摘要
目的探究升麻环氧醇苷联合顺铂对肺癌A549细胞增殖与凋亡的作用及机制。方法选择A549、BEAS-2B细胞系作为研究对象,采用CCK-8法检测升麻环氧醇苷、顺铂对A549细胞活力的影响,以及升麻环氧醇苷对BEAS-2B细胞活力的影响,计算半数抑制浓度(IC_(50))和20%抑制浓度(IC_(20));使用升麻环氧醇苷和顺铂对A549细胞的IC_(20)进行后续实验。将A549细胞分为对照组、顺铂组(5.09μmol/L)、升麻环氧醇苷组(4.39μmol/L)、顺铂+升麻环氧醇苷组(5.09μmol/L+4.39μmol/L)。4组细胞分别加入相应药物处理48 h后,采用EdU-488荧光探针法检测A549细胞阳性细胞率;膜联蛋白V/碘化丙啶染色法检测A549细胞凋亡情况;Bodipy 493/503染色法观察A549细胞脂滴生成;蛋白质印迹法检测A549细胞蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、固醇调节元件结合蛋白1(SREBP1)、脂肪酸合成酶(FASN)、乙酰辅酶A羧化酶1(ACC1)、三磷酸腺苷柠檬酸裂解酶(ACLY)、细胞周期蛋白E1(Cyclin E1)、细胞周期蛋白依赖性激酶2(CDK2)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、剪切型胱天蛋白酶-3(Cleaved-Caspase-3)蛋白表达;共聚焦免疫荧光法检测A549细胞SREBP1平均荧光强度。结果升麻环氧醇苷对A549细胞的IC_(50)为(22.80±0.93)μmol/L、IC_(20)为(4.39±0.73)μmol/L,顺铂对A549细胞的IC_(50)为(31.57±1.53)μmol/L、IC_(20)值为(5.09±0.78)μmol/L。升麻环氧醇苷对BEAS-2B细胞的IC_(50)为(26.60±1.41)μmol/L。与对照组比较,顺铂组、升麻环氧醇苷组、顺铂+升麻环氧醇苷组EdU阳性细胞率降低,细胞坏死率和总凋亡率升高,脂滴生成减少,p-Akt、mTOR、SREBP1、FASN、ACC1、ACLY、Cyclin E1、CDK2、Bcl-2蛋白表达降低,Bax和Cleaved-Caspase-3蛋白表达升高,SREBP1平均荧光强度降低(P<0.05)。与顺铂组比较,升麻环氧醇苷组细胞坏死率升高,脂滴生成减少,SREBP1、Cyclin E1、Bax和Cleaved-Caspase-3蛋白表达降低,CDK2、Bcl-2蛋白表达升高(P<0.05);顺铂+升麻环氧醇苷组细胞坏死率降低、总凋亡率升高,脂滴生成减少,p-Akt、mTOR、SREBP1、FASN、ACC1、ACLY、Cyclin E1、CDK2、Bcl-2蛋白表达降低,Bax和Cleaved-Caspase-3蛋白表达升高,SREBP1平均荧光强度降低(P<0.05)。与升麻环氧醇苷组比较,顺铂+升麻环氧醇苷组细胞坏死率降低、总凋亡率升高,脂滴生成减少,p-Akt、mTOR、SREBP1、FASN、ACC1、ACLY、Cyclin E1、CDK2、Bcl-2蛋白表达降低,Bax和Cleaved-Caspase-3蛋白表达升高,SREBP1平均荧光强度降低(P<0.05)。结论升麻环氧醇苷联合顺铂可能通过抑制磷脂酰肌醇3激酶/Akt/mTOR信号通路,调控SREBP1表达,影响A549细胞脂质代谢途径,从而抑制其增殖。
Objective To investigate the effect and mechanism of a combination of cimigenoside and cisplatin on the proliferation and apoptosis of lung cancer A549 cells.Methods Selecting A549 and BEAS-2B cell lines as the research objects,the CCK-8 method was employed to detect the cell viability of cimigenoside and cisplatin on A549 cells,as well as the cell viability of cimigenoside on BEAS-2B cells,and calculate the half maximal inhibitory concentration(IC_(50))and 20%inhibitory concentration(IC_(20)).Follow-up experiments were conducted on A549 cells using a IC_(20) of cimigenoside and cisplatin.A549 cells were divided into control,cisplatin(5.09μmol/L),cimigenoside(4.39μmol/L),and cisplatin+cimigenoside groups(5.09μmol/L+4.39μmol/L).The four groups were treated with the corresponding drugs for 48 h.The EdU-488 fluorescent probe method was used to detect the positive cell rate of A549 cells,and Annexin V/PI staining was used to detect the apoptosis status of A549 cells.Bodipy 493/503 staining was used to observe lipid droplet formation in A549 cells.Western blotting was used to detect protein kinase B(Akt),phosphorylated Akt(p-Akt),mammalian target of rapamycin(mTOR),steroid regulatory element-binding protein 1(SREBP1),fatty acid synthase(FASN),acetyl CoA carboxylase 1(ACC1),adenosine triphosphate