甜菊多糖诱导弥漫性大B淋巴瘤细胞U2940凋亡的作用及其机制  

Effect of stevia polysaccharide on apoptosis of diffuse large B-cell lymphoma cell line U2940 and its molecular mechanism

作　　者：杨林军[1] 汪正一 王建 张瑾[1] 金晓燕[1] Yang Linjun;Wang Zhengyi;Wang Jian;Zhang Jin;Jin Xiaoyan(Department of Surgical Oncology,Taizhou Municipal Hospital,Taizhou 318000,China)

机构地区：[1]台州市立医院肿瘤外科,台州318000

出　　处：《中华实验外科杂志》2024年第12期2760-2763,共4页Chinese Journal of Experimental Surgery

基　　金：浙江省科技厅公益技术研究计划项目(LGF20H280008);台州市科技计划项目(23ywb62、20ywa38)。

摘　　要：目的探讨甜菊多糖对大B淋巴瘤细胞U2940的作用及机制。方法分别用0~64 μg/ml甜菊多糖作用于U2940细胞, 细胞计数试剂盒(CCK-8)法分析该细胞增殖活性, 确定药物有效浓度;乳酸脱氢酶(LDH)释放实验证实甜菊多糖的细胞毒作用, JC-1染色观察细胞线粒体膜电位, 流式检测细胞凋亡率, 实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)确定细胞内基因转录和表达水平。观察甜菊多糖对荷弥漫性大B淋巴瘤(DLBCL)小鼠肿瘤生长的抑制作用和小鼠生存期影响。采用配对样本t检验和χ^(2)检验来统计分析。结果甜菊多糖实验组对U2940细胞的增殖活性低于对照组, 16 μg/ml和64 μg/ml的甜菊多糖在作用24 h后抑制U2940细胞的增殖低于对照组(0.24±0.01比0.31±0.01, t=6.31, P<0.05;0.20±0.03比0.31±0.01, t=4.42, P<0.05), 同时该药诱导U2940细胞凋亡, 实验组Ras/Raf/丝裂原细胞外激酶(MEK)/细胞外信号调节激酶(ERK)、磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)和核因子-κB(NF-κB)通路及下游B细胞淋巴瘤/白血病-2(bcl-2)、bcI-XL、MCL-1基因转录低于对照组, (0.44±0.02比1.00±0.06, t=6.11, P<0.05;0.51±0.09比1.00±0.03, t=3.77, P<0.05;0.39±0.06比1.00±0.04, t=2.78, P<0.05), 实验组凋亡直接相关蛋白半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、Caspase-8和Caspase-9等的表达高于对照组, (3.69±0.08比1.00±0.03, t=6.75, P<0.05;2.39±0.10比1.00±0.02, t=6.45, P<0.05;3.67±0.08比1.00±0.03, t=6.32, P<0.05);抑瘤实验证实甜菊多糖跟对照组比较可抑制小鼠瘤组织的生长并延长荷瘤小鼠生存期(54 d比14 d, t=4.66, P<0.05)。结论甜菊多糖可通过影响Ras/Raf/MEK/ERK、P13K/Akt/mTOR和NF-κB信号通路, 抑制U2940细胞增殖并诱导其凋亡, 有显著的抗DLBCL作用。ObjectiveTo investigate the effect and mechanism of stevia polysaccharide on large B lymphoma cell line U2940.MethodsU2940 cells were treated with stevia polysaccharides at the concentration of 0-64μg/ml,and the proliferation activity of U2940 cells was determined by cell counting kit-8(CCK-8).The cytotoxicity of stevia polysaccharides was confirmed by lactate dehydrogenase(LDH)release assay,JC-1 staining was used to observe mitochondrial membrane potential,flow cytometry was used to detect apoptosis rate,and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting were used to determine gene transcription and expression level.In addition,the inhibitory effect of stevia polysaccharide on tumor growth and survival time of diffuse large B-cell lymphoma(DLBCL)-bearing mice were also observed.Paired sampl t test andχ^(2) test were used for statistical analysis.ResultsThe proliferation activity of U2940 cells in the experimental group was lower than that in the control group.After 24 h,16μg/ml and 64μg/ml stevia polysaccharide inhibited the proliferation of U2940 cells less than that in the control group(0.24±0.01 vs.0.31±0.01,t=6.31,P<0.05;0.20±0.03 vs.0.31±0.01,t=4.42,P<0.05),and the apoptosis of U2940 cells was induced by this drug.The transcription of Ras/Raf/mitogen extracellular kinase(MEK)/extracellular signal-regulated kinase(ERK),phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)and nuclear factor-κB(NF-κB)pathway and downstream bcI-2,bcI-XL and MCL-1 genes in the experimental group was lower than that in the control group(0.44±0.02 vs.1.00±0.06,t=6.11,P<0.05;0.51±0.09 vs.1.00±0.03,t=3.77,P<0.05;0.39±0.06 vs.1.00±0.04,t=2.78,P<0.05).The expressions of cysteinyl aspartate-specific protease(Caspase)-3,Caspase-8 and Caspase-9,which are directly related to apoptosis,in the experimental group were higher than those in the control group,(3.69±0.08 vs.1.00±0.03,t=6.75,P<0.05);(2.39±0.10 vs.1.00±0.02,t=6.45,P<0.0

关 键 词：甜菊多糖 弥漫大B细胞淋巴瘤 信号通路 增殖活性 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]