尿石素C对人急性髓系白血病HL-60细胞增殖、凋亡和自噬的影响及其机制  

Effect of urolithin C on proliferation,apoptosis and autophagy of human acute myeloid leukemia HL-60 cells and its mechanism

作　　者：于国兴 张鑫 杜恒玮 崔冰洁 高娜 刘翠兰 杜静 YU Guoxing;ZHANG Xin;DU Hengwei;CUI Bingjie;GAO Na;LIU Cuilan;DU Jing(Department of Hematology,Binzhou Medical University Hospital,Binzhou 256603,China;Medical Research Center,Binzhou Medical University Hospital,Binzhou 256603,China;Department of Gynecology,Binzhou Medical University Hospital,Binzhou 256603,China)

机构地区：[1]滨州医学院附属医院血液内科,山东滨州256603 [2]滨州医学院附属医院医学研究中心,山东滨州256603 [3]滨州医学院附属医院妇科,山东滨州256603

出　　处：《吉林大学学报（医学版）》2024年第4期908-916,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(82373097);国家自然科学基金青年基金项目(31900441);山东省泰山学者青年专家和齐鲁卫生与健康杰出青年人才项目(tsqn202103191);山东省卫健委中医药科技基金项目(Q-2022121)。

摘　　要：目的:探讨尿石素C(UC)对急性髓系白血病(AML)HL-60细胞增殖、凋亡和自噬的影响,阐明其相关作用机制。方法:HL-60细胞分为不同浓度(0、20、40、60、80及100μmol·L^(-1))尿石素A(UA)、尿石素B(UB)和UC组,采用CCK-8法检测各组细胞增殖活性,光学显微镜观察不同浓度UC组细胞形态表现;HL-60细胞分为不同浓度(0、20、40及80μmol·L^(-1))UC组和3-甲基腺嘌呤(3-MA)联合不同浓度(0、20、40及80μmol·L^(-1))UC组,采用CCK-8法检测各组细胞增殖活性。HL-60细胞分为对照组和不同浓度(20、40及80μmol·L^(-1))UC组,采用活/死细胞染色法检测各组死细胞率,流式细胞术检测各组细胞凋亡率,细胞自噬染色检测试剂盒[单丹磺酰戊二胺(MDC)法]检测各组细胞自噬情况,实时荧光定量PCR(RT-qPCR)法检测各组细胞中苄氯素1(Beclin 1)、自噬相关蛋白9(ATG9)和自噬相关蛋白7(ATG7)mRNA表达水平,Western blotting法检测各组细胞中含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、活化的Caspase-3(Cleaved Caspase-3)、微管相关蛋白1轻链3(LC-3)、细胞外信号调节激酶(ERK)、磷酸化ERK(p-ERK)、AMP依赖的蛋白激酶(AMPK)和磷酸化AMPK(p-AMPK)蛋白表达水平。结果:CCK-8法,培养24、48和72 h,分别与0μmol·L^(-1) UA、UB和UC组比较,不同浓度UA、UB和UC组细胞增殖活性均降低(P<0.05或P<0.01),且呈浓度和时间依赖性;48 h时,与UA和UB比较,UC半数抑制浓度(IC50)最低。细胞形态表现,与对照组比较,随着UC浓度升高,细胞间连接和细胞数量减少,细胞碎片增加。CCK-8法,与40和80μmol·L^(-1) UC组比较,3-MA联合40和80μmol·L^(-1) UC组细胞增殖活性升高(P<0.05或P<0.01)。活/死细胞染色,与对照组比较,40和80μmol·L^(-1) UC组死细胞率升高(P<0.01)。流式细胞术,与对照组比较,80μmol·L^(-1) UC组细胞凋亡率升高(P<0.01)。MDC法,与对照组比较,随着UC浓度升高,不同浓度UC组细胞绿色荧光逐渐增强。RT-qPCR法,与对�Objective:To discuss the effect of urolithin C(UC)on the proliferation,apoptosis,and autophagy of the acute myeloid leukemia(AML)HL-60 cells,and to clarify its mechanism.Methods:The HL-60 cells were divided into different concentrations(20,40,60,80,and 100μmol·L^(-1))of urolithin A(UA)groups,urolithin B(UB)groups,and UC groups.CCK-8 assay was used to detect the proliferation activity of the cells in various groups;the morphology of the cells in different concentrations of UC groups was observed under optical microscope.The HL-60 cells were divided into different concentrations(0,20,40,and 80μmol·L^(-1))of UC groups and 3-methyladenine(3-MA)combined with different concentrations(0,20,40,and 80μmol·L^(-1))of UC groups.CCK-8 assay was used to detect the proliferation activities of the cells in various groups.The HL-60 cells were divided into control group(0μmol·L^(-1))and different concentrations(20,40,and 80μmol·L^(-1))of UC groups.The live/dead cell staining method was used to detect the dead rates of the cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups;the autophagy of the cells was detected by autophagy staining kit(monodansylcadaverine,MDC)method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of Beclin 1,autophagy related gene 9(ATG9),and autophagy related gene 7(ATG7)mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of cysteinyl aspartate specific proteinase-3(Caspase-3),cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3),microtubule-associated protein 1 light 3(LC-3),extracellular regulated protein kinases(ERK),phosphorylated ERK(p-ERK),AMP-activated protein kinase(AMPK),and phosphorylated AMPK(p-AMPK)in the cells in various groups.Results:The CCK-8 assay results showed that after cultured for 24,48,and 72 h,compared with 0μmol·L^(-1) UA,UB,and UC groups,the proliferation activities of the cells in different concentrations

关 键 词：尿石素C 白血病细胞 细胞增殖 细胞凋亡 细胞自噬 

分 类 号：R733.71[医药卫生—肿瘤] R979.19[医药卫生—临床医学]