| 马冬云,李蓓蕾,李明亮.咪达唑仑通过调控 AMPK/mTORC1通路影响子宫内膜癌细胞增殖和凋亡的研究[J].安徽医药,2025,29(3):487-493. |
| 咪达唑仑通过调控 AMPK/mTORC1通路影响子宫内膜癌细胞增殖和凋亡的研究 |
| Midazolam affects the proliferation and apoptosis of endometrial cancer cells by regulating the AMPK/mTORC1 pathway |
| |
| DOI：10.3969/j.issn.1009-6469.2025.03.012 |
| 中文关键词: 咪达唑仑 腺苷酸活化蛋白激酶 /哺乳动物雷帕霉素靶蛋白复合物 1通路 子宫内膜癌 增殖 凋亡 |
| 英文关键词: Midazolam AMP-activated protein kinase/mammalian target of rapamycin complex 1 pathway Endometrial cancer Proliferation Apoptosis |
| 基金项目: |
|
| 摘要点击次数: 365 |
| 全文下载次数: 172 |
| 中文摘要: |
| 目的探究咪达唑仑( MDZ)通过调控腺苷酸活化蛋白激酶 /哺乳动物雷帕霉素靶蛋白复合物 1(AMPK/mTORC1)通路影响子宫内膜癌细胞增殖和凋亡情况。方法 2022年 3—12月，以人子宫内膜癌 HEC-1B、Ishikawa、HHUA细胞株为研究对象，分别添加 0、10、20、40、80 μmol/L MDZ培养 48 h，细胞计数试剂盒( CCK-8)检测细胞增殖情况并计算其半数抑制浓度(IC50)；选择 HEC-1B细胞，用 0、10、20、40、80 μmol/L MDZ培养 48 h，流式细胞术检测细胞凋亡情况；蛋白质印迹法检测细胞中 AMPK、磷酸化腺苷酸活化蛋白激酶( p-AMPK)蛋白表达情况。细胞分为空白组(正常培养)、 MDZ组(添加 40 μmol/L MDZ)、 MDZ+AMPK抑制剂组(添加 40 μmol/L MDZ+10 μmol/L Compound C)，分组处理后培养 48 h，CCK-8检测细胞增殖情况；流式细胞术检测细胞凋亡情况；蛋白质印迹法检测细胞中 AMPK、p-AMPK、mTORC1、胱天蛋白酶 3(caspase3)、活化胱天蛋白酶 3(c-caspase3)、 p70 S6激酶( p70S6K)、磷酸化 p70S6K(p-p70S6K)蛋白表达情况。通过皮下注射 HEC-1B细胞与 MDZ、 MDZ和 AMPK抑制剂 Compound C的单细胞悬液建立异种移植瘤小鼠模型，观察其对肿瘤生长的影响；免疫组织化学染色检测移植瘤组织细胞增殖核抗原( Ki-67)表达；蛋白质印迹法检测移植瘤组织中 AMPK/mTORC1通路相关蛋白表达。结果随着 MDZ浓度的增加， HEC-1B、Ishikawa、HHUA细胞的增殖抑制率升高，且具有浓度依赖性。 MDZ对 HEC-1B、Ishikawa、 HHUA细胞的 IC50分别为( 37.62±0.93)μmol/L、(55.31±1.12)μmol/L、(61.37±1.38)μmol/L，MDZ对 HEC-1B细胞作用效果更好，因此，后期选择 HEC-1B作为研究对象。分别与 0、10 μmol/L MDZ相比， 20、40、80 μmol/L MDZ处理细胞凋亡率、细胞中 p-AMPK/AMPK蛋白水平升高( P<0.05)；与 20 μmol/L MDZ相比， 40、80 μmol/L MDZ处理细胞凋亡率、细胞中 p-AMPK/ AMPK蛋白水平升高( P<0.05)；与 40 μmol/L MDZ相比， 80 μmol/L MDZ处理细胞凋亡率升高( P<0.05)。与空白组相比， MDZ组细胞增殖抑制率、凋亡率、细胞中 p-AMPK/AMPK、c-caspase3/caspase3蛋白水平升高( P<0.05)，细胞中 mTORC1、p-p70S6K/ p70S6K蛋白水平降低( P<0.05)；与 MDZ组相比， MDZ+AMPK抑制剂组细胞增殖抑制率、凋亡率、细胞中 p-AMPK/AMPK、 c-caspase3/caspase3蛋白水平降低( P<0.05)，细胞中 mTORC1、p-p70S6K/p70S6K蛋白水平升高( P<0.05)。与空白组比较， MDZ组肿瘤体积和肿瘤质量显著降低， Ki-67表达减弱， p-AMPK/AMPK水平显著升高， mTORC1、p-p70S6K/p70S6K水平显著降低( P<0.05)；与 MDZ组比较， MDZ+AMPK抑制剂组上述各项指标被逆转( P<0.05)。结论 MDZ能够激活 AMPK/mTORC1通路从而抑制子宫内膜癌细胞增殖和促进细胞凋亡。 |
| 英文摘要: |
| Objective To explore the effect of midazolam (MDZ) on the proliferation and apoptosis of endometrial cancer cells by reg-ulating the AMP-activated protein kinase/mammalian target of rapamycin complex 1 (AMPK/mTORC1) pathway. Methods From March to December 2022, human endometrial cancer HEC-1B, Ishikawa, HHUA cell line were used as the research objects, and werecultured for 48 hours after adding 0, 10, 20, 40, 80 μmol/L MDZ, cell counting kit-8 (CCK-8) was applied to detect cell proliferation, and the half inhibitory concentration (IC50) was calculated; HEC-1B cells were selected and cultured with 0, 10, 20, 40, 80 μmol/LMDZ for 48 hours, flow cytometry was applied to detect cell apoptosis; Western blot was applied to detect the protein expression ofAMPK and phosphorylated adenylate activates protein kinase (p-AMPK) in cells. Cells were