木犀草素对博来霉素诱导的肺上皮细胞增殖、凋亡及Hippo/Yes相关蛋白通路的影响

Effect of luteolin on the proliferation, apoptosis and Hippo/Yes-related protein pathway of lung epithelial cells induced by bleomycin

  • 摘要:
      目的  探究木犀草素(Lut)对博来霉素(BLM)诱导的人肺正常上皮细胞(BEAS-2B细胞)增殖、凋亡及Hippo/Yes相关蛋白(YAP)通路的影响。
      方法  体外培养BEAS-2B细胞,使用4、8、12、24 μg/mL BLM诱导12、24、48 h,筛选BLM诱导浓度和时间。将BEAS-2B细胞分为对照组、BLM组以及Lut低、中、高浓度组。各组细胞培养24 h后,倒置显微镜观察细胞形态学变化;采用CCK-8法检测细胞增殖能力;采用流式细胞术检测细胞凋亡率;采用蛋白免疫印迹法(Western blot)检测Hippo/YAP通路相关蛋白-哺乳动物STE20样蛋白激酶1(MST1)、大肿瘤抑制因子1(LAST1)和YAP磷酸化水平。
      结果  BLM诱导24 h后BEAS-2B细胞的半数抑制浓度(IC50)约为8 μg/mL,所以本研究选取8 μg/mL BLM诱导细胞24 h。与对照组相比,BLM组细胞较小,边缘不规则,细胞间隙增加,细胞增殖能力、MST1、LAST1和YAP蛋白磷酸化水平显著降低(P < 0.05),凋亡率显著升高(P < 0.05);与BLM组相比,Lut低、中、高浓度组细胞形态趋于正常,细胞增殖能力、MST1、LAST1和YAP磷酸化水平依次升高(P < 0.05),凋亡率依次降低(P < 0.05)。
      结论  Lut可能通过激活BEAS-2B细胞中Hippo/YAP信号通路,提高BLM诱导后细胞的增殖能力,抑制细胞凋亡。

     

    Abstract:
      Objective  To explore the effect of luteolin(Lut)on the proliferation, apoptosis and the Hippo/Yes-related protein (YAP) pathway of human lung epithelial cell (BEAS-2B cells) induced by bleomycin(BLM).
      Methods  BEAS-2B cells were cultured in vitro, and induced with 4, 8, 12 and 24 μg/mL BLM for 12, 24 and 48 h, respectively, to determine the BLM induction concentration and time. The BEAS-2B cells were divided into control group, BLM group, and Lut low, medium and high concentration groups. After 24 hours of cell culture in each group, the morphological changes of the cells were observed with an inverted microscope; the cell proliferation ability was detected by the CCK-8 method; the rate of cell apoptosis was detected by flow cytometry; Western blot was used to detect Hippo/YAP pathway related proteins-mammalian Sterile20-like 1(MST1), the large tumour suppressor 1 (LAST1) and YAP phosphorylation levels.
      Results  The half maximal inhibitory concentration (IC50) of BEAS-2B cells after 24 hours of BLM induction was about 8 μg/mL, so 8 μg/mL BLM was selected to induce cells for 24 hours. Compared with the control group, the BLM group had smaller cells, irregular edges, increased intercellular space, the cell proliferation ability, MST1, LAST1 and YAP phosphorylation levels significantly decreased, and apoptosis rate significantly increased (P < 0.05); compared with the BLM group, the cell morphology of the Lut low, medium, and high concentration groups tended to be normal, and the cell proliferation ability, MST1, LAST1 and YAP protein phosphorylation levels increased successively (P < 0.05), and the rate of apoptosis decreased successively (P < 0.05).
      Conclusion  Lut may activate the Hippo/YAP signaling pathway in BEAS-2B cells, increase the proliferation of BEAS-2B cells induced by BLM, and inhibit cell apoptosis.

     

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