黄芪甲苷对肾癌细胞系(769-P)增殖、凋亡的影响  

Effects of astragaloside IV on renal cell carcinoma cell proliferation and apoptosis in vitro

作　　者：施志超[1] SHI Zhi-chao(Department of Clinical Pharmacy,Lishui People's Hospital,Lishui,Yunnan 323000,China)

机构地区：[1]浙江省丽水市人民医院临床药学科,丽水323000

出　　处：《浙江中西医结合杂志》2022年第10期898-902,共5页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省丽水市科技局项目(No.2020SJZC039)。

摘　　要：目的探讨黄芪甲苷对肾癌细胞(769-P)增殖、凋亡的作用机制。方法人源肾癌细胞(769-7)在标准细胞培养条件(37℃,5%CO_(2))下培养传代,取对数生长期细胞进行实验。使用0、5、10、20、40和80μM浓度黄芪甲苷溶液与肾癌细胞(769-P)共同培育24 h或48 h后,使用CCK-8法检测黄芪甲苷对肾癌细胞的细胞毒性作用。使用活性氧(ROS)探针(DCFH-DA)检测ROS水平,使用Annexin V FITC/PI双染法检测细胞凋亡比例,使用线粒体膜电位探针(JC-1)检测线粒体膜电位,使用分光光度法检测活性胱天蛋白酶3(Caspase 3)和胱天蛋白酶9(Caspase 9)水平。结果5、10、20、40和80μM黄芪甲苷干预48 h后,769-P细胞活性较对照组显著降低[(85.37±4.60)%、(63.73±3.51)%、(42.43±5.65)%、(26.57±2.41)%和(18.47±3.23)%比(100.00±0.00)%,P<0.05或P<0.01]。与对照组比较,黄芪甲苷10、20和40μM处理769-P细胞24 h后,ROS荧光强度明显增强[(180274.98±3150.01)、(210165.51±15648.06)和(264301.56±8331.92)比(153166.22±7782.05),P<0.05或P<0.01],线粒体膜电位JC-1荧光强度比值(FL1-A/FL2-A)明显上升[(2.12±0.07)、(2.61±0.35)和(3.15±0.30)比(1.79±0.16),P<0.05或P<0.01]。与对照组比较,黄芪甲苷10、20和40μM处理769-P细胞48 h后,凋亡比例明显上升[(51.23±4.66)%、(63.01±3.22)%和(85.50±3.60)%比(1.38±0.65)%,P均<0.05],Casapse 3活性明显上升[(2.81±0.46)、(3.93±0.72)和(6.08±1.58)比(1.00±0.00),P均<0.05],Casapse 9活性明显上升[(1.44±0.19)、(1.73±0.25)和(2.67±0.12)比[(1.00±0.00),P<0.05或P<0.01]。结论黄芪甲苷以浓度依赖性的方式抑制769-P细胞的增殖活性,对769-P细胞产生细胞毒性,促进ROS过度表达,降低线粒体膜电位,增加Caspase 3/9活性,最终诱导细胞凋亡。黄芪甲苷抑制769-P细胞增殖,可以诱导细胞凋亡,其可能的机制与激活Caspase通路有关。Objective To assess the effect and the underlying molecular events of astragalosideⅣon renal cell carcinoma cell proliferation and apoptosis.Methods A human renal cell carcinoma cell line 769-P was cultured and treated with 0,5,10,20,40 and 80μM astragalosideⅣsolutions for 24 or 48 h to analyze the cytotoxic effects of astragalosideⅣusing cell viability CCK-8 assay.The ROS level was detected using the ROS probe DCFH-DA,tumor cell apoptosis was assayed using Annexin V FITC/PI double staining method,and the mitochondrial membrane potential was detected using mitochondrial membrane potential probe JC-1.Activation of Caspase 3 and Caspase 9 was visualized by using spectrophotometry.Results Treatment of 769-P cells with 5,10,20,40,and 80μM of astragalosideⅣfor 48 h significantly reduced cell viability compared with that of the control cells(85.37±4.60,63.73±3.51,42.43±5.65,26.57±2.41,and 18.47±3.23 vs.100.00±0.00%;P<0.05 or P<0.01).The 24 h-treatments also significantly enhanced the ROS fluorescence intensity(180274.98±3150.01,210165.51±15648.06,and 264301.56±8331.92 vs.153166.22±7782.05;P<0.05 or P<0.01)and the fluorescence intensity ratio of mitochondrial membrane potential JC-1(FL1-A/FL2-A)(2.12±0.07,2.61±0.35,and 3.15±0.30 vs.1.79±0.16;P<0.05 or P<0.01).Furthermore,compared with the control cells,tumor cell apoptosis after treated with 10,20 and 40μM astragalosideⅣfor 48 h was significantly increased(51.23±4.66,63.01±3.22,and 85.50±3.60 vs.1.38±0.65%;all P<0.05).Caspase 3 and 9 activity was also increased significantly(2.81±0.46,3.93±0.72,and 6.08±1.58 vs.1.00±0.00;1.44±0.19,1.73±0.25,and 2.67±0.12 vs.1.00±0.00;P<0.05 or P<0.01,respectively;all P<0.05).Conclusion Astragaloside IV was able to reduce renal cancer 769-P cell viability in a dose-dependent manner,promoted tumor cells to overexpress ROS,but decreased the mitochondrial membrane potential to increase Caspase 3/9 activity and apoptosis.

关 键 词：黄芪甲苷 肾癌 769-P 凋亡 

分 类 号：R285[医药卫生—中药学]