人参皂苷Rg3联合肿瘤抑素19肽对肝癌HepG2细胞凋亡的影响及机制  

Effects and mechanism of ginsenoside Rg3 combined with tumstatin 19 peptide on apoptosis of HepG2 cells in liver cancer

作　　者：衣同辉[1] 吴艳敏[1] 刘睿[2] 张春晶[1] 姚淑娟[1] 师岩[1] 李淑艳[1] Yi Tonghui;Wu Yanmin;Liu Rui;Zhang Chunjing;Yao Shujuan;Shi Yan;Li Shuyan(School of Medical Technology,Qiqihar Medical University,Heilongjiang 161006,China;The First Affiliated Hospital of Qiqihar Medical University,Heilongjiang 161041,China)

机构地区：[1]齐齐哈尔医学院医学技术学院,黑龙江161006 [2]齐齐哈尔医学院附属第一医院

出　　处：《北京中医药大学学报》2020年第7期575-582,共8页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.81641134);黑龙江省教育厅基金项目(No.2016-KYYWF-0838)。

摘　　要：目的研究人参皂苷Rg3联合肿瘤抑素19肽对肝癌HepG2细胞凋亡的影响及作用机制。方法实验为两因素两水平2×2析因设计,因素A为人参皂苷Rg3,水平为0 mg/L和40 mg/L,因素B为肿瘤抑素19肽,水平为0 mg/L和200 mg/L。CCK-8试剂盒检测肝癌HepG2细胞增殖情况;Hoechst33342染色检测细胞凋亡的形态学变化;流式细胞术检测HepG2细胞凋亡率。荧光探针JC-1检测细胞的膜电位改变。Western blot法检测细胞抑癌基因磷酸酶和张力蛋白同源蛋白(PTEN)、磷酸化的磷脂酰肌醇-3激酶(p-PI3K)和磷酸化的丝氨酸/苏氨酸激酶(p-Akt)蛋白的表达变化。天冬氨酸蛋白水解酶(Caspase)活性测定试剂盒检测细胞Caspase-3和Caspase-9的活性。结果人参皂苷Rg3、肿瘤抑素19肽及联合用药对HepG2细胞的增殖均有抑制作用,且两者具有正协同效应;CCK-8测定人参皂苷Rg3的IC50为45.06 mg/L,肿瘤抑素19肽IC50为214.03 mg/L。Hoechst33342染色可见Rg3和19肽单用或合用时均出现细胞凋亡,并且联合用药细胞凋亡形态变化明显。流式细胞术检测细胞凋亡趋势与Hoechst33342染色结果一致,联合用药凋亡率最高。荧光探针JC-1检测表明单用或合用时细胞膜电位下降,产生绿色荧光,联合用药产生绿色荧光最为明显。联合用药更为显著上调PTEN蛋白表达,下调p-PI3K和p-Akt蛋白表达,显著增高Caspase-3和Caspase-9活性(P<0.001)。结论人参皂苷Rg3和肿瘤抑素19肽具有抑制肝癌HepG2细胞增殖,诱导其凋亡的作用,且两者具有正协同效应。其作用机制可能是通过PTEN/PI3K/Akt信号通路实现的。Objective To study the effects and mechanism of ginsenoside Rg3 combined with tumstatin 19 peptide on apoptosis of liver cancer HepG2 cells.Methods The two-level factorial-analysis design was used in the experiment with factor A of ginsenoside Rg3 with two levels of 0 mg/L and 40mg/L,and factor B of tumstatin 19 peptide with two levels of 0 mg/L and 200 mg/L.The proliferation of HepG2 cells was measured by using the CCK-8 kit.Hoechst33342 staining was used to detect the morphological changes of the apoptosis of HepG2 cells.The apoptosis rates of HepG2 cells were detected with flow cytometry.The cell membrane potential change of HepG2 cells was measured by fluorescence probe JC-1.The expression ofphosphatase and tensin homology deleted on chromosometen(PTEN),phosphated phosphatidylinositol phosph atidylinositol 3-kinase(p-PI3K)and p-Akt was evaluated by using Western blot assay.The activities of caspase-3 and caspase-9 were measured with the Caspase-activity assay kit.Results Ginsenoside Rg3,tumstatin 19 peptide and the combined therapyhad in hibitory effect on the proliferation of HepG2 cells.Ginsenoside Rg3,tumstatin 19 peptide had a positive synergistic effect.The IC50 of Rg3 was 45.06 mg/L,and that of 19 peptide was 214.03mg/L.Results of Hoechst33342 staining showed that changes of apoptosis in combined therapy was more evident.The results of flow cytometry were consistent with Hoechst33342 staining,and the apoptosis rate was the highest in combined therapy.Fluorescence probe JC-1 showed that combined therapy emitted the strongest green fluorescence.The combined therapy was more significant in up-regulating the expression of PTEN protein and down-regulating the expression of p-PI3K and p-Akt protein.The activities of caspase-3 and caspase-9 enzyme were significantly increased in combined therapy(P<0.001).Conclusion Ginsenoside Rg3 and tumstatin 19 peptide can inhibit proliferation and induce apoptosis of HepG2 cells.They manifest a positive synergistic effect.The synergistic effect might be achieved through th

关 键 词：人参皂苷RG3 肿瘤抑素19肽 肝癌 凋亡 PTEN /PI3K /Akt信号通路 

分 类 号：R285.5[医药卫生—中药学]