倪连丽 闫爽 肖怀 巫秀美 何苗 李玥

摘 要 目的：探討美洲大蠊提取物对人非小细胞肺癌细胞A549增殖、凋亡的影响及其可能机制。方法：将美洲大蠊干燥虫体以90%乙醇冷浸提取后，经聚酰胺柱色谱以水-甲醇梯度洗脱，得20%、30%、40%、50%、60%、70%、80%、90%甲醇洗脱部位（YS-A～H）。采用MTT法筛选活性部位，并检测不同剂量活性部位作用后的细胞增殖抑制率;采用流式细胞术检测不同剂量活性部位作用后的细胞凋亡、细胞周期和线粒体膜电位变化情况。结果：YS-A～H的半数抑制浓度分别为（95.25±8.42）、（129.93±7.24）、（221.28±12.68）、（275.39±14.87）、（276.76±16.32）、（31.90±5.34）、（163.15±6.97）、（122.81±8.36）μg/mL，以YS-F的活性最强。经YS-F 3、9、27、81 μg/mL作用24、48、72 h后，各时间点的细胞增殖抑制率均显著升高，且药物作用48、72 h时的细胞增殖抑制率显著高于同组24 h，作用72 h时的细胞增殖抑制率均显著高于同组48 h（P<0.01）。除YS-F 3 μg/mL作用24 h、YS-F 9 μg/mL作用72 h对坏死晚期细胞百分比，YS-F 3 μg/mL作用24 h对G2/M期细胞比例以及YS-F 3 μg/mL作用48 h对细胞线粒体膜电位降低率均无显著影响（P>0.05）外，其余各剂量组各时间点凋亡早期、凋亡晚期和坏死早期、坏死晚期细胞百分比以及Sub-G0/G1期、S期细胞比例均显著升高，G0/G1期、G2/M期细胞比例均显著降低（P<0.01）;且药物作用48、72 h时各剂量组凋亡早期、凋亡晚期和坏死早期、坏死晚期细胞百分比（除YS-F 9 μg/mL作用72 h时的坏死晚期细胞百分比外）以及Sub-G0/G1期、G2/M期（除48 h YS-F 3、9 μg/mL组外）细胞比例均显著高于同组24 h，而G0/G1期、S期、G2/M期（除48 h YS-F 9 μg/mL组外）细胞比例均显著低于同组24 h（P<0.01）;药物作用72 h时各剂量组凋亡早期、凋亡晚期和坏死早期、坏死晚期细胞百分比（除YS-F 27 μg/mL作用72 h时的凋亡晚期和坏死早期细胞百分比以及YS-F 3、9 μg/mL作用72 h时的坏死晚期细胞百分比显著降低外）以及S期（除72 h YS-F 3 μg/mL组外）、Sub-G0/G1期细胞比例均显著高于同组48 h，而G0/G1期、G2/M期细胞比例均显著低于同组48 h（P<0.01）。经YS-F 9、27、81 μg/mL作用48 h后，细胞线粒体膜电位降低率均显著升高，且YS-F 27、81 μg/mL组显著高于YS-F 9 μg/mL组，YS-F 81 μg/mL组显著高于YS-F 27 μg/mL组。结论：YS-F可通过阻滞细胞从S期向G2/M期转化、降低线粒体膜电位等途径来抑制A549细胞的增殖并促进其凋亡，且这种作用具有时间或剂量依赖性。

