Yang Yue, Kim Bohkyung, Park Young-Ki, Koo Sung I, Lee Ji-Young
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA.
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA.
Biochim Biophys Acta. 2015 Jan;1850(1):178-85. doi: 10.1016/j.bbagen.2014.10.014. Epub 2014 Oct 23.
Non-alcoholic steatohepatitis (NASH) is a subset of non-alcoholic fatty liver disease, the most common chronic liver disease in the U.S. Fibrosis, a common feature of NASH, results from the dysregulation of fibrogenesis in hepatic stellate cells (HSCs). In this study, we investigated whether astaxanthin (ASTX), a xanthophyll carotenoid, can inhibit fibrogenic effects of transforming growth factor β1 (TGFβ1), a key fibrogenic cytokine, in HSCs.
Reactive oxygen species (ROS) accumulation was measured in LX-2, an immortalized human HSC cell line. Quantitative realtime PCR, Western blot, immunocytochemical analysis, and in-cell Western blot were performed to determine mRNA and protein of fibrogenic genes, and the activation of Smad3 in TGFβ1-activated LX-2 cells and primary mouse HSCs.
In LX-2 cells, ROS accumulation induced by tert-butyl hydrogen peroxide and TGFβ1 was abolished by ASTX. ASTX significantly decreased TGFβ1-induced α-smooth muscle actin (α-SMA) and procollagen type 1, alpha 1 (Col1A1) mRNA as well as α-SMA protein levels. Knockdown of Smad3 showed the significant role of Smad3 in the expression of α-SMA and Col1A1, but not TGFβ1, in LX-2 cells. ASTX attenuated TGFβ1-induced Smad3 phosphorylation and nuclear translocation with a concomitant inhibition of Smad3, Smad7, TGFβ receptor I (TβRI), and TβRII expression. The inhibitory effect of ASTX on HSC activation was confirmed in primary mouse HSCs as evidenced by decreased mRNA and protein levels of α-SMA during activation.
Taken together, ASTX exerted anti-fibrogenic effects by blocking TGFβ1-signaling, consequently inhibiting the activation of Smad3 pathway in HSCs.
This study suggests that ASTX may be used as a preventive/therapeutic agent to prevent hepatic fibrosis.
非酒精性脂肪性肝炎（NASH）是非酒精性脂肪性肝病的一个亚型，是美国最常见的慢性肝病。纤维化是NASH的一个常见特征，由肝星状细胞（HSC）中纤维生成的失调引起。在本研究中，我们调查了叶黄素类胡萝卜素虾青素（ASTX）是否能抑制关键促纤维化细胞因子转化生长因子β1（TGFβ1）对HSC的促纤维化作用。
在永生化人HSC细胞系LX-2中测量活性氧（ROS）积累。进行定量实时PCR、蛋白质印迹、免疫细胞化学分析和细胞内蛋白质印迹，以确定促纤维化基因的mRNA和蛋白质，以及TGFβ1激活的LX-2细胞和原代小鼠HSC中Smad3的激活情况。
在LX-2细胞中，ASTX消除了叔丁基过氧化氢和TGFβ1诱导的ROS积累。ASTX显著降低了TGFβ1诱导的α-平滑肌肌动蛋白（α-SMA）和I型前胶原α1（Col1A1）mRNA以及α-SMA蛋白水平。敲低Smad3显示Smad3在LX-2细胞中α-SMA和Col1A1的表达中起重要作用，但在TGFβ1表达中不起作用。ASTX减弱了TGFβ1诱导的Smad3磷酸化和核转位，同时抑制了Smad3、Smad7、TGFβ受体I（TβRI）和TβRII的表达。ASTX对HSC激活的抑制作用在原代小鼠HSC中得到证实，表现为激活过程中α-SMA的mRNA和蛋白质水平降低。
综上所述，ASTX通过阻断TGFβ1信号传导发挥抗纤维化作用，从而抑制HSC中Smad3途径的激活。
本研究表明ASTX可作为预防/治疗剂用于预防肝纤维化。
