老鼠簕生物碱A对胰腺癌L3.6细胞增殖和凋亡的影响  

Effects of 4-hydroxy-2(3H)-benzoxazolone on proliferation and apoptosis of pancreatic cancer L3.6 cells

作　　者：梁嘉敏 杨思淇 胡文浩 李旭艳 王锂韫 LIANG Jia-min;YANG Si-qi;HU Wen-hao;LI Xu-yan;WANG Li-yun(Western Guangdong Characteristic Biology and Medicine Engineering and Research Center,Zhanjiang 524048,China;Guangdong Engineering Technology Research Center of Protection and Utilization of Blue Carbon Resources,Zhanjiang 524048,China;Mangrove Institute,Lingnan Normal University,Zhanjiang 524048,China;School of Life Sciences and Technology,Lingnan Normal University,Zhanjiang 524048,China;Western Guangdong Provincial Engineering Technology Research Center of Seafood Resource Sustainable Utilization,Zhanjiang 524048,China)

机构地区：[1]粤西特色生物医药工程技术研究中心,广东湛江524048 [2]广东省粤西蓝碳资源保护与利用工程技术研究中心,广东湛江524048 [3]岭南师范学院红树林研究院,广东湛江524048 [4]岭南师范学院生命科学与技术学院,广东湛江524048 [5]广东省粤西海鲜资源可持续利用工程技术研究中心,广东湛江524048

出　　处：《中国中药杂志》2024年第9期2355-2363,共9页China Journal of Chinese Materia Medica

基　　金：粤西特色生物医药工程技术研究中心开放课题(2022-K13);岭南师范学院红树林研究院开放课题(YBXM08);湛江市科学技术局海洋青年人才创新项目(2021E05024);粤西海洋中药及南药高值化开发利用创新团队项目(2021KCXTD039);湛江海鲜食材资源可持续利用重点实验室项目(2015A06008);大学生创新创业项目(X202210579049,X202210579055)。

摘　　要：探讨老鼠簕生物碱A[4-hydroxy-2(3H)-benzoxazolone,HBOA]对胰腺癌细胞增殖和凋亡的影响及其分子机制。0~1.0 mmol·L^(-1) HBOA处理体外培养的胰腺癌L3.6细胞,采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)法测定细胞活力并计算半数抑制浓度IC50,筛选药物作用浓度和时间。利用倒置显微镜、吖啶橙(acridine orange,AO)染色观察细胞形态,活细胞计数、细胞集落形成实验检测细胞增殖、自我更新能力,流式细胞术检测细胞周期进程和细胞凋亡比率,扫描电镜观察细胞凋亡形态,qPCR检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白A(cyclinA1、cyclinA2)、细胞周期蛋白依赖性激酶2(cyclin dependent kinase 2,CDK2)、细胞周期蛋白依赖性激酶抑制因子1A(cyclin dependent kinase inhibitor 1A,P21)mRNA表达,流式细胞术检测细胞活性氧(reactive oxygen species,ROS)、脂质过氧化物、线粒体膜电位水平,Western blot检测蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路活性。与对照组相比,HBOA处理组细胞活力显著下降,筛选出0.4、0.6 mmol·L^(-1) HBOA干预48 h为后续实验剂量与时间;与对照组相比,0.4、0.6 mmol·L^(-1) HBOA处理组细胞增殖和集落形成能力被抑制,PCNA、cyclinA1、cyclinA2、CDK2 mRNA表达水平显著降低,P21 mRNA表达水平显著升高,细胞周期阻滞于S期,细胞凋亡率显著增加、出现凋亡小体,ROS水平显著升高,脂质过氧化物水平显著升高,线粒体膜电位降低,其中0.6 mmol·L^(-1)处理组显著降低,p-Akt、p-mTOR蛋白水平显著降低。该研究表明HBOA抑制胰腺癌L3.6细胞增殖,诱导细胞凋亡,其机制可能与ROS增加、Akt/mTOR通路活性抑制有关。This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA)on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism.The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L^(-1).The cell viability was detected by the cell counting kit-8(CCK-8)method,and the half inhibitory concentration(ICso)was analyzed to determine the drug concentration and time.The cell morphology was observed under an inverted microscope and by acridine orange(AO)staining.The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments.The cell cycle progression and cell apoptosis rate were detected by flow cytometry.The morphology of cell apoptosis was observed by scanning electron microscopy.The mRNA expression of proliferating cell nuclear antigen(PCNA),cyclinAl,cyclinA2,cyclin dependent kinase 2(CDK2),and cyclin dependent kinase inhibitor 1A(P21)were determined by qPCR.The level of reactive oxygen species(ROS),lipid peroxide,and mitochondrial membrane potential were measured by flow cytometry.The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway was detected by Western blot.Compared with the control group,the cells treated with HBOA exhibited a significant decrease in viability.Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L^(-1),0.6 mmol·L^(-1),and 48 h.Compared with the control group,groups with HBOA of 0.4 mmol·L^(-1)and O.6 mmol·L^(-1) showed a significant suppression in cell proliferation and colony formation ability,down-regulated mRNA of PCNA,cyclinA1,cyclinA2,and CDK2,up-regulated P21 mRNA,S-phase cell cycle arrest,and increased cell apoptosis rate.There was an appearance of apoptotic bodies,increased ROS and lipid peroxide,decreased mitochondrial membrane potential(with a significant decrease in O.6 mmol·L^(-1)group),and down-regulated p-Akt and p-mTOR proteins.The results show that HBOA inhibits the proliferation of pancreatic

关 键 词：老鼠簕生物碱A 胰腺癌细胞 细胞增殖 细胞凋亡 

分 类 号：R285[医药卫生—中药学]