Department of Pharmacology, Tianjin Medical University, TianJin, China.
PLoS One. 2011;6(10):e26500. doi: 10.1371/journal.pone.0026500. Epub 2011 Oct 28.
Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen. p53, the "guardian of the genome", is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apoptosis, senescence and DNA repair. Artesunate is a derivative of Artemisinin, Scholars had found it had more extensive pharmacological effects past 10 years. However, little is known about the expression of p53 in the effects of Artesunate on induction of apoptosis and inhibition of proliferation in rat HSCs.
METHODOLOGY/PRINCIPAL FINDINGS: Isolated and cultured rat primary HSCs in the flask for 10 days to make cells activated. HSCs were divided into two groups: experimental groups and control groups, experimental groups included with various concentrations of Artesunate (125, 150, 175, 200, 225 µmol/L) for 24, 48 and 72 hours. Analysis of MTT revealed that activated HSCs treated with various concentrations of Artesunate (150-225 µmol/L) were inhibited on dose and time-effect relationships; Concentration of hydroxyproline in supernatant was detected by digestive method; Analysis of flow cytometry demonstrated that Artesunate could arrest cell cycle in G1 and induce apoptosis; The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33258 dye; The expression of p53 were up-regulated showed by western blotting and RT-PCR.
Artesunate could inhibit HSCs proliferation in dose-dependent and time-dependent manners in vitro through increase the expression of p53.
肝星状细胞（HSCs）的激活通过增加胶原的产生在肝硬化的发展中起重要作用。p53 是“基因组的守护者”，是一种转录因子，可与数百个基因的启动子区域结合，从而激活或抑制基因表达。因此，p53 通过诱导细胞周期停滞、细胞凋亡、衰老和 DNA 修复来发挥肿瘤抑制作用。青蒿琥酯是青蒿素的衍生物，学者们发现它在过去 10 年具有更广泛的药理作用。然而，关于 p53 在青蒿琥酯诱导大鼠 HSCs 凋亡和抑制增殖中的表达知之甚少。
方法/主要发现：将分离并培养的大鼠原代 HSCs 在培养瓶中培养 10 天，使其活化。将 HSCs 分为两组：实验组和对照组，实验组包括用不同浓度的青蒿琥酯（125、150、175、200、225µmol/L）处理 24、48 和 72 小时。MTT 分析显示，用不同浓度的青蒿琥酯（150-225µmol/L）处理的活化 HSCs 呈剂量和时间依赖性抑制；用消化法检测上清液中羟脯氨酸的浓度；用流式细胞术分析显示，青蒿琥酯可使细胞周期停滞在 G1 期并诱导细胞凋亡；用 Hoechst 33258 染料对凋亡细胞的核形态变化进行 DNA 染色评估；用 Western blot 和 RT-PCR 显示 p53 的表达上调。
青蒿琥酯可通过上调 p53 的表达，在体外以剂量和时间依赖的方式抑制 HSCs 的增殖。
