胡桃醌对骨肉瘤细胞凋亡和焦亡的影响  

Effect of Juglone on apoptosis and pyroptosis of osteosarcoma cells

作　　者：赵杰瑞 籍铭辛 张钰涵 陈姝彤 郭玉苗 张巍 彭鹏 ZHAO Jierui;JI Mingxin;ZHANG Yuhan;CHEN Shutong;GUO Yumiao;ZHANG Wei;PENG Peng(Faculty of Chinese Medicine,Macao University of Science and Technology,Macao 999078,China;Department of Orthopedics and Traumatology,Affiliated Hospital,Faculty of Chinese Medicine,Macao University of Science and Technology,Zhuhai 519003,China;Department of Basic Medical Laboratory,School of Basic Medical Science,Jilin Medical University,Jilin 132000,China;Department of Endocrinology,Affiliated Hospital,Changchun University of Chinese Medicine,Changchun 130012,China;Department of Pathogen Biology Laboratory,School of Basic Medicine,Beihua University,Jilin 132000,China;Department of Basic Medical Laboratory,School of Basic Medical Science,School of Medical Science,Yanbian University,Yanji 133000,China)

机构地区：[1]澳门科技大学中医药学院,中国澳门999078 [2]澳门科技大学中医药学院附属珠海医院骨伤二科,广东珠海519003 [3]吉林医药学院基础医学院基础医学实验室,吉林吉林132000 [4]长春中医药大学附属医院内分泌科,吉林长春130012 [5]北华大学基础医学院病原生物学实验室,吉林吉林132000 [6]延边大学医学院基础医学院基础医学实验室,吉林延吉133000

出　　处：《吉林大学学报(医学版)》2025年第2期420-427,共8页Journal of Jilin University:Medicine Edition

基　　金：广东省卫健委医学科学技术研究基金资助项目(A2021004);吉林省科技厅科技发展计划项目(YDZJ202301ZYTS111);吉林省教育厅“十三五”科学技术研究项目(JJKH20220464KJ)。

摘　　要：目的:探讨胡桃醌通过天冬氨酸特异性的半胱氨酸蛋白水解酶3 (Caspase-3)/蛋白焦孔素E (GSDME)介导焦亡途径对骨肉瘤(OS)细胞(U2OS和MG63细胞)凋亡的影响。方法:体外培养U2OS和MG63细胞,将细胞分为对照组、不同浓度(5、10和20μmol·L^(-1))胡桃醌处理组和Caspase-3抑制剂Z-DEVD-FMK组(10μmol·L^(-1)胡桃醌+30μmol·L^(-1) Z-DEVD-FMK)。采用细胞计数试剂盒8 (CCK-8)法检测各组细胞存活率,流式细胞术检测各组细胞程序性死亡率,乳酸脱氢酶(LDH)释放实验检测各组细胞LDH释放率,Western blotting法检测细胞中B细胞淋巴瘤2 (Bcl-2)、Bcl-2相关X蛋白(Bax)、聚二磷酸腺苷核糖聚合酶(PARP)和裂解的Caspase-3 (cleaved-Caspase-3)等凋亡相关蛋白以及蛋白焦孔素E全长(GSDME-F)和蛋白焦孔素E的N末端(GSDME-N)等焦亡相关活性蛋白的表达水平,酶联免疫吸附试验(ELISA)法检测各组细胞上清液中白细胞介素1β (IL-1β)和白细胞介素18 (IL-18)水平。结果:与对照组比较,5、10和20μmol·L^(-1)胡桃醌组细胞存活率明显降低(P<0.05或P<0.01),U2OS和MG63细胞的半数抑制浓度(IC_(50))分别为8.4及10.2μmol·L^(-1);与对照组比较,5和10μmol·L^(-1)胡桃醌组细胞凋亡率和LDH释放率明显升高(P<0.05或P<0.01);与对照组比较,5和10μmol·L^(-1)胡桃醌组细胞中Bax,cleaved-Caspase-3和cleaved-PARP蛋白表达水平明显升高(P<0.01),Bcl-2蛋白表达水平明显降低(P<0.01)。与对照组比较,5和10μmol·L^(-1)胡桃醌组细胞上清液中IL-1β和IL-18水平均升高(P<0.01),cleaved-Caspase-3和GSDME-N蛋白表达水平升高(P<0.01),GSDME-F蛋白表达水平差异无统计学意义(P>0.05)。与10μmol·L^(-1)胡桃醌组比较,Z-DEVD-FMK组细胞中cleaved-Caspase-3和GSDME-N蛋白表达水平明显降低(P<0.01),GSDME-F蛋白表达水平差异无统计学意义(P>0.05)。结论:胡桃醌可诱导U2OS和MG63细胞凋亡,并引起Caspase-3/GSDME介导的细胞焦亡。Objective:To investigate the effects of Juglone on the apoptosis of osteosarcoma(OS)cells(U2OS and MG63 cells)through the cysteinyl aspartate specific proteinase-3(Caspase-3)/gasdermin E(GSDME)-mediated pyroptosis pathway.Methods:The U2OS and MG63 cells were cultured in vitro and divided into control group,different concentrations(5,10 and 20μmol·L^(-1))of Juglone groups and Caspase-3 inhibitor Z-DEVD-FMK group(10μmol·L^(-1) Juglone+30μmol·L^(-1) Z-DEVD-FMK).The survival rates of cells in various groups were assessed by cell counting kit-8(CCK-8)assay,and the apoptotic rates were detected by flow cytometry.Lactate dehydrogenase(LDH)release assay was used to measure the release rates of LDH from the cells.Western blotting method was used to detect the expression levels of apoptosis-related proteins including B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved-Caspase-3 and poly(ADP-ribose)polymerase(PARP)and pyroptosis-related proteins including GSDME full form(GSDME-F)and GSDME N-terminal(GSDME-N).The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in the cell supernataut in various groups were measured by enzyme-linked immunosorbent assay(ELISA)method.Results:Compared with control group,the survival rates of cells in 5,10,and 20μmol·L^(-1) Juglone groups were significantly decreased(P<0.05 or P<0.01),and the 50%inhibitory concentration(IC50)values of U2OS cells and MG63 cells were 8.4 and 10.2μmol·L^(-1),respectively.Compared with control group,the apoptotic rates and LDH release rates of U2OS and MG63 cells in 5 and 10μmol·L^(-1) Juglone groups were significantly increased(P<0.05 or P<0.01).Compared with control group,the expression levels of Bax,cleaved-Caspase-3,and cleaved-PARP proteins in 5 and 10μmol·L^(-1) Juglone groups were significantly increased(P<0.01),while the expression levels of Bcl-2 protein were significantly decreased(P<0.01).Compared with control group,the levels of IL-1βand IL-18 in cell supernatant in 5 and 10μmol·L^(-1) Juglone groups were increased(

关 键 词：胡桃醌 骨肉瘤 细胞凋亡 细胞焦亡 B细胞淋巴瘤2 B细胞淋巴瘤2相关X蛋白 

分 类 号：R979.1[医药卫生—药品]