摘要
【目的】探讨青蒿琥酯抗急性髓系白血病(AML)的作用。【方法】取对数生长期AML细胞株MV4-11,用不同浓度青蒿琥酯分别处理24、48、72 h,四甲基偶氮唑盐(MTT)法测定细胞增殖抑制率,选择青蒿琥酯处理48 h对MV4-11细胞的半数抑制浓度(IC50)进行后续实验。将细胞随机分为对照组、青蒿琥酯组、复合物C组与青蒿琥酯+复合物C组,MTT法测定细胞存活率,流式细胞技术检测细胞周期与凋亡情况,定量聚合酶链反应(q PCR)法检测细胞中B淋巴细胞瘤-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)的mRNA水平,蛋白免疫印迹(Western Blot)法检测细胞腺苷酸活化蛋白激酶(AMPK)通路相关蛋白的表达水平。【结果】青蒿琥酯可以抑制MV4-11细胞增殖,呈时间、剂量依赖性,处理48、72 h对MV4-11细胞的IC50分别为1.58、1.39μg/mL。与对照组比较,青蒿琥酯组细胞存活率降低,凋亡率升高,G0/G1期细胞增加,S期、G2/M期细胞减少,Bax mRNA相对表达量升高,Bcl-2 m RNA相对表达量降低,p-AMPK、Bax蛋白表达水平上调,p-mTOR、Bcl-2蛋白表达水平下调(均P<0.05);与对照组比较,复合物C组细胞存活率升高,凋亡率降低,G0/G1期细胞减少,S期、G2/M期细胞增加,Bax mRNA相对表达量降低,Bcl-2 mRNA相对表达量升高,p-AMPK、Bax蛋白表达水平下调,p-mTOR、Bcl-2蛋白表达水平上调(均P<0.05);与对照组比较,青蒿琥酯+复合物C组p-AMPK蛋白表达水平上调,p-mTOR蛋白表达水平下调(均P<0.05)。与青蒿琥酯组比较,复合物C组与青蒿琥酯+复合物C组细胞存活率升高,凋亡率降低,G0/G1期细胞减少,S期、G2/M期细胞增加,Bax m RNA相对表达量降低,Bcl-2 mRNA相对表达量升高,p-AMPK、Bax蛋白表达水平下调,p-mTOR、Bcl-2蛋白表达水平上调(均P<0.05)。与复合物C组比较,青蒿琥酯+复合物C组细胞存活率降低,凋亡率升高,G0/G1期细胞增加,S期、G2/M期细胞减少,Bax mRNA相对表达量升高,Bcl-2 mRNA相对表达量降低,p-AMPK、Bax蛋白表达水平上调,p-mTOR、Bcl-2蛋白表达水平下调(均P<0.05)。【结论】青蒿琥酯对MV4-11细胞具有抑制增殖、诱导凋亡的作用,其机制可能与激活AMPK信号通路有关。
Objective To investigate the effect of artesunate against acute myeloid leukemi(AML).Methods The AML cell line MV4-11 at logarithmic growth phase was treated with different concentrations of artesunate for 24,48 and 72 h.The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium(MTT)method,and the median inhibitory concentration(IC50)of artesunate treatment for 48 h on MV4-11 cells was selected for subsequent tests.Cells were randomly divided into control group,artesunate group,compound C group and artesunate+compound C group.Cell survival was measured by MTT,cell cycle and apoptosis were detected by flow cytometry(FCM),mRNA levels of B-lymphoma-2 gene(Bcl-2)and Bcl-2-associated X protein(Bax)were detected by quantitative polymerase chain reaction(qPCR),and the expression levels of adenosine-activated protein kinase(AMPK)signaling pathway related proteins were detected by Western Blot.Results Artesunate inhibited the proliferation of MV4-11 cells in a time-and dose-dependent manner,with IC50 of 1.58 and 1.39μg/mL for MV4-11 cells at 48 h and 72 h of treatment respectively.Compared with the control group,the cell survival rate in the artesunate group was decreased,the apoptosis rate was increased,the G0/G1 phase cells were increased,the S and G2/M phase cells were decreased,the relative mRNA expression level of Bax was increased and the relative mRNA expression level of Bcl-2 was decreased,the protein expression level of p-AMPK and Bax was upregulated,and the protein expression levels of p-m TOR and Bcl-2 was downregulated(P<0.05);compared with the controll group,the cell survival rate in compound C group was increased,and apoptosis rate was decreased,G0/G1 phase cells were decreased,S and G2/M phase cells were increased,relative mRNA expression level of Bax was decreased,relative m RNA expression level of Bcl-2 was increased,p-AMPK and Bax protein expression levels were downregulated,p-mTOR and Bcl-2 protein expression levels were upregulated(P<0.05);compared with the control group,the p-AMPK protein expression level was upregulated and p-mTOR protein expression level was downregulated(P<0.05)in artesunate+compound C group.Compared with the artesunate group,the cell survival rate in compound C group and artesunate+compound C group was increased,and the apoptosis rate was decreased,the G0/G1 phase cells were decreased and the S and G2/M phase cells were increased(P<0.05),the relative m RNA expression level of Bax was decreased,the relative mRNA expression level of Bcl-2 was increased,the protein expression levels of p-AMPK and Bax were downregulated and the protein expression levels of p-mTOR and Bcl-2 were upregulated(P<0.05).Compared with the compound C group,the cell survival rate of artesunate+compound C group was decreased,the apoptosis rate was increased,the G0/G1 phase cells were increased,the S phase and G2/M phase cells were decreased,the relative mRNA expression level of Bax was increased,the relative m RNA expression level of Bcl-2 was decreased,the protein expression levels of p-AMPK and Bax were upregulated,the protein expression levels of p-mTOR and Bcl-2 were downregulated(P<0.05).Conclusion Artesunate inhibits proliferation and induces apoptosis in MV4-11 cells,and the mechanism may be related to the activation of AMPK signaling pathway.
作者
赵小强
吴雅莉
张曼
程英英
杨海平
ZHAO Xiao-Qiang;WU Ya-Li;ZHANG Man;CHENG Ying-Ying;YANG Hai-Ping(Dept.of Hematology,the First Affiliated Hospital of Henan University of Science and Technology(Clinical Medical School,Henan University of Science and Technology),Luoyang 471000 Henan,China)
出处
《广州中医药大学学报》
CAS
2022年第3期631-637,共7页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
河南省医学科技攻关项目(编号:LHGJ20190546)。