关键词 美洲大蠊;非小细胞肺癌;A549细胞;增殖;凋亡;细胞周期;线粒体膜电位

ABSTRACT   OBJECTIVE： To investigate the effects of Periplaneta americana extract on the proliferation and apoptosis of human non-small cell lung cancer A549 cells as well as its possible mechanism. METHODS： The dry bodies of P. americana were soaked with 90% ethanol and eluted with gradient water-methanol by polyamide column chromatography. The 20%， 30%， 40%， 50%， 60%， 70%， 80%， 90% methanol elution sites （YS-A-H） were obtained. MTT method was used to screen the active site， and the inhibition rate of different doses of active site was detected. Flow cytometry was adopted to detect cell apoptosis， cell cycle and mitochondrial membrane potential of cells after treated with different doses of active site. RESULTS： Half inhibition concentrations of YS-A-H were （95.25±8.42）， （129.93±7.24）， （221.28±12.68）， （275.39±14.87）， （276.76±16.32）， （31.90±5.34）， （163.15±6.97）， （122.81±8.36） μg/mL， respectively. YS-F had the strongest activity. After treated with 3， 9， 27， 81 μg/mL YS-F for 24， 48， 72 h， cell proliferation inhibitory rate was increased significantly at different time points; after treated for 48， 72 h， that was significantly higher than same group after treated for 24 h; after 72 h treatment， that was significantly higher than same group after 48 h treatment （P<0.01）.  There was no significant effect of 24 h treatment of 3 μg/mL YS-F and 72 h treatment of 9 μg/mL YS-F on the percentage of cells in the late stage of necrosis， 24 h treatment of 3 μg/mL YS-F on the percentage of cells in G2/M phase and 48 h treatment of 3 μg/mL YS-F on the reduction rate of mitochondrial membrane potential （P>0.05）. The percentage of cells in the early stage of apoptosis， the late stage of apoptosis and the early stage of necrosis， the late stage of necrosis， as well as the percentage of cells in the Sub-G0/G1 and S phase at each time point were significantly increased in other different doses groups， while the percentage of cells in G0/G1 and G2/M phase was decreased significantly （P<0.01）. In each dose group， the percentage of cells in the early stage of apoptosis， the late stage of apoptosis and the early stage of necrosis， the late stage of necrosis （except for the percentage of cells in the late stage of necrosis treated with YS-F 9 μg/mL for 72 h） and the percentage of cells in Sub-G0/G1 phase， G2/M phase （except for YS-F 27， 81 μg/mL for 48 h） after treated for 48， 72 h were significantly higher than same group after 24 h of treatment; the percentage of cells in G0/G1 phase， S phase and G2/M phase （except for YS-F 9 μg/mL for 48 h） after treated for 48， 72 h were significantly lower than same group after 24 h of treatment （P<0.01）; the percentage of cells in the early stage of apoptosis， the late stage of apoptosis and the early stage of necrosis， the late stage of necrosis （except for the percentage of cells in the late stage of apoptosis and early stage of necrosis when treated with YS-F 27 μg/mL for 72 h， the percentage of cells in the late stage of necrosis when treated with YS-F 3，9 μg/mL for 72 h were decreased significantly） and the percentage of cells in S phase （except for YS-F 3 μg/mL for 72 h） and Sub-G0/G1 phase after treated for 72 h were significantly higher than same group after 48 h of treatment， while the percentage of cells in G0/G1 and G2/M phase were significantly lower than same group after 48 h of treatment （P<0.01）. After treated with YS-F 9， 27， 81 μg/mL for 48 h， the reduction rate of cell mitochondrial membrane potential was increased significantly; YS-F 27， 81 μg/mL groups were significantly higher than YS-F 9 μg/mL group， and YS-F 81 μg/mL group was significantly higher than YS-F 27 μg/mL group. CONCLUSIONS： YS-F can inhibit the proliferation and promote the apoptosis of A549 cells by  preventing cell transformation from S phase to G2/M phase， and reducing mitochondrial membrane potential， in time-dependent or dose-dependent manner.

KEYWORDS   Periplaneta americana; Non-small cell lung cancer cell; A549 cells; Proliferation; Apoptosis; Cell cycle; Mitochondrial membrane potential

美洲大蠊（Periplaneta americana L.）属昆虫纲蜚蠊科昆虫[1]，最早记载于《神农本草经》，“主血瘀症坚，寒热，破积聚，喉咽闭，内寒无子”[2]。现代药理研究表明，美洲大蠊具有抗肿瘤、抑菌、修复受损组织等作用[3-5];其提取物对呼吸系统肿瘤细胞具有明显的抑制作用[6]。张丹等[7]报道，美洲大蠊多肽提取物可通过上调人肝癌细胞SMMC-7721中促凋亡蛋白Bax的表达、下调抗凋亡蛋白Bcl-2的表达，从而发挥促进肝癌细胞凋亡的作用;CHEN PP等[8]研究发现，由美洲大蠊提取物制得的康复新液可通过激活内质网应激和自噬来促进胃癌细胞的凋亡。……