CAJ | 학술논문

目的探讨丹皮酚对体外培养人黑素瘤A375细胞增殖和凋亡的影响及其作用机制.方法 CCK8法检测0.5,1,2,4,8 mmol/L丹皮酚作用24,48和72 h后A375细胞增殖水平.用0、1.25、2.5和5 mmol/L浓度丹皮酚作用24 h后,以Annexin-V/PI法观察A375细胞凋亡的变化、分别测定caspase 3,caspase 8和caspase 9的活性,并以Western印迹检测p53,NF-κB及相关蛋白水平的变化.结果与空白对照组比较,0.5,1,2,4,8 mmol/L丹皮酚作用24,48和72 h后均对A375细胞的增殖有抑制作用,且与时间、浓度呈依赖性.1.25、2.5、5 mmol/L丹皮酚在作用24 h时,A375细胞早期凋亡率由对照组的(3.11±0.53)％分别上升至(13.74±1.73)％、(25.95±0.57)％、(46.44±0.81)％,各组与对照组间差异均有统计学意义(P＜0.05或＜0.01).2.5 mmol/L和5 mmol/L作用组的caspase 3,caspase 8,caspase 9活性升高,且与对照组间比较,差异均有统计学意义(P＜ 0.05或＜0.01).p53及Bax的蛋白水平随药物浓度的增加而升高,NF-κB、bcl-2、bcl-XL的蛋白水平随药物浓度增加而降低.结论丹皮酚对黑素瘤A375细胞具有抑制增殖和诱导凋亡的作用.可通过细胞内和细胞外两条途径发挥促凋亡作用,其机制可能与调节p53及NF-κB基因有关.
목적 탐토단피분대체외배양인흑소류A375세포증식화조망적영향급기작용궤제.방법 CCK8법검측0.5,1,2,4,8 mmol/L단피분작용24,48화72 h후A375세포증식수평.용0、1.25、2.5화5 mmol/L농도단피분작용24 h후,이Annexin-V/PI법관찰A375세포조망적변화、분별측정caspase 3,caspase 8화caspase 9적활성,병이Western인적검측p53,NF-κB급상관단백수평적변화.결과 여공백대조조비교,0.5,1,2,4,8 mmol/L단피분작용24,48화72 h후균대A375세포적증식유억제작용,차여시간、농도정의뢰성.1.25、2.5、5 mmol/L단피분재작용24 h시,A375세포조기조망솔유대조조적(3.11±0.53)％분별상승지(13.74±1.73)％、(25.95±0.57)％、(46.44±0.81)％,각조여대조조간차이균유통계학의의(P＜0.05혹＜0.01).2.5 mmol/L화5 mmol/L작용조적caspase 3,caspase 8,caspase 9활성승고,차여대조조간비교,차이균유통계학의의(P＜ 0.05혹＜0.01).p53급Bax적단백수평수약물농도적증가이승고,NF-κB、bcl-2、bcl-XL적단백수평수약물농도증가이강저.결론 단피분대흑소류A375세포구유억제증식화유도조망적작용.가통과세포내화세포외량조도경발휘촉조망작용,기궤제가능여조절p53급NF-κB기인유관.
Objective To study the effect of paeonol on the proliferation and apoptosis of A375 human melanoma cells and its mechanism.Methods Cell counting kit-8 (CCK8) was used to evaluate the proliferative activity of A375 cells treated with paeonol of 0.5,1,2,4,8 mmol/L for 24,48 and 72 hours respectively.Subsequently,A375 cells were treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours followed by double staining with annexin V and propidium iodide for the detection of cell apoptosis,fluorometric assay for the estimation of caspase 3,caspase 8 and caspase 9 activity,and Western blot for the determination of the levels of p53,nuclear factor-κB proteins and some of their target proteins.The A375 cells receiving no treatment served as the blank control group.Statistical analysis was carried out by t test.Results Within the investigated concentration and time ranges,paeonol significantly inhibited the proliferative activity of A375 cells in a concentration-and timedependent manner.Compared with the blank control group,a significant increase was observed in the early apoptosis rate in A375 cells treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours (13.74％-± 1.73％,25.95％ ± 0.57％ and 46.44％ ± 0.81％ vs.3.11％ ± 0.53％,P ＜ 0.05 or 0.01),as well as in the activity of caspase 3,8 and 9 in A375 cells treated with paeonol of 2.5 and 5 mmol/L for 24 hours (P ＜ 0.05 or 0.01).After 24-hour treatment,the protein levels of p53 and Bax were elevated,but those of nuclear factor-κB,Bcl-2 and Bcl-XL were decreased in A375 cells with the increase of paeonol concentration.Conclusions Paeonol can inhibit the proliferation but induce the apoptosis of A375 cells,and the apoptosis-inducing effect may be realized through intrinsic and extrinsic pathways by modulating nuclear factor-κB and p53 genes.