柴胡皂苷D通过Akt/mTOR通路调控胰腺癌Panc-1细胞凋亡及自噬  

Saikosaponin D regulates apoptosis and autophagy of pancreatic cancer Panc-1 cells via Akt/mTOR pathway

作　　者：关月宏 刘桂梅 刘雨思 兰琳博 郑蕊 刘晓斌[1] GUAN Yue-hong;LIU Gui-mei;LIU Yu-si;LAN Lin-bo;ZHENG Rui;LIU Xiao-bin(Department of Immunology,School of Medicine,Yan′an University,Yan′an 716000,China)

机构地区：[1]延安大学医学院免疫学教研室,陕西延安716000

出　　处：《中国中药杂志》2023年第19期5278-5284,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82260489);陕西省科技厅项目(2022JQ-931);陕西省教育厅专项科研基金项目(11JK0669)。

摘　　要：该研究旨在探讨柴胡皂苷D(saikosaponin D)对胰腺癌Panc-1细胞增殖、凋亡和自噬的影响及其作用机制。采用细胞增殖及细胞毒性检测试剂盒(CCK-8)测定7、10、13、16、19、22、25、28μmol·L^(-1)柴胡皂苷D对Panc-1细胞增殖活性的影响;分别设置对照组(0μmol·L^(-1))、柴胡皂苷D低浓度组(10μmol·L^(-1))和柴胡皂苷D高浓度组(16μmol·L^(-1)),采用平板克隆形成实验检测柴胡皂苷D对Panc-1细胞克隆形成率的影响;采用苏木素-伊红(HE)染色观察柴胡皂苷D作用后细胞形态的变化;采用Hoechst 33258荧光染色检测柴胡皂苷D对细胞凋亡的影响;采用细胞自噬染色检测试剂盒(MDC法)检测柴胡皂苷D对细胞自噬的影响;采用蛋白免疫印迹(Western blot)法和免疫细胞化学法检测柴胡皂苷D对B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(caspase-3)、剪切的caspase-3(cleaved caspase-3)、自噬相关蛋白Beclin1、微管相关蛋白轻链3(LC3)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)表达水平及分布的影响。结果显示,与对照组相比,柴胡皂苷D显著抑制Panc-1细胞增殖,且呈时间和浓度依赖性;柴胡皂苷D作用后细胞克隆形成率显著降低。与对照组相比,柴胡皂苷D处理组细胞萎缩变小,并伴有凋亡小体形成;细胞凋亡率显著升高,自噬小泡堆积显著增多;Bax、cleaved caspase-3、Beclin1、LC3Ⅱ/LC3Ⅰ的表达显著升高,Bcl-2、caspase-3、p-Akt/Akt、p-mTOR/mTOR的表达显著降低,均主要分布在细胞质中。因此,柴胡皂苷D可通过抑制Akt/mTOR通路抑制胰腺癌Panc-1细胞增殖并诱导其凋亡及自噬。This study aims to investigate the effect and mechanism of saikosaponin D on the proliferation,apoptosis,and autophagy of pancreatic cancer Panc-1 cells.The cell counting kit(CCK-8) was used to examine the effects of 7,10,13,16,19,22,25,and 28 μmol·L^(-1) saikosaponin D on the proliferation of Panc-1 cells.Three groups including the control(0 μmol·L^(-1)),low-concentration(10 μmol·L^(-1)) saikosaponin D,and high-concentration(16 μmol·L^(-1)) saikosaponin D groups were designed.The colony formation assay was employed to measure the effect of saikosaponin D on the colony formation rate of Panc-1 cells.The cells treated with saikosaponin D were stained with hematoxylin-eosin(HE),and the changes of cell morphology were observed.Hoechst 33258 fluorescent staining was used to detect the effect of saikosaponin D on the cell apoptosis.The autophagy staining assay kit with MDC was used to examine the effect of saikosaponin D on the autophagy of Panc-1 cells.Western blot and immunocytochemistry(ICC) were employed to examine the effect of saikosaponin D on the expression levels and distribution of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cysteine-aspartic acid protease-3(caspase-3),cleaved caspase-3,autophagy-associated protein Beclin1,microtubule-associated protein light chain 3(LC3),protein kinase B(Akt),phosphorylated protein kinase B(p-Akt),mammalian target of rapamycin(mTOR),and phosphorylated mammalian target of rapamycin(p-mTOR).The results showed that compared with the control group,saikosaponin D decreased the proliferation rate of Panc-1 cells in a dose-dependent and time-dependent manner.The colony formation rate of the cells significantly decreased after saikosaponin D treatment.Compared with the control group,the cells treated with saikosaponin D became small,accompanied by the formation of apoptotic bodies.The saikosaponin D groups showed increased apoptosis rate and autophagic vesicle accumulation.Compared with the control group,saikosaponin D up-regulated the expression of Bax,cleaved

关 键 词：柴胡皂苷D 胰腺癌 凋亡 自噬 AKT/MTOR 

分 类 号：R285[医药卫生—中药学]