安石榴苷调节PI3K/Akt/mTOR信号通路对急性髓系白血病细胞自噬和凋亡的影响

Effect of Punicalagin on Autophagy and Apoptosis in Acute Myeloid Leukemia Cells by Regulating the PI3K/Akt/mTOR Signaling Pathway

目的:探讨安石榴苷(PUN)调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对急性髓系白血病(AML)细胞自噬和凋亡的影响。方法:体外培养人AML细胞(HL-60),随机分为Control组(正常培养)、L-PUN、M-PUN、H-PUN组(20、40、60μg/mL PUN)、740Y-P组(60μg/mL PUN+30μmoL/L PI3K/Akt/mTOR信号通路激活剂740Y-P)。四甲基偶氮唑蓝(MTT)法和平板克隆法检测各组细胞增殖情况;单丹磺酰戊二胺(MDC)染色检测各组细胞自噬发生情况;流式细胞仪检测各组细胞凋亡情况;蛋白免疫印迹(WB)法检测各组细胞中凋亡相关蛋白(Bcl-2、Bax)、自噬相关蛋白(P62、LC3-Ⅱ/Ⅰ比值、BECN1)、PI3K/Akt/mTOR信号通路相关蛋白(p-PI3K、p-Akt、p-mTOR)表达情况。结果:与Control组相比,L-PUN组、M-PUN组、H-PUN组HL-60细胞的存活率、克隆数、p-PI3K、p-Akt、p-mTOR、P62、Bcl-2蛋白表达降低,而细胞凋亡率、自噬小体阳性率、LC3-Ⅱ/Ⅰ比值、BECN1、Bax蛋白表达升高(P<0.05);与H-PUN组相比,740Y-P组细胞的存活率、克隆数、p-PI3K、p-Akt、p-mTOR、P62、Bcl-2蛋白表达升高,而细胞凋亡率、自噬小体阳性率、LC3-Ⅱ/Ⅰ比值、BECN1、Bax蛋白表达水平降低(P<0.05)。结论:PUN通过抑制PI3K/Akt/mTOR信号通路,抑制HL-60细胞增殖,促进其凋亡与自噬的发生,减缓AML的进展。

Objective:To investigate the effect of punicalagin(PUN)on autophagy and apoptosis in acute myeloid leukemia(AML)cells by regulating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway.Methods:Human AML cells(HL-60)were cultured in vitro and randomly separated into control group(normal culture),L-PUN,M-PUN,H-PUN groups(20,40,60μg/mL PUN),and 740 Y-P group(60μg/mL PUN+30μmol/L PI3K/Akt/mTOR signaling pathway activator 740 Y-P).Methylthiazolyltetrazolium(MTT)method and plate cloning method were applied to detect cell proliferation in each group.MDC staining was applied to detect the occurrence of autophagy of cells in various groups.Flow cytometry was applied to detect cell apoptosis in each group.Protein immunoblotting(WB)was applied to detect the expression of apoptosis related proteins(Bcl-2,Bax),autophagy related proteins(P62,LC3-II/I ratio,BECN1),and PI3K/Akt/mTOR signaling pathway related proteins(p-PI3K,p-Akt,p-mTOR)of cells in each group.Results:Compared with the control group,the survival rate,clone count,p-PI3K,p-Akt,p-mTOR,P62,and Bcl-2 protein expression of HL-60 cells in the L-PUN group,M-PUN group,and H-PUN group decreased,while the apoptosis rate,autophagosome positive rate,LC3-II/I ratio,BECN1,and Bax protein expression increased(P<0.05).Compared with the H-PUN group,the survival rate,clone number,p-PI3K,p-Akt,p-mTOR,P62,and Bcl-2 protein expression of the 740 Y-P group cells increased,while the apoptosis rate,autophagosome positive rate,LC3-II/I ratio,BECN1,and Bax protein expression levels decreased(P<0.05).Conclusion:PUN inhibits the PI3K/Akt/mTOR signaling pathway,inhibits HL-60 cell proliferation,promotes apoptosis and autophagy,and slows down the progression of AML.