聚肌胞苷酸增强木犀草素抑制Nalm6细胞增殖的作用机制  

Mechanism of Polyinosinic Polycytidylic Acid Promoting the Inhibitory Effect of Luteolin on Proliferation of Nalm6 Cell

作　　者：蒋海静 敬林 赵克天 刘阳[2] 罗兴燕[2] JIANG Haijing;JING Lin;ZHAO Ketian;LIU Yang;LUO Xingyan(School of Pharmacy and Bioengineering,Chongqing University of Technology,Chongqing 400054,China;Development and Regeneration Key Laboratory of Sichuan Province,Chengdu Medical College,Chengdu 610500,China)

机构地区：[1]重庆理工大学药学与生物工程学院,重庆400054 [2]成都医学院发育与再生四川省重点实验室,成都610500

出　　处：《中国现代应用药学》2024年第16期2206-2212,共7页Chinese Journal of Modern Applied Pharmacy

基　　金：发育与再生四川省重点实验室基金项目(SYS20-08);四川省科技厅应用基础面上项目(2022NSFSC0580,2023NSFSC0624)。

摘　　要：目的研究聚肌胞苷酸[polyinosinic polycytidylic acid,Poly(I:C)]增强木犀草素(luteolin,LTN)抑制急性淋巴细胞白血病细胞株Nalm6增殖的作用机制。方法采用CCK-8法和流式细胞术检测LTN单独作用或Poly(I:C)与LTN联合作用对Nalm6细胞增殖和凋亡的影响,以及程序性凋亡抑制剂(programmed apoptosis inhibitor,ZVAD)和坏死性细胞死亡抑制剂(Nec-1)对联合作用的影响;采用流式细胞术分析在LTN单独作用或Poly(I:C)与LTN联合作用下,不同时间点对Nalm6细胞凋亡的影响。24 h的细胞周期情况以及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和Caspase 8蛋白表达水平。结果与LTN组相比,Poly(I:C)与LTN联合作用可显著抑制Nalm6细胞增殖(P<0.05);ZVAD可完全阻断Poly(I:C)与LTN联用后的抑制增殖作用(P<0.05)。Poly(I:C)与LTN联合作用细胞3 h后细胞发生凋亡,24 h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)与LTN联用后的促Nalm6细胞凋亡。蛋白免疫印迹结果显示,与LTN单独作用相比,Poly(I:C)与LTN联合作用Nalm6细胞6 h,通过显著激活PARP、Caspase 3和Caspase 8蛋白启动凋亡程序,但不影响NF-κB和PI3K-AKT信号通路;LTN可单独激活p-ERK1/2磷酸化,通过活化MEK/ERK信号通路抑制细胞增殖。结论Poly(I:C)通过激活PARP、Caspase 3和Caspase 8诱导细胞凋亡而非细胞坏死,从而增强LTN对细胞增殖的抑制作用。OBJECTIVE To investigate the mechanism of polyinosinic polycytidylic acid[Poly(I:C)]enhancing luteolin(LTN)to inhibit the proliferation of acute lymphoblastic leukemia cell line Nalm6.METHODS Using CCK-8 method and flow cytometry to detect the proliferation and apoptosis of Nalm6 cells treated with LTN alone or combined with Poly(I:C),and explored the effect of programmed apoptosis inhibitor(ZVAD)and necrotic cell death inhibitor(Nec-1)on the combined treatment.Flow cytometry was performed to analyze the effects of LTN alone or combined with Poly(I:C)on cell apoptosis of Nalm6 cells at different time points,cell cycle states at 24 h,and TLR3 expression.The protein expression of p-IRF3,p-mTOR,p-NFκB-65,pp70S6K,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 could completely block the proliferation-inhibiting effects of Poly(I:C)combined with LTN in Nalm6 cells(P<0.05).Under Poly(I:C)combined with LTN treatment,cell apoptosis occurred at 3 h and reached the late stage of apoptosis after 24 h.Cell cycle results showed that Poly(I:C)significantly promoted LTN-induced DNA breakage(P<0.05).Neutralization of TLR3 pathway did not affect the apoptosis-enhancing effects of Poly(I:C)combined with LTN in Nalm6 cells.Western blotting analysis showed that compared with LTN alone,Poly(I:C)combined with LTN activated PARP,Caspase 3 and Caspase 8 to initiate apoptosis of Nalm6 cells for 6 h,but did not affect the NF-κB and PI3K-AKT signaling pathways.LTN alone activated p-ERK1/2 phosphorylation,inhibiting cell proliferation by activating the MEK/ERK signaling pathway.CONCLUSION Poly(I:C)induced apoptosis by activating PARP,Caspase 3 and Caspase 8 rather than necrosis,to enhance the proliferation-inhibiting effects of LTN on proliferation of Nalm6 cell.

关 键 词：急性淋巴细胞白血病 聚肌胞苷酸 木犀草素 细胞凋亡 

分 类 号：R966[医药卫生—药理学]