Department of Cell Biology, Vrije Universiteit Brussel, Brussels, Belgium.
Br J Pharmacol. 2010 Jan 1;159(2):304-15. doi: 10.1111/j.1476-5381.2009.00477.x. Epub 2009 Dec 18.
Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts.
Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca(2+) release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments.
In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca(2+) release. Finally, it promoted wound-induced cell migration.
By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.
肌球蛋白-II 特异性 ATP 酶抑制剂 blebbistatin 已被用于抑制含有非肌肉肌球蛋白的无脊椎动物和哺乳动物肌肉制剂的收缩。激活的肝星状细胞具有收缩特性，在肝纤维化和门静脉高压的病理生理学中发挥重要作用。因此，肝星状细胞被认为是治疗靶细胞。本研究旨在研究 blebbistatin 在小鼠肝星状细胞向收缩型肌成纤维细胞转化过程中的作用。
通过相差显微镜评估 blebbistatin 对细胞形态的影响。通过双重免疫荧光染色研究细胞应力纤维和黏着斑，并通过荧光显微镜观察。通过硅酮皱纹形成测定法和胶原凝胶收缩测定法检测收缩力的产生。通过 Fluo-4 测量内皮素-1 引起的细胞内 Ca(2+)释放。通过划痕愈合实验测量细胞迁移。
在培养激活的肝星状细胞中，发现 blebbistatin 改变了细胞形态和功能。在 blebbistatin 存在下，星状细胞变小，获得树突状形态，肌球蛋白 IIA 含量减少的应力纤维和含 vinculin 的黏着斑减少。此外，blebbistatin 抑制硅酮皱纹形成，减少胶原凝胶收缩并阻断内皮素-1 诱导的细胞内 Ca(2+)释放。最后，它促进了伤口诱导的细胞迁移。
通过抑制肌球蛋白 II ATP 酶，blebbistatin 对激活的肝星状细胞的形态和功能有深远的影响。我们的数据表明，肌球蛋白 II 可能是治疗肝纤维化和门静脉高压的治疗靶点。
