咪达唑仑对人肝癌细胞增殖能力的影响

Midazolam suppresses HCC cell proliferation via ERK pathway

目的探讨静脉麻醉药咪达唑仑对人肝癌细胞增殖能力的影响。方法首先通过CCK-8增殖实验观察递增浓度20、40、60、80和100μmol/L的咪达唑仑分别各自作用24、48和72 h后对人肝癌细胞的影响;通过流式细胞仪检测浓度为50μmol/L(Mida50组)和100μmol/L(Mida100组)的咪达唑仑以及PBS对照组(Control组)作用于人肝癌细胞发生的凋亡和细胞周期的变化;通过免疫印迹方法观察咪达唑仑对ERK通路的影响。结果咪达唑仑浓度为20μmol/L递增至100μmol/L作用24 h时,人肝癌细胞株Huh7细胞增殖生存率由(88. 55±5. 64)%下降至(47. 71±4. 45)%,差异有统计学意义(P <0. 05),且浓度为100μmol/L的咪达唑仑作用24、48和72 h的增殖生存率分别为(47. 71±4. 45)%、(25. 69±6. 52)%和(18. 10±2. 95)%,差异有统计学意义(P <0. 05)。流式细胞实验显示Mida50和Mida100组的早期凋亡和晚期凋亡比率分别为(7. 07±0. 41)%和(8. 36±0. 48)%以及(7. 58±0. 47)%和(13. 44±0. 67)%。细胞周期示Control组、Mida50组、Mida100组S期比例分别为(22. 19±1. 07)%、(27. 57±2. 15)%和(38. 11±1. 25)%。免疫印迹结果显示咪达唑仑作用于人肝癌细胞会使p-ERK下调。结论咪达唑仑在体外细胞实验中能抑制人肝癌细胞株Huh7细胞的增殖并呈浓度相关和时间相关,其中可能与咪达唑仑促进其凋亡和细胞周期S期停滞相关,相关机制可能是通过ERK通路实现的。

Objective To investigate the effects of Midazolam on the proliferation of human hepatocellular carcinoma（ HCC） cells. Methods CCK8 proliferation assay was used to observe the anti-tumor effects of Midazolam on HCC cells. Midazolam treatment were given at different concentrations,including 20,40,60,80 and 100 μmol/L,and for 24 h,48 h and 72 h. The cell apoptosis and cell cycle of HCC were studied by flow cytometry. Western blotting was adopted to study the influence of Midazolam on ERK pathways. Results The cell viabilities of huh7 cells inhibited by Midazolam with concentrations of 20 μmol/L and 100 μmol/L were（ 88. 55 ± 5. 64） % and（ 47. 71 ± 4. 45） % in a concentration-dependent manner,and they were（ 47. 71 ± 4. 45） %,（ 25. 69 ± 6. 52） % and（ 18. 10 ± 2. 95） % inhibited by Midazolam of 100 μmol/L for 24 h,48 h and 72 h,respectively（ P〈0. 05）. Midazolam induced apoptosis,and suppressed cell cycle progression of huh7 cells. The percentages of early apoptosis cells in Group Mida50 and Mida100 were（ 7. 07 ± 0. 41） % and（ 7. 58 ± 0. 47） %,respectively; as the late apoptosis percentages were（ 8. 36 ± 0. 48） % and（ 13. 44 ± 0. 67） %,respectively. The accumulation rates of live cells in S phase in Group Control,Mida50 and Mida100 were（ 22. 19 ± 1. 07） %,（ 27. 57 ± 2. 15） % and（ 38. 11 ± 1. 25） %,respectively. Meanwhile,western blotting showed Midazolam induced down-regulation of p-ERK. Conclusion Midazolam is able to inhibit the proliferation of HCC cells,and to induce apoptosis and suppress cell cycle. ERK pathways may play a crucial role in this process.