摘要
目的探讨重楼皂苷Ⅱ(polyphyllinⅡ,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(activating transcription factor 4,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。
OBJECTIVE To investigate the effect of polyphyllinⅡ(PPⅡ)on apoptosis of osteosarcoma cells and its mechanism.METHODS Osteosarcoma(OS)U2OS and HOS cell lines were selected as the research object.CCK-8 method was used to detect the effect of PPⅡon cell viability.Colony forming assay was used to detect the effect of PPⅡon colony formation ability.EdU incorporation assay was used to detect the effect of PPⅡon DNA synthesis.Annexin V-FITC/PI staining assay was used to detect the effect of PPⅡon cell apoptosis.JC-1 assay was used to determine the effect of PPⅡon 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-eIF2αwere analyzed by Western blot.RESULTS PPⅡsignificantly inhibited the viability of OS cells in a time-and dose-dependent manner,dose-dependently inhibited the colony formation and DNA synthesis,significantly promoted apoptosis rate,decreased MMP(P<0.05 or P<0.01),and increased Bax/Bcl-2 protein 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 PPⅡpromotes OS cell apoptosis by regulating mitochondrial pathway and mediating endoplasmic reticulum(ER)stress.
作者
施亚敏
李新存
聂安政
朱春胜
SHI Yamin;LI Xincun;NIE Anzheng;ZHU Chunsheng(The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2024年第1期29-38,共10页
Chinese Pharmaceutical Journal
基金
国家自然科学基金青年科学基金项目资助(82204701)
河南省医学科技攻关计划联合共建项目资助(2018020106)。
关键词
重楼皂苷Ⅱ
骨肉瘤
细胞凋亡
线粒体途径
内质网应激
polyphyllinⅡ
osteosarcoma
apoptosis
mitochondrial pathway
endoplasmic reticulum stress
