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

作　　者：马冬云 李蓓蕾 李明亮 MA Dongyun;LI Beilei;LI Mingliang(Department of Anesthesiology,The First Affiliated Hospital of Shaoyang University,Shaoyang,Hu'nan 422000,China)

机构地区：[1]邵阳学院附属第一医院麻醉科,湖南邵阳422000

出　　处：《安徽医药》2025年第3期487-493,I0001,共8页Anhui Medical and Pharmaceutical Journal

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、80μmol/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、80μmol/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40μmol/L MDZ)、MDZ+AMPK抑制剂组(添加40μmol/L MDZ+10μmol/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、10μmol/L MDZ相比,20、40、80μmol/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20μmol/L MDZ相比,40、80μmol/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40μmol/L MDZ相比,80μmol/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�Objective To explore the effect of midazolam(MDZ)on the proliferation and apoptosis of endometrial cancer cells by regulating 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 were cultured for 48 hours after adding 0,10,20,40,80μmol/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 0,10,20,40,80μmol/L MDZ for 48 hours,flow cytometry was applied to detect cell apoptosis;Western blot was applied to detect the protein expression of AMPK and phosphorylated adenylate activates protein kinase(p-AMPK)in cells.Cells were assigned into blank group(normal culture),MDZ group(40μmol/L MDZ added),MDZ+AMPK inhibitor group(40μmol/L MDZ+10μmol/L Compound C added),and after grouping and culture for 48 hours,CCK-8 was applied to detect cell proliferation;flow cytometry was applied to detect cell apoptosis;Western 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 subcutaneous injection of single-cell suspensions of the HEC-1B cells,the MDZ,the MDZ and AMPK inhibitor Compound C,and their effects on tumor growth were observed;immunohistochemical staining was applied to detect the expression of cell proliferating nuclear antigen(Ki-67)in the transplanted tumor tissue;Western blotting was applied to detect the expression of AMPK/mTORC1 pathway-related 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 HHUA cells was(37.62±0.93)μmol/L,(55.31±1.12)μmol/L and(61.37±1

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 

分 类 号：R737.33[医药卫生—肿瘤]