并促进HBV感染性的增加,从而有助于终末期肝病的进展。
Hepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue.
For in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks.
We found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite \"fibrotic\" stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice.
Based on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression.