摘要
目的探究姜黄素对葡萄膜黑色素瘤(UM)恶性生物学行为的抑制作用及机制。方法应用含不同浓度(0、20、40和80μmol/L)姜黄素培养液培养M23细胞48 h,于倒置显微镜下观察细胞形态学改变;采用细胞计数试剂盒8(CCK-8)法检测细胞存活率;分别采用平板克隆形成实验、流式细胞术、细胞划痕实验及Transwell实验检测M23细胞集落形成、凋亡、迁移及侵袭情况;采用实时荧光定量PCR法检测细胞中Wnt/β-catenin通路相关基因c-Myc、细胞周期蛋白D1(Cyclin D1)、Survivin及基质金属蛋白酶9(MMP-9)mRNA相对表达情况;采用Western blot法检测Wnt/β-catenin通路相关蛋白c-Myc、Cyclin D1、Survivin、MMP-9、β-连环蛋白(β-catenin)、糖原合成酶激酶3β(GSK-3β)、磷酸化GSK-3β(p-GSK-3β)及轴抑制蛋白2(Axin2)蛋白相对表达量。另取20只6周龄雌性BALB/c小鼠,左后腹皮下脂肪垫注射M23细胞悬浮液建立小鼠M23体内移植肿瘤模型,将造模成功小鼠按照随机数字表法随机平均分为模型组、姜黄素低剂量组、姜黄素中剂量组和姜黄素高剂量组,每组5只,分别腹腔内注射0、10、20和40 mg/kg姜黄素生理盐水溶液,连续注射30 d后剥离皮下瘤体并称质量。结果0μmol/L姜黄素组、20μmol/L姜黄素组、40μmol/L姜黄素组和80μmol/L姜黄素组细胞存活率分别为(100.00±0.00)%、(83.78±4.59)%、(66.09±3.92)%和(47.16±3.63)%,细胞集落形成数分别为128.67±9.18、100.33±8.73、58.67±6.55和31.67±4.92,细胞凋亡率分别为(1.33±0.29)%、(14.53±2.04)%、(27.23±3.56)%和(44.73±4.36)%,细胞迁移率分别为(89.76±4.57)%、(65.43±3.70)%、(34.83±2.19)%和(18.82±1.99)%,细胞侵袭数分别为148.33±8.18、125.33±7.41、73.67±6.34、45.67±5.31,各组M23细胞存活率、集落形成数、细胞凋亡率、细胞迁移率、细胞侵袭数总体比较差异均有统计学意义(F=125.321、97.941、72.516、277.097、139.006,均P<0.001)。随姜黄素作用浓度的增大,细胞存活率、集落形成数、细胞迁移率、细胞侵袭数均明显降低,细胞凋亡率均明显增加,组间两两比较差异均有统计学意义(均P<0.05)。随姜黄素浓度的增大,细胞中c-Myc、Cyclin D1、Survivin和MMP-9 mRNA和蛋白及β-catenin和p-GSK-3β蛋白相对表达水平均明显降低,Axin2蛋白相对表达水平明显升高,组间比较差异均有统计学意义(均P<0.05)。小鼠荷瘤质量随姜黄素作用剂量的增加而降低,组间比较差异均有统计学意义(均P<0.05)。结论姜黄素可抑制UM细胞M23增生、迁移、侵袭等恶性生物学行为,抑制肿瘤生长,促进细胞凋亡,其作用机制可能与阻断Wnt/β-catenin通路激活有关。
Objective To explore the inhibitory effect of curcumin on the malignant biological behavior of uveal melanoma(UM)and its possible mechanism.Methods M23 cells were cultured in curcumin medium with different concentrations(0,20,40 and 80μmol/L)for 48 hours,respectively.The morphological changes of cells were observed under an inverted microscope.The cell survival rate was detected by the cell counting kit-8(CCK-8)method.The apoptosis,colony formation,migration and invasion of cells were detected by flow cytometry,plate clone formation experiment,cell scratch experiment and Transwell assay,respectively.The relative expressions of genes related to Wnt/β-catenin pathway,c-Myc,Cyclin D1,Survivin and matrix metallo proteinase 9(MMP-9)mRNA in cells were detected by real-time fluorescence quantitative PCR.The relative expressions of proteins related to Wnt/β-catenin pathway,c-Myc,Cyclin D1,Survivin,MMP-9 andβ-catenin,glycogen synthase kinase 3β(GSK-3β),phosphorylated GSK-3β(p-GSK-3β)and axis inhibition protein 2(Axin2)proteins were detected by Western blot.Another 20 female BALB/c mice were selected and injected with M23 cell suspension under the subcutaneous fat pad in the left posterior abdomen to establish the in vivo M23 transplanted tumor model.The mice successfully modeled were randomly divided into model group,low-dose curcumin group,medium-dose curcumin group and high-dose curcumin group according to the random number table method,which was intraperitoneally injected with 0,10,20 and 40 mg/kg curcumin physiological saline solution respectively.After a continuous injection for 30 days,the subcutaneous tumor was stripped and weighed.The animal experiment process followed the 3Rs principle of animal research and was approved by the Laboratory Animal Ethics Committee of Inner Mongolia Baotou Steel Hospital(No.2021MER-023).Results The cell survival rate,the number of colony formation,the apoptosis rate,the cell invasion rate and the cell migration rate were(100.00±0.00)%,128.67±9.18,(1.33±0.29)%,(89.76±4.57)%and 148.33±8.18 in 0μmol/L curcumin group,(83.78±4.59)%,100.33±8.73,(14.53±2.04)%,(65.43±3.70)%and 125.33±7.41 in 20μmol/L curcumin group,(66.09±3.92)%,58.67±6.55,(27.23±3.56)%,(34.83±2.19)%and 73.67±6.34 in 40μmol/L curcumin group,and(47.16±3.63)%,31.67±4.92,(44.73±4.36)%,(18.82±1.99)%and 45.67±5.31 in 80μmol/L curcumin group.There were statistically significant differences in the survival rate,colony formation number,cell apoptosis rate,migration rate and invasion rate of M23 cells among the four groups(F=125.321,97.941,72.516,277.097,139.006;all at P<0.001).With the increase of curcumin concentration,the cell survival rate,colony formation number,cell migration rate and cell invasion number decreased obviously,and the cell apoptosis rate increased obviously,and the pairwise comparisons showed significant differences(all at P<0.05).With the increase of curcumin concentration,the relative expression levels of c-Myc,Cyclin D1,Survivin,MMP-9 mRNA and proteins,β-catenin and p-GSK-3βproteins decreased significantly,while the relative expression level of Axin2 protein increased significantly,showing significant differences in pairwise comparisons(all at P<0.05).The tumor tissue weight of mice decreased with the increase of curcumin dosage,and the pairwise comparisons were statistically significant(all at P<0.05).Conclusions Curcumin can inhibit the proliferation,migration,invasion and other malignant biological behaviors of UM M23 cells,inhibit tumor growth and promote cell apoptosis.Its mechanism may be related to blocking the activation of Wnt/β-catenin pathway.
作者
盛小红
王利明
赵鑫
辛向阳
Sheng Xiaohong;Wang Liming;Zhao Xin;Xin Xiangyang(Department of Ophthalmology,Inner Mongolia Baotou Steel Hospital,Baotou 014010,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2024年第1期29-37,共9页
Chinese Journal Of Experimental Ophthalmology
基金
内蒙古自治区自然科学基金(2020MS08094)。
