积雪草酸通过ROS介导的细胞凋亡抑制胃癌细胞增殖并诱导保护性自噬  

Asiatic acid inhibits the proliferation via ROS-mediated apoptosis and induces protective autophagy in gastric cancer cells

作　　者：杜军[1] 陈玉忠[1] 李雷[1] 王凯 马家驰 DU Jun;CHEN Yuzhong;LI Lei;WANG Kai;MA Jiachi(Department of Oncology,First Affiliated Hospital of Bengbu Medical College,Bengbu 233000,China)

机构地区：[1]蚌埠医学院第一附属医院肿瘤外科,蚌埠233000

出　　处：《山西医科大学学报》2020年第4期288-295,共8页Journal of Shanxi Medical University

基　　金：蚌埠医学院科技发展基金资助项目(BYKF1879);蚌埠医学院胃癌多学科诊疗创新团队项目(BYKC201907);安徽高校自然科学研究项目(KJ2019A0396)。

摘　　要：目的研究积雪草酸对BGC-823和HGC-27人胃癌细胞增殖的抑制作用,并对其作用机制进行初步探讨.方法BGC-823和HGC-27胃癌细胞中分别加入浓度为0,25,50,100μmol/L的积雪草酸处理12,24,48,72 h.MTT法检测细胞增殖的影响;流式检测细胞凋亡率的变化;Western blot检测凋亡标志蛋白cleaved Caspase3、cleaved Caspase9、cleaved PARP、自噬标志蛋白LC3、P62以及线粒体和细胞质中细胞色素C的表达;ROS检测试剂盒检测ROS水平;GSH检测试剂盒检测GSH的含量.同时给与积雪草酸和ROS清除剂N-乙酰半胱氨酸,采用MTT考察BGC-823和HGC-27胃癌细胞的生存率.同时给与积雪草酸和自噬抑制剂氯喹或3-甲基腺嘌呤考察自噬在积雪草酸抗BGC-823和HGC-27胃癌细胞增殖中的作用.结果MTT结果显示,积雪草酸(0,25,50,100μmol/L)作用于BGC-823和HGC-27胃癌细胞后,细胞生存率随时间和剂量依赖性地降低(P<0.05).流式结果显示,积雪草酸(0,25,50,100μmol/L)作用于BGC-823和HGC-27胃癌细胞后,细胞凋亡率明显增加(P<0.05).Western blot显示,积雪草酸(0,25,50,100μmol/L)处理BGC-823和HGC-27胃癌细胞后,cleaved Caspase3、cleaved Caspase9、cleaved PARP的表达显著上调(P<0.05).积雪草酸(0,25,50,100μmol/L)作用于BGC-823和HGC-27胃癌细胞后,细胞中的ROS水平增加(P<0.05),GSH水平降低(P<0.05).MTT结果显示,与单独积雪草酸处理相比,5 mmol/L ROS清除剂N-乙酰半胱联合50μmol/L积雪草酸处理BGC-823和HGC-27胃癌细胞生存率明显升高(P<0.05).Western blot显示,积雪草酸浓度依赖性地上调BGC-823和HGC-27胃癌细胞中LC3-Ⅱ的表达(P<0.05),并下调P62的表达(P<0.05).MTT结果显示,与单独积雪草酸处理相比,20μmol/L自噬抑制剂氯喹或25μmol/L 3-甲基腺嘌呤联合50μmol/L积雪草酸处理BGC-823和HGC-27胃癌细胞生存率明显降低(P<0.05).结论积雪草酸可通过ROS介导的细胞凋亡抑制胃癌细胞增殖并诱导保护性自噬.Objective To investigate the inhibitory effect of asiatic acid on the proliferation of human gastric cancer BGC-823 and HGC-27 cells and its mechanism.Methods BGC-823 and HGC-27 cells were treated with 0,25,50,100μmol/L asiatic acid for 12,24,48,72 h respectively.MTT assay was used to detect the cell proliferation.Cell apoptosis rate was detected by flow cytometry.The expression levels of apoptotic marker proteins cleaved Caspase3,cleaved Caspase9,cleaved PARP,autophagy marker proteins LC3,P62 and cytochrome C in mitochondria and cytoplasm were detected by Western blot.ROS detection kit was used to detect ROS level,and glutathione detection kit was used to detect the content of GSH.The effect of ROS scavenging agent NAC combined with asiatic acid on the proliferation of BGC-823 and HGC-27 gastric cancer cells was investigated using MTT assay.The effect of autophagy on the proliferation of BGC-823 and HGC-27 gastric cancer cells was investigated after treated with asiatic acid and autophagy inhibitors chloroquine or 3-methyladenine.Results MTT results showed that the survival rate of gastric cancer cells was decreased in a time-and dose-dependent manner in BGC-823 and HGC-27 gastric cancer cells after treatment with asiatic acid(0,25,50,100μmol/L)(P<0.05).Flow cytometry results showed that the apoptosis rate of gastric cancer cells was significantly increased after treatment with asiatic acid(0,25,50,100μmol/L)(P<0.05).Western blot showed that the expression levels of cleaved Caspase3,cleaved Caspase9 and cleaved PARP were significantly up-regulated in BGC-823 and HGC-27 cells treated with asiatic acid(0,25,50,100μmol/L)(P<0.05).ROS level was increased in BGC-823 and HGC-27 cells treated with asiatic acid(0,25,50,100μmol/L)(P<0.05),while GSH level was decreased(P<0.05).MTT results showed that the cell survival rate was significantly higher after treatment with 5 mmol/L ROS scavenging agent NAC and 50 mol/L asiatic acid than that after treatment with asiatic acid(P<0.05).Western blot showed that the expression

关 键 词：积雪草酸 ROS 凋亡 自噬 增殖 胃癌 

分 类 号：R735.2[医药卫生—肿瘤]