重楼皂苷A直接靶向活化AMPK诱导结直肠癌细胞自噬

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摘要目的：探讨重楼皂苷A （Polyphyllin A, PPA）对结直肠癌细胞自噬的影响及其潜在机制。方法：选用结直肠癌RKO和HRT18细胞株作为细胞模型。用不同浓度PPA处理RKO细胞和HRT18细胞，MTT法检测细胞活力，筛选最佳实验浓度；集落形成实验检测细胞克隆能力；免疫印迹法检测微管相关蛋白LC3=Ⅱ表达、腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）和雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）表达及其磷酸化；免疫荧光检测自噬体形成；分子对接分析PPA与AMPK相互间作用。结果：与0 μmol/L组相比，PPA各浓度组细胞活力明显降低（P<0.05）。与0 μmol/L组相比，PPA组两株细胞克隆形成能力明显降低（P<0.05）；随着PPA浓度逐渐增加，两株细胞中LC3-Ⅱ表达明显上调（P<0.05），自噬体发生聚集，总AMPK、mTOR蛋白无明显变化（P>0.05），而磷酸化AMPK升高、磷酸化mTOR蛋白降低（P<0.05）；PPA与AMPK存在直接结合作用。结论：PPA通过直接靶向活化AMPK诱导结直肠癌细胞发生自噬。

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	彭鹏1
	2
	杨书胜1
	3
	向雨晨1
	3
	文君1
	2
	司渊 1
	3
	刘莹1
	2
	3

关键词 ：重楼皂苷A, 结直肠癌, 自噬, 腺苷酸活化蛋白激酶

Abstract：Objective: To investigate the effect of Polyphyllin A （PPA） on autophage of colorectal cancer （CRC） cells and its potential mechanism. Methods:Colorectal cancer RKO and HRT18 cells were selected as cell model. RKO and HRT18 cells were treated with different concentrations of PPA, MTT assay was used to detect the cell viability, and to screen the optimal experimental concentration. Colony formation assay was used to detect cell cloning ability. Western blotting was used to detect the expression of microtubule-associated protein Ⅱ light chain-3 （LC3Ⅱ）, expression of AMPactivated protein kinase （AMPK） and mammalian target of rapamycin （mTOR） and its phosphorylation. In addition, immunofluorescence was used to detect autophagosome formation. Molecular docking was used to analyze the interaction of PPA and AMPK. Results: Compared with the 0 μmol/L group, the cell viability in each concentration group of PPA was significantly reduced（P<0.05）. Compared with the 0 μmol/L group, the cloning ability of the two cell lines was significantly reduced （P<0.05）. With the gradual increase of PPA concentration, the expression of LC3Ⅱ in the two cell lines gradually increased, and autophagosomes aggregated. The expression of total AMPK and mTOR protein did not change significantly（P＞0.05）, while the phosphorylation of AMPK was increased and the phosphorylation of mTOR was decreased （P<0.05）. The molecular docking showed that PPA directly combined with AMPK. Conclusion: PPA induced autophagy in CRC cells by directly targeting and activating AMPK.

收稿日期: 2020-12-17

基金资助:国家自然科学基金资助项目（82072928，81802387）；国家级大学生创新训练计划项目（202110929004）；湖北医药学院研究生科技创新项目（YC2021104）

通讯作者: 刘莹（通讯作者），教授，硕士生导师，E-mail：liu_ying@ hbmu.edu.cn

作者简介: 彭鹏（1993—），男，硕士研究生

引用本文:

彭鹏1,2，杨书胜1,3，向雨晨1,3，文君1,2，司渊 1,3，刘莹1,2,3. 重楼皂苷A直接靶向活化AMPK诱导结直肠癌细胞自噬[J]. 江苏大学学报:医学版, 2021, 31(04): 290-295. PENG Peng1,2, YANG Shusheng1,3, XIANG Yuchen1,3, WEN Jun1,2, SI Yuan1,3, LIU Ying1,2,3.

Polyphyllin A directly activates AMPK to induce autophagy in colorectal cancer cells

. Journal of Jiangsu University(Medicine Edition), 2021, 31(04): 290-295.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2021/V31/I04/290

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