Poly(I:C)增强木犀草素抑制Nalm6细胞增殖作用机制研究

Effect of PolyS(I:C) on inhibitory effect of Luteolin on proliferation of Nalm6 cell

关键词:  急性淋巴细胞白血病  poly(I:C)  木犀草素  细胞凋亡

Key words:  acute lymphoblastic leukemia  poly(I:C)  luteolin  cell apoptosis

摘要:

目的 研究Poly(I:C)促进木犀草素(LTN)抑制急性淋巴细胞白血病细胞株Nalm6增殖的可能作用机制。方法 CCK-8法和流式细胞术检测Poly(I:C)、LTN、程序性凋亡抑制剂(ZVAD)以及坏死性细胞死亡抑制剂(Nec-1)单独或联合作用Nalm6细胞增殖和凋亡情况；采用流式细胞术分析Poly(I:C)联合 LTN作用于Nalm6细胞3h,6h,12h,24h的凋亡情况,Nalm6细胞24h的周期情况以及Nalm6的TLR3表达；蛋白免疫印迹检测细胞中p-IRF3、p-mTOR、p-NFκB-65、p-p70S6K 、p-ERK1/2、PARP、Caspase 3、Caspase 8蛋白表达水平。结果 与LTN组相比,Poly(I:C)与LTN联合使用后可显著抑制Nalm6细胞增殖(P<0.05)；ZVAD可完全阻碍Poly(I:C)增强木犀草素抑制Nalm6细胞增殖(P＜0.001)；LTN与Poly(I:C)作用细胞3h后细胞发生细胞凋亡,24h后进入到凋亡后期；细胞周期结果显示,Poly(I:C)可显著促进LTN诱发的DNA断裂(P<0.05)；中和TLR3信号通路不影响Poly(I:C)增强LTN诱导Nalm6细胞凋亡；蛋白免疫印迹结果显示Poly(I:C)和LTN单独或联合作用Nalm6细胞6h后PARP、Caspase 3,Caspase 8蛋白激活启动凋亡程序,但不影响NF-κB,PI3K-AKT的信号通路,LTN可单独激活p-ERK1/2磷酸化从而活化MEK/ERK信号通路抗细胞增殖。结论 Poly(I:C)通过Caspase 8的激活诱导细胞凋亡而非细胞坏死,增强LTN抑制细胞增殖能力。

Abstract:

ABSTRACT: OBJECTIVE To study the mechanism of Poly(I:C) promoting luteolin (LTN) to inhibit the proliferation of acute lymphoblastic leukemia cell line Nalm6. METHODS The proliferation and apoptosis of Nalm6 cells treated with LTN alone or in combination Poly(I:C), programmed apoptosis inhibitor (ZVAD), necrotic cell death inhibitor (Nec-1) were detected by CCK-8 method and flow cytometry; The apoptosis of Nalm6 cells treated with Poly(I:C) or/and LTN at 3h, 6h, 12h and 24h, the cell cycle of Nalm6 at 24h and the expression of TLR3 of Nalm6 were analyzed by flow cytometry; The protein expression levels of p-IRF3, p-mTOR, p-NFκB-65, p-p70S6K, p-ERK1/2, PARP, Caspase 3 and Caspase 8 were detected by western blotting. RESULTS Compared with LTN group, Poly(I:C) combined with LTN group significantly inhibited the proliferation of Nalm6 cells (P<0.05); ZVAD can completely blocked the proliferation-inhibiting effects of Poly(I:C) and LTN in Nalm6 cells (P<0.001). After treatment with LTN and Poly(I:C), cell apoptosis occurred for 3h and reached the late stage of apoptosis after 24h. Cell cycle results showed that Poly(I:C) significantly promoted LTN-induced DNA breakage (P<0.05). The neutralization of TLR3 pathway did not affect the Poly(I:C) enhanced LTN induced apoptosis of Nalm6 cells. Protein western blotting results showed that PARP, Caspase 3 and Caspase 8 were activated by Poly(I:C) and LTN alone or in combination for 6h to initiate apoptosis, but did not affect the NF-κB and PI3K-AKT signaling pathways. LTN alone activates p-ERK1/2 phosphorylation to activate the MEK/ERK signaling pathway against cell proliferation. CONCLUSION Poly(I:C) induced apoptosis through Caspase 8 activation but not cell necrosis to enhanced inhibitory effect of LTN on proliferation of Nalm6 cell. . KEY WORDS: acute lymphoblastic leukemia; poly(I:C); luteolin; cell apoptosis