香菇多糖在体外对白血病HL-60细胞凋亡及PI3K/AKT信号通路的影响

目的:研究香菇多糖在体外对人白血病HL-60细胞凋亡的调节作用及其对PI3K/AKT信号通路的影响。方法:分别用浓度为0 mg/L、15 mg/L、30 mg/L和45 mg/L的香菇多糖作用于体外培养至对数期的HL-60细胞,24 h、48 h和72 h后用MTT法检测香菇多糖对HL-60细胞活力的抑制作用。用流式细胞术检测香菇多糖对HL-60细胞凋亡的影响,Western blot检测cleaved PARP、cleaved caspase-9、cleaved caspase-3和cleaved caspase-8、cytochrome C、PI3K、AKT和p-AKT的蛋白水平。用浓度为5 mg/L的PI3K抑制剂LY294002处理HL-60细胞72 h后,检测细胞的凋亡情况。结果:香菇多糖(15 mg/L、30 mg/L和45 mg/L)作用24 h、48 h和72 h后,HL-60细胞的活力受到抑制(P<0.05),且具有浓度依赖性和时间依赖性。香菇多糖(15 mg/L、30 mg/L和45 mg/L)作用72 h后,以浓度依赖性的方式促进HL-60细胞的凋亡(P<0.05)。30 mg/L香菇多糖诱导HL-60细胞凋亡过程中,cleaved PARP、cleaved caspase-9和cleaved caspase-3及胞浆cytochrome C的蛋白水平随着处理时间的延长而明显升高,而caspase-8没有变化(P<0.05);PI3K、AKT和p-AKT的蛋白水平随着香菇多糖浓度的增加而明显降低(P<0.05)。PI3K通路抑制剂LY294002处理HL-60细胞与香菇多糖处理产生类似的凋亡效果(P<0.05)。结论:香菇多糖可通过抑制PI3K/AKT信号通路诱导白血病HL-60细胞凋亡。

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM: To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3 K/AKT signaling pathway in HL-60 cells in vitro.METHODS: Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3 K, AKT and p-AKT were determined by Western blot. After treatment with PI3 K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS: The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners(P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan(15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner(P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time(P<0.05), but caspase-8 did not show any change. The protein levels of PI3 K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration(P<0.05). Treatment of HL-60 cells with LY294002, a PI3 K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan(P<0.05). CONCLUSION: Lentinan induces HL-60 cell apoptosis by inhibiting PI3 K/AKT signaling pathway.