Center of Research & Development of New Drugs, Guangxi Traditional Chinese Medical University, Nanning 530001, Guangxi Zhuang Autonomous Region, China.
J Ethnopharmacol. 2011 Jan 27;133(2):765-72. doi: 10.1016/j.jep.2010.11.001. Epub 2010 Nov 10.
This study was aimed to investigate the effects of Progallin A isolated from the acetic ether part of the leaves of Phyllanthus emblica L. on apoptosis in human hepatocellular carcinoma BEL-7404 cells and its possible mechanisms, and to measure the immune toxicity of Progallin A in vitro.
Progallin A was isolated from the acetic ether part of the leaves of Phyllanthus emblica L. with column chromatography. The proliferation of spleen lymphocytes and the viability of BEL-7404 cells were assessed with MTT assay. Inverted microscope, light microscope and fluorescence microscope were utilized to observe the morphological changes of BEL-7404 cells respectively. AnnexinV/PI double labeling and TUNEL assay were used to detect early apoptosis and DNA fragmentations of BEL-7404 cells respectively. In addition, cell cycle distribution was analyzed by using flow cytometry (FCM). Bax and Bcl-2 protein levels were determined by immunofluorescence staining and western blot respectively.
The results showed that Progallin A had low immune toxicity and the proliferation of BEL-7404 cells was strongly inhibited by Progallin A in a time- and dose-dependent manner and that the characteristics of apoptosis of BEL-7404 cells were observed. The results also showed that apoptosis rates and the number of apoptotic cells significantly increased with prolongation of the action time. The results of flow cytometry (FCM) indicated that Progallin A induced significant G1/M and G2/M arrest of BEL-7404 cells. Immunofluorescence staining and western blot showed that the expression of Bax was found to be noticeably up-regulated and the expression of Bcl-2 was down-regulated significantly.
These results indicate that Progallin A has low immune toxicity in vitro and induces apoptosis of human hepatocellular carcinoma BEL-7404 cells which is related to the G1/M and G2/M arrest, and it exerts its apoptotic effect by up-regulation of Bax expression and down-regulation of Bcl-2 expression.
本研究旨在探讨从余甘子叶的乙酸乙酯部分分离得到的 Progallin A 对人肝癌 BEL-7404 细胞凋亡的影响及其可能的机制，并测量 Progallin A 的体外免疫毒性。
Progallin A 采用柱层析从余甘子叶的乙酸乙酯部分分离得到。MTT 法检测脾淋巴细胞增殖和 BEL-7404 细胞活力。倒置显微镜、光镜和荧光显微镜分别观察 BEL-7404 细胞形态变化。AnnexinV/PI 双标记和 TUNEL 检测分别检测 BEL-7404 细胞的早期凋亡和 DNA 片段化。此外，流式细胞术（FCM）分析细胞周期分布。免疫荧光染色和 Western blot 分别测定 Bax 和 Bcl-2 蛋白水平。
结果表明，Progallin A 具有低免疫毒性，Progallin A 呈时间和剂量依赖性强烈抑制 BEL-7404 细胞增殖，并观察到 BEL-7404 细胞凋亡的特征。结果还表明，随着作用时间的延长，凋亡率和凋亡细胞数显著增加。FCM 结果表明，Progallin A 诱导 BEL-7404 细胞发生明显的 G1/M 和 G2/M 期阻滞。免疫荧光染色和 Western blot 显示，Bax 表达明显上调，Bcl-2 表达明显下调。
这些结果表明，Progallin A 在体外具有低免疫毒性，诱导人肝癌 BEL-7404 细胞凋亡，与 G1/M 和 G2/M 期阻滞有关，通过上调 Bax 表达和下调 Bcl-2 表达发挥其凋亡作用。
