Thirunavukkarasu Chinnasamy, Watkins Simon, Harvey Stephen A K, Gandhi Chandrashekhar R
Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, E-1542 BST, 200 Lothrop street, Pittsburgh, PA 15213, USA.
J Hepatol. 2004 Oct;41(4):567-75. doi: 10.1016/j.jhep.2004.06.023.
BACKGROUND/AIMS: During liver injury, reactive oxygen species (ROS) are produced by the resident macrophages (Kupffer cells) and infiltrating blood cells such as neutrophils. ROS cause transformation of desmin-positive quiescent hepatic stellate cells (HSCs) into the proliferating activated phenotype that expresses alpha-smooth muscle actin (alpha-SMA). The highly fibrogenic and contractile activated HSCs (aHSCs) produce various cytokines and growth factors, and play important role in the pathophysiology of chronic liver disease. However, apoptotic aHSCs are also observed during active fibrogenesis in the injured liver. Therefore, we investigated the mechanisms of apoptosis of aHSCs in relation to ROS.
HSCs, isolated from normal rat liver, were activated in culture and effects of superoxide were determined between subcultures 3 and 5.
Treatment with superoxide caused apoptosis of aHSCs as determined by flow cytometry, TUNEL assay and DNA laddering analysis. The mechanisms of superoxide-induced apoptosis involved release of cytochrome c, increased Bax expression, increased caspase-3 activity, and hydrolysis of polyADP-ribose polymerase. Superoxide also increased the expression of antiapoptotic Bcl-xL and nuclear translocation of NFkappaB. Caspase-3 inhibitor (DEVD-fmk) and antioxidants (N-acetylcysteine, vitamin E and superoxide dismutase) inhibited superoxide-induced apoptosis.
Superoxide-induced apoptosis of aHSCs may be a novel mechanism of limiting chronic fibrotic liver injury.
背景/目的：在肝损伤过程中，活性氧（ROS）由驻留巨噬细胞（库普弗细胞）和浸润的血细胞（如中性粒细胞）产生。ROS导致结蛋白阳性的静止肝星状细胞（HSCs）转变为表达α-平滑肌肌动蛋白（α-SMA）的增殖活化表型。高度纤维化且具有收缩性的活化HSCs（aHSCs）产生多种细胞因子和生长因子，并在慢性肝病的病理生理学中发挥重要作用。然而，在受损肝脏的活跃纤维化过程中也观察到凋亡的aHSCs。因此，我们研究了与ROS相关的aHSCs凋亡机制。
从正常大鼠肝脏分离的HSCs在培养中被激活，并在传代培养3至5次之间测定超氧化物的作用。
通过流式细胞术、TUNEL分析和DNA梯状分析确定，超氧化物处理导致aHSCs凋亡。超氧化物诱导凋亡的机制包括细胞色素c的释放、Bax表达增加、caspase-3活性增加以及聚ADP-核糖聚合酶的水解。超氧化物还增加了抗凋亡蛋白Bcl-xL的表达和NFκB的核转位。caspase-3抑制剂（DEVD-fmk）和抗氧化剂（N-乙酰半胱氨酸、维生素E和超氧化物歧化酶）抑制超氧化物诱导的凋亡。
超氧化物诱导的aHSCs凋亡可能是限制慢性纤维化肝损伤的一种新机制。