citrate lyase(ACLY),Cyclin E1,cyclin-dependent kinase 2(CDK2),B-cell lymphoma 2(Bcl-2),Bcl-2 related X protein(Bax),and Cleaved-Caspase-3 protein expression in A549 cells.Confocal immunofluorescence was used to detect the average fluorescence intensity of SREBP1 in A549 cells.Results The IC_(50) and IC_(20) values of cimigenoside on A549 cells were 22.80±0.93μmol/L and 4.39±0.73μmol/L,respectively.The IC_(50) and IC_(20) values of cisplatin on A549 cells were 31.57±1.53μmol/L and 5.09±0.78μmol/L,respectively.The IC_(50) of cimigenoside for BEAS-2B cells was 26.60±1.41μmol/L.Compared with the control group,the EdU-positive cell rate decreased,the cell necrosis rate and total apoptosis rate increased,lipid droplet formation reduced,p-Akt,mTOR,SREBP1,FASN,ACC1,ACLY,Cyclin E1,CDK2,and Bcl-2 protein expression decreased,Bax and Cleaved-Caspase-3 protein expression increased,and the average SREBP1 fluorescence intensity decreased(P<0.05)in the cisplatin,cimigenoside,and cisplatin+cimigenoside groups.Compared with the cisplatin group,the cell necrosis rate increased,and lipid droplet formation was reduced,and SREBP1,Cyclin E1,Bax,and Cleaved-Caspase-3 protein expression decreased,whereas CDK2 and Bcl-2 protein expression increased in the cimigenoside group(P<0.05).The cisplatin+cimigenoside group showed a decrease in cell necrosis rate,an increase in total apoptosis rate,a reduction in lipid droplet formation,a decrease in p-Akt,mTOR,SREBP1,FASN,ACC1,ACLY,Cyclin E1,CDK2,and Bcl-2 protein expression,an increase in Bax and Cleaved-Caspase-3 protein expression,and a decrease in the average fluorescence intensity of SREBP1(P<0.05).Compared with the cimigenoside group,the cisplatin+cimigenoside group showed a decrease in cell necrosis rate,an increase in total apoptosis rate,a reduction in lipid droplet formation,a decrease in p-Akt,mTOR,SREBP1,FASN,ACC1,ACLY,Cyclin E1,CDK2,and Bcl-2 protein expression,an increase in Bax and Cleaved-Caspase-3 protein expression,and a decrease in the average fluorescence intensity of SREBP1(P<0.05).Conclusion The combination of cisplatin and cimigenoside can inhibit the phosphoinositide 3-kinase/Akt/mTOR signaling pathway,regulate SREBP1 expression,and affect the lipid metabolism pathway of A549 cells,thereby inhibiting their proliferation.
作者
李贺
刘文俊
王淳
LI He;LIU Wenjun;WANG Chun(School of Basic Medical Sciences,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;Teaching and Experiment Center,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China)
出处
《北京中医药大学学报》
北大核心
2025年第10期1377-1389,共13页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金项目(No.82174254)
辽宁省科技计划联合项目(No.2023JH2/101700210)
辽宁省教育厅科技创新团队项目(No.LJ222510162018)
辽宁中医药大学中医药科技创新多学科交叉团队项目(No.2025-Dxkjc-15-02)
辽宁中医药大学中西医结合学院“岐济”人才支撑计划项目(No.2023QJ01001)。
