目的 探讨重楼皂苷Ⅱ(polyphyllin Ⅱ,PPⅡ)对骨肉瘤(osteosarcoma,OS)细胞凋亡的影响及作用机制.方法 选取OS的U2OS和HOS细胞株作为研究对象.细胞计数试剂盒-8(CCK-8)法检测PPⅡ对细胞活力的影响;克隆形成实验检测PPⅡ对细胞克隆形成能力的影响;5-乙炔基-2'-脱氧尿苷(EdU)渗入实验检测PPⅡ对细胞DNA合成的影响;膜联蛋白V-异硫氰基荧光素/碘化丙啶(annexin V-FITC/PI)双染法检测PPⅡ对细胞凋亡率的影响;JC-1荧光探针检测PPⅡ对线粒体膜电位变化的影响;Western blot 法检测 PPⅡ 对 B 淋巴细胞瘤 2(B-cell lymphoma-2,Bcl-2)、Bcl-2 相关 X 蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸天冬氨酸蛋白酶3(cysteinyl aspartate specific proteinase 3,Caspase 3)、Caspase 7、Caspase 9、切割型半胱氨酸天冬氨酸蛋白酶3(cleaved cysteinyl aspartate specific proteinase 3,cleaved Caspase 3)、cleaved Caspase 7、cleaved Caspase 9、多腺苷二磷酸多聚酶[poly(ADP-ribose)polymerase,PARP]、切割型多腺苷二磷酸多聚酶[cleaved poly(ADP-ribose)polymerase,cleaved PARP]、蛋白激酶 R 样内质网激酶(protein kinase R like endoplasmic reticulum kinase,PERK)、真核生物翻译起始因子2α(eukaryotic translation initiation factor 2α,eIF2α)、C/EBP 同源蛋白(C/EBP homologous protein,CHOP)、激活转录因子 4(acti-vating transcription factor4,ATF4)、p-PERK和p-eIF2α蛋白表达水平的影响.结果 PPⅡ显著性抑制OS细胞活力,并呈时间和剂量相关性;抑制细胞克隆形成和DNA合成;显著性增加细胞凋亡率,降低线粒体膜电位(P<0.05或P<0.01);显著性提高 Bax/Bcl-2 蛋白比值,升高 cleaved Caspase 3、cleaved Caspase 7、cleaved Caspase 9、cleaved PARP、p-PERK、p-eIF2α、ATF4 和CHOP蛋白表达水平(P<0.05或P<0.01).结论 PPⅡ通过线粒体和内质网应激途径诱导OS细胞凋亡.
Polyphyllin Ⅱ Induces Apoptosis of Osteosarcoma Cells Through Mitochondrial Pathway and Endoplasmic Reticulum Stress
OBJECTIVE To investigate the effect of polyphyllin Ⅱ(PP Ⅱ)on apoptosis of osteosarcoma cells and its mecha-nism.METHODS Osteosarcoma(OS)U2OS and HOS cell lines were selected as the research object.CCK-8 method was used to detect the effect of PP Ⅱ on cell viability.Colony forming assay was used to detect the effect of PP Ⅱ on colony formation ability.EdU incorporation assay was used to detect the effect of PPⅡ on DNA synthesis.Annexin V-FITC/PI staining assay was used to detect the effect of PP Ⅱ on cell apoptosis.JC-1 assay was used to determine the effect of PP Ⅱ on the mitochondrial membrane potential(MMP).The protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cysteinyl aspartate specific proteinase 3(Caspase 3),Caspase 7,Caspase 9,cleaved cysteinyl aspartate specific proteinase 3(cleaved Caspase 3),cleaved Caspase 7,cleaved Caspase 9,poly(ADP-ribose)polymerase(PARP),cleaved poly(ADP-ribose)polymerase(cleaved PARP),protein kinase R like endoplasmic reticulum kinase(PERK),eukaryotic translation initiation factor 2α(eIF2α),C/EBP homologous protein(CHOP),activating transcription factor 4(ATF4),p-PERK,and p-eIF2α were analyzed by Western blot RESULTS PPⅡ significantly inhibited the viability of OS cells in a time-and dose-dependent manner,dose-dependently inhibited the colony forma-tion and DNA synthesis,significantly promoted apoptosis rate,decreased MMP(P<0.05 or P<0.01),and increased Bax/Bcl-2 pro-tein ratio and the protein expression levels of cleaved Caspase 3,cleaved Caspase 7,cleaved Caspase 9,cleaved PARP,p-PERK,p-eIF2α,ATF4,CHOP(P<0.05 or P<0.01).CONCLUSION PP Ⅱ promotes OS cell apoptosis by regulating mitochondrial pathway and mediating endoplasmic reticulum(ER)stress.