Department of Internal Medicine I, University of Bonn, Sigmund-Freud Straße 25, D-53105 Bonn, Germany.
J Hepatol. 2012 Dec;57(6):1220-7. doi: 10.1016/j.jhep.2012.07.033. Epub 2012 Aug 6.
BACKGROUND & AIMS: Rho-kinase activation mediates cell contraction and increases intrahepatic resistance and consequently portal pressure in liver cirrhosis. Systemic Rho-kinase inhibition decreases portal pressure in cirrhosis, but also arterial pressure. Thus, liver-specific Rho-kinase inhibition is needed. The delivery of Rho-kinase inhibitor to activated hepatic stellate cells reduces fibrosis. It might also relax these contractile cells and therewith decrease intrahepatic resistance. We tested this hypothesis by performing acute experiments in cirrhotic rats.
Cirrhosis models were CCl(4)-intoxication and bile duct ligation. Three hours after injection of the Rho-kinase inhibitor (Y26732) coupled with a carrier (mannose-6-phosphate modified human serum albumin), which targets activated hepatic stellate cells, hemodynamics were analyzed by the colored microsphere technique and direct pressure measurements. The delivery site and effect of Rho-kinase inhibitor were investigated by immunohistochemical stainings, as well as Western blot. Experiments with Rho-kinase inhibitor coupled with unmodified human serum albumin served as untargeted control.
In both models of cirrhosis, the carrier coupled Rho-kinase inhibitor lowered the portal pressure and decreased the hepatic-portal resistance. Immunohistochemical desmin-staining showed the carrier in hepatic stellate cells. The targeted therapy decreased the expression of the phosphorylated substrate of Rho-kinase (moesin) and abolished myosin light chains phosphorylation in fibrotic septae (collagen-staining). The targeted Rho-kinase inhibitor showed no major extrahepatic effects. By contrast, the untargeted Rho-kinase inhibitor elicited severe systemic hypotension.
Activated hepatic stellate cells are crucially involved in portal hypertension in cirrhosis. Targeting of Rho-kinase in hepatic stellate cells not only decreased fibrosis, as previously shown, but also lowers portal pressure acutely without major systemic effects as demonstrated in this study.
Rho 激酶的激活介导了细胞收缩，并增加了肝硬化时的肝内阻力，进而导致门静脉压力升高。全身性 Rho 激酶抑制可降低肝硬化时的门静脉压力，但也会降低动脉血压。因此，需要肝特异性 Rho 激酶抑制。将 Rho 激酶抑制剂递送至活化的肝星状细胞可减少纤维化。它还可能使这些收缩细胞松弛，并由此降低肝内阻力。我们通过在肝硬化大鼠中进行急性实验来检验这一假说。
使用 CCl4 中毒和胆管结扎两种方法建立肝硬化模型。在 Rho 激酶抑制剂（Y26732）与靶向活化肝星状细胞的载体（甘露糖-6-磷酸修饰的人血清白蛋白）偶联 3 小时后，通过彩色微球技术和直接压力测量分析血液动力学。通过免疫组织化学染色和 Western blot 研究 Rho 激酶抑制剂的递药部位和作用。将 Rho 激酶抑制剂与未修饰的人血清白蛋白偶联作为非靶向对照。
在两种肝硬化模型中，载体偶联的 Rho 激酶抑制剂均降低了门静脉压力和肝门静脉阻力。肝星状细胞中的免疫组织化学染色显示了载体。靶向治疗降低了 Rho 激酶磷酸化底物（肌球蛋白结合蛋白）的表达，并消除了纤维间隔（胶原染色）中的肌球蛋白轻链磷酸化。靶向 Rho 激酶抑制剂未显示出主要的肝外作用。相比之下，非靶向 Rho 激酶抑制剂引起严重的全身性低血压。
活化的肝星状细胞在肝硬化门静脉高压中起着至关重要的作用。肝星状细胞中 Rho 激酶的靶向治疗不仅如先前所示减少了纤维化，而且还如本研究所示，在没有明显全身作用的情况下急性降低了门静脉压力。
