双氢青蒿素经AMPK/mTOR通路逆转卵巢癌细胞A2780/DDP顺铂耐药的机制研究  

REVERSAL EFFECT AND ITS MECHANISM OF DIHYDROARTEMISININ ON CISPLATIN RESISTANCE IN OVARIAN CANCER CELL LINES A2780/DDP BY AMPK/MTOR PATHWAY

作　　者：童焰[1] 肖沁 赖智清 刘朝霞[1] TONG Yan;XIAO Qin;LAI Zhiqing;LIU Zhaoxia(The Second Affiliated Hospital of Nanchang University,Nanchang,Jiangxi 330006,China)

机构地区：[1]南昌大学第二附属医院,江西南昌330006

出　　处：《井冈山大学学报(自然科学版)》2025年第1期51-57,共7页Journal of Jinggangshan University (Natural Science)

基　　金：国家自然科学基金项目(82260298);江西省中医院管理局科技计划项目(2021A228)。

摘　　要：为了探讨双氢青蒿素(dihydroartemisinin,DHA)通过AMPK/mTOR通路逆转卵巢癌细胞A2780/DDP顺铂耐药的分子机制。通过体外培养人卵巢癌A2780和顺铂耐药A2780/DDP细胞,给予不同浓度的DHA(5、10、20、40、80μmol/L),培养48 h后,采用CCK-8法计算不同浓度DHA对A2780和A2780/DDP细胞增殖的抑制作用,选取最适的DHA浓度进行后续实验;选取单药DDP组(0.625、1.25、2.5、5、10 mg/L),最适DHA浓度与顺铂各浓度联用,计算顺铂对A2780和A2780/DDP细胞的IC50,并计算DHA对A2780和A2780/DDP细胞的耐药逆转倍数;DAPI法检测DHA对A2780/DDP细胞凋亡的影响;转染AMPKαsiRNA后,Western blot法检测DHA对A2780/DDP细胞AMPKα、m TOR、p-mTOR、HIF-1α、P-gp蛋白的影响。实验结果显示,随DHA浓度升高,A2780/DDP细胞的生长受到明显抑制,且具有浓度依赖性,10μmol/L是DHA最佳逆转耐药浓度;DDP+DHA组中顺铂对A2780/DDP的IC50值为4.95 mg/L,单用DDP组的IC50值为20.33 mg/L,DHA的逆转耐药倍数为4.11;DDP+DHA组呈现出凋亡形态,体积缩小,出现核固缩或碎裂,呈亮蓝色,且细胞凋亡率明显增加;敲低AMPK基因的表达后,p-mTOR、HIF-1α、P-gp表达明显增加,A2780/DDP细胞的生长速度明显增加。DHA可能通过调控AMPK/mTOR信号通路,下调HIF-1α、P-gp的表达,抑制A2780/DDP细胞的增殖并促进其凋亡,逆转耐药的发生。In order to explore the molecular mechanism of dihydroartemisinin(DHA)reversing the cisplatin resistance of ovarian cancer cells A2780/DDP through AMPK/mTOR pathway.Human ovarian cancer A2780 and cisplatin-resistant A2780/DDP cells were cultured with DHA under different concentrations(5,10,20,40,80μmol/L)in vitro.The CCK-8 method was used to calculate the inhibitory effect of DHA on the proliferation of A2780 and A2780/DDP cells after 48 hours,and the most appropriate DHA concentration was selected for the subsequent experiments.There were single DDP groups(0.625,1.25,2.5,5 and 10 mg/L)and the groups of DHA combined with cisplatin of each concentration.The IC50 of cisplatin and the resistance reversal fold of DHA on A2780 and A2780/DDP cells were calculated.DAPI assay was used to detect the effect of DHA on apoptosis of A2780/DDP.Effect of DHA on AMPKα,m TOR,p-m TOR,HIF-1α,and P-gp in A2780/DDP cells were detected by Western blot after treated with AMPKαsi RNA and the agonist of AMPK.The results showed that the growth of A2780/DDP cells was significantly inhibited with increasing DHA concentration in a concentration-dependent manner.And the optimal concentration of DHA to reverse drug resistance was 10μmol/L.The IC50 of cisplatin against A2780/DDP in the DDP+DHA group was 4.95 mg/L and 20.33 mg/L in the DDP group.And the resistance reversal fold of DHA was 4.11.The DDP+DHA group showed apoptotic morphology with reduced size,nuclear consolidation or fragmentation,bright blue color and significantly increased apoptosis rate.After knocking down the expression of AMPK gene,the expression of m TOR phosphorylation,HIF-1αand P-gp was increased,and the growth rate of A2780/DDP cells was significantly increased.Meanwhile,after using m TOR,HIF-1αand P-gp agonists respectively,the expression of their downstream HIF-1αand P-gp increased significantly,and the growth rate of A2780/DDP cells increased significantly compared with the DHA group.DHA may inhibit the proliferation and promote the apoptosis of A2780/DDP cells

关 键 词：双氢青蒿素 卵巢癌 顺铂耐药 AMPK/mTOR信号通路 

分 类 号：Q256[生物学—细胞生物学] Q354