Xie Bu-Shan, He Xing-Xing, Ai Zheng-Lin, Yao Shu-Kun
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, P.R. China.
Graduate School, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, P.R. China.
Mol Med Rep. 2014 Jun;9(6):2547-53. doi: 10.3892/mmr.2014.2107. Epub 2014 Apr 2.
Matrine, one of the main components extracted from Sophora flavescens, has exhibited pharmacological effects on the differentiation in rat liver oval cells. However, its function and mechanism have not yet been fully elucidated. To further investigate them, an in vitro model was established using a rat liver oval cell line called WB-F344 and treated with matrine. Initially, a significant increase in the number of monodansylcadaverine-positive cells and in the levels of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, which is a specific marker for detecting autophagy, was observed in matrine-treated cells. This indicated that autophagy was stimulated by matrine, which was further confirmed by transmission electron microscopy. Additionally, the apoptotic oval cells were easily detected under matrine treatment using an Annexin-V-fluorescein isothiocyanate/propidium iodide assay, indicating that autophagy and apoptosis were synchronously induced by matrine. A decrease in B-cell lymphoma (Bcl-2) mRNA expression, but an increase in Bcl2-associated X protein (Bax) mRNA expression were observed in matrine-treated cells, which led to an upregulation of the Bax/Bcl-2 ratio, a molecular marker for determining the extent of apoptosis. Next, the molecular mechanism of matrine-induced autophagy and apoptosis was analyzed in WB-F344 cells. β-catenin degradation was downregulated by matrine and rapamycin, a foregone chemical agonist of autophagy, whereas it was upregulated by 3-methyladenine, a specific inhibitor of autophagy. Additionally, β-catenin activation induced an increase in LC3-II levels and reversed the Bax/Bcl-2 mRNA ratio under matrine treatment, whereas inhibition of β-catenin by RNA interference induced a decrease of the LC3-II amount and of the Bax/Bcl-2 mRNA ratio. Finally, matrine treatment attenuated p53; however, with little or no change in LC3-II levels, but a decrease in β-catenin levels occurred in WB-F344 cells upon treatment with pifithrin-α, a chemical inhibitor of p53, revealing that p53, interfering with β-catenin, may not be involved in matrine-induced autophagy in WB-F344 cells. These results demonstrate that β-catenin is involved in matrine-induced autophagy and apoptosis in WB-F344 cells, while β-catenin is negatively regulated by autophagy and positively by p53, indicating that β-catenin may be involved in the crosstalk between autophagy and apoptosis in WB-F344 cells.
苦参碱是从苦参中提取的主要成分之一，已显示出对大鼠肝卵圆细胞分化的药理作用。然而，其功能和机制尚未完全阐明。为了进一步研究它们，使用一种名为WB-F344的大鼠肝卵圆细胞系建立了体外模型并用苦参碱进行处理。最初，在苦参碱处理的细胞中观察到单丹磺酰尸胺阳性细胞数量显著增加以及微管相关蛋白1A/1B轻链3（LC3）-II水平升高，LC3-II是检测自噬的特异性标志物。这表明苦参碱刺激了自噬，这通过透射电子显微镜进一步得到证实。此外，使用膜联蛋白-V-异硫氰酸荧光素/碘化丙啶检测法在苦参碱处理下很容易检测到凋亡的卵圆细胞，表明苦参碱同步诱导了自噬和凋亡。在苦参碱处理的细胞中观察到B细胞淋巴瘤（Bcl-2）mRNA表达下降，但Bcl2相关X蛋白（Bax）mRNA表达增加，这导致Bax/Bcl-2比值上调，Bax/Bcl-2比值是确定凋亡程度的分子标志物。接下来，在WB-F344细胞中分析了苦参碱诱导自噬和凋亡的分子机制。苦参碱和雷帕霉素（一种已知的自噬化学激动剂）下调了β-连环蛋白的降解，而3-甲基腺嘌呤（一种自噬特异性抑制剂）上调了β-连环蛋白的降解。此外，β-连环蛋白激活在苦参碱处理下诱导LC3-II水平升高并逆转了Bax/Bcl-2 mRNA比值，而通过RNA干扰抑制β-连环蛋白则导致LC3-II量和Bax/Bcl-2 mRNA比值下降。最后，苦参碱处理使p53减弱；然而，在用pifithrin-α（一种p53化学抑制剂）处理后，WB-F344细胞中LC3-II水平几乎没有变化，但β-连环蛋白水平下降，这表明p53干扰β-连环蛋白，可能不参与苦参碱诱导的WB-F344细胞自噬。这些结果表明，β-连环蛋白参与了苦参碱诱导的WB-F344细胞自噬和凋亡，而β-连环蛋白受到自噬的负调控和p53的正调控，表明β-连环蛋白可能参与了WB-F344细胞自噬和凋亡之间的相互作用。