assigned into blank group (normal culture),MDZ group (40 μmol/L MDZ added), MDZ+AMPK inhibitor group (40 μmol/L MDZ + 10 μmol/L Compound C added), and after group-ing and culture for 48 hours, CCK-8 was applied to detect cell proliferation; flow cytometry was applied to detect cell apoptosis; West-ern blot was applied to detect the protein expression of AMPK, p-AMPK, mTORC1, caspase 3, cleaved caspase 3 (c-caspase 3), p70 S6 kinase (p70S6K), and phosphorylated p70S6K (p-p70S6K) in cells. The xenograft tumor model was established in nude mice by subcu-taneous injection of single-cell suspensions of the HEC-1B cells, the MDZ, the MDZ and AMPK inhibitor Compound C, and their ef-fects on tumor growth were observed; immunohistochemical staining was applied to detect the expression of cell proliferating nuclearantigen (Ki-67) in the transplanted tumor tissue; Western blotting was applied to detect the expression of AMPK/mTORC1 pathway-re-lated proteins in transplanted tumor tissues.Results With the increase of MDZ concentration, the proliferation inhibition rate of HEC-1B, Ishikawa and HHUA cells increased, in a concentration dependent manner. The IC50 of MDZ on HEC-1B, Ishikawa and HHUAcells was (37.62±0.93) μmol/L, (55.31±1.12) μmol/L and (61.37±1.38) μmol/L, respectively. The effect of MDZ on HEC-1B cells was best, therefore, HEC-1B was selected as the study subject in the later study. Compared with 0, 10 μmol/L MDZ, the apoptosis rate and the level of p-AMPK/AMPK protein were higher in the cells treated with 20, 40, 80 μmol/L MDZ (P<0.05); compared with 20 μmol/L MDZ, the apoptosis rate and the level of p-AMPK/AMPK protein were higher in the cells treated with 40, 80 μmol/L MDZ (P<0.05); compared with 40 μmol/L MDZ, the apoptosis rate was higher in the cells treated with 80 μmol/L MDZ (P<0.05). Compared with the blank group, the cell proliferation inhibition rate, apoptosis rate, and the protein levels of p-AMPK/AMPK and c-caspase3/caspase3 were higher in the MDZ group (P<0.05), the protein levels of mTORC1 and p-p70S6K/p70S6K in cells were lower (P<0.05); compared with the MDZ group, the cell proliferation inhibition rate, apoptosis rate, and the protein levels of p-AMPK/AMPK and c-caspase3/cas-pase3 were lower in the MDZ+AMPK inhibitor group (P<0.05), the protein levels of mTORC1 and p-p70S6K/p70S6K in cells were higher (P<0.05). Compared with the blank group, the tumor volume and tumor weight in the MDZ group were significantly lower, the ex-pression of Ki-67 was lower, the level of p-AMPK/AMPK was significantly higher, and the levels of mTORC1 and p-p70S6K/p70S6K were significantly lower (P<0.05). Compared with the MDZ group, the above indexes were reversed in the MDZ+AMPK inhibitor group (P<0.05).Conclusion MDZ can activate AMPK/mTORC1 pathway to inhibit the proliferation and promote cell apoptosis of endometri-al cancer cells. |
|
查看全文
查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
