前胡提取物角型吡喃骈香豆素对U266细胞增殖和凋亡的影响

Effect of angular pyranocoumarin isolated from peucedanum praeruptorum on the proliferation and apoptosis of U266 cells

目的探讨白花前胡提取物角型吡喃骈香豆素（＋／一）-4＇-O-acetyl-3’-O-angeloyl-cis-khellactone（APC）对骨髓瘤细胞株U266细胞增殖和凋亡的影响。方法采用石油醚法提取APC，高效液相色谱检测其纯度，磁共振谱法进行结构鉴定。采用不同浓度的APC（0、10、20、30、40μg／m1）作用于U266细胞不同时间（24、48h），CCK-8法检测其对细胞增殖的影响；作用于U266细胞24h后，采用AnnexinV／PI流式细胞术及Hochest33342荧光染色法检测细胞凋亡；Westernblot法检测caspase-3、caspase-8、AKT、p-AKT、ERK、p-ERK蛋白活性的变化；RT-PCR法检测人端粒酶逆转录酶（hTERT）亚基表达的变化。结果高效液相色谱检测APC纯度为98．8％。APC可抑制U266细胞的增殖，抑制率呈一定的浓度和时间依赖性，与空白对照组比较，抑制率差异均有统计学意义（P值均〈0．01）；10、20、30、40μg／ml APC作用24h后U266细胞凋亡率分别为（5．63±0．21）％、（16．07±2．27）％、（24．83±1．65）％、（43．46±2．91）％，与空白对照组[（2．50±0．13）％]比较，差异均有统计学意义（P值均〈0．05）；caspase-3、caspase-8蛋白表达随药物浓度增加呈上调趋势，AKT、ERK变化不明显，P-AKT、p-ERK蛋白表达随药物浓度增加而明显下调；随APC药物浓度增加，hTERTmRNA的表达下调。结论APC能抑制U266细胞增殖并诱导其凋亡，其凋亡机制可能与caspase-8、caspase．3、p-AKT、p-ERK蛋白表达变化以及hTERT亚基表达下调相关。

Objective To investigate the effects of angular pyranocoumarin （±）-4＇-O- acetyl-3＇-O- angeloyl-cis-khellactone （APC） extracted from peucedanum praeruptoruon on the proliferation and apoptosis of U266 cells, and to explore its related mechanism. Methods APC was extracted by petroleum ether technique, and its purity was tested by high performance liquid chromatography, and its chemical structure was identified by magnetic resonance spectroscopy. U266 cells were treated with APC in various concentrations （0, 10, 20, 30, 40 μg/ml） for different durations（24 and 48 h）. The inhibitive effect of APC on cell growth was detected by CCK-8 method. After U266 cells were incubated with APC （0, 10, 20, 30, 40 μg/ml） for 24 h, the apoptosis of cells were observed by flow cytometry stained with Annexin V/PI and Hochest33342; the expression levels of caspase-3, 8, ERK, p-ERK, AKT and p-AKT protein were assayed by Western blot; the expression of hTERT mRNA was measured by RT-PCR. Results The purity of APC identified by magnetic resonance imaging was 98.8%. The proliferation of U266 cells was inhibited, and the apoptosis was induced in a time- and/or dose- dependent manner after treatment with APC. APC could upregulate the caspase-8, 3 protein expression and downregulate the p-ERK, p-AKT protein expression along with the increase of APC dose. APC also could downregulate the hTERT mRNA expression. Conclusion Angular pyranocoumarin APC could inhibit the proliferation and induce the apoptosis of U266 cells. The probable mechanism might be achieved by upregulating caspase-8, 3 protein expression and downregulating p-ERK, P-AKT protein and the hTERT mRNA expression.