Department of Internal Medicine III, RWTH Aachen University, Aachen, Germany.
Institute for Experimental Molecular Imaging, RWTH Aachen University, Aachen, Germany.
Hepatology. 2017 Oct;66(4):1242-1257. doi: 10.1002/hep.29275. Epub 2017 Aug 26.
Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45 cells after single injection. Acute CCl -mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45 leukocytes. Pretreatment with CcnE1-siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl for 4-6 weeks. Co-treatment with CcnE1-siRNA once a week was sufficient to continuously block CcnE1 expression and cell-cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1-siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury.
Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257).
肝纤维化的起始和进展需要静止的肝星状细胞（HSCs）的增殖和激活。细胞周期蛋白 E1（CcnE1）是细胞周期依赖性激酶 2（Cdk2）的调节亚基，控制细胞周期再进入。我们最近表明，CcnE1 的基因失活可防止 HSCs 的激活、增殖和存活，并可防止肝纤维化。本研究的目的是使用基于 RNA 干扰（RNAi）的方法将这些发现转化为临床前应用。CcnE1-siRNA（小干扰 RNA）可有效抑制鼠和人 HSC 细胞系以及原代 HSCs 中的 CcnE1 基因表达，导致增殖减少和细胞死亡增加。在 C57BL/6 野生型（WT）小鼠中，使用基于脂质体的载体递送稳定的 siRNA 后，单次注射后约 95%的 HSCs、70%的肝细胞和 40%的 CD45 细胞被靶向。WT 小鼠的急性 CCl 介导的肝损伤诱导内源性 CcnE1 表达和存活的肝细胞和非实质细胞（包括 CD45 白细胞）的增殖。用 CcnE1-siRNA 预处理可使 CcnE1 诱导恢复至健康小鼠的基线水平，这与肝损伤减少、肝细胞和白细胞增殖减少以及整体炎症反应减弱相关。为了诱导肝纤维化，WT 小鼠接受 CCl 挑战 4-6 周。每周一次用 CcnE1-siRNA 联合治疗足以持续阻断肝细胞和非实质细胞的 CcnE1 表达和细胞周期活性，导致肝纤维化和炎症明显改善。重要的是，CcnE1-siRNA 在慢性肝损伤发作后应用也可防止肝纤维化进展。
体内使用 RNAi 靶向 CcnE1 是可行的，具有很强的抗纤维化活性。（Hepatology 2017；66：1242-1257）。
