参白解毒方抑制HCT116细胞增殖的药效及机制

Efficacy and Mechanism of Shenbai Jiedu Prescription Against Proliferation of HCT116 Cells

目的:探讨参白解毒方(SBJDF)抑制结直肠癌(CRC)细胞HCT116增殖的药效及机制。方法:利用参白解毒方(0、0.25、0.5、1、2、4 g·L^(-1))处理HCT116细胞48 h,噻唑蓝(MTT)比色法检测参白解毒方对HCT116细胞活力的影响,分别设置空白组、参白解毒方(1、2、4 g·L^(-1))组,倒置显微镜观察参白解毒方对HCT116细胞形态的影响,克隆形成实验观察参白解毒方对HCT116细胞增殖能力的影响,JC-1探针检测参白解毒方对HCT116细胞线粒体膜电位(Δψm)的影响,流式细胞仪检测HCT116细胞周期分布和细胞凋亡率,蛋白免疫印迹法(Western blot)分析参白解毒方对细胞周期,抗凋亡以及核转录因子-κB(NF-κB)信号通路相关蛋白的影响。结果:参白解毒方有效抑制HCT116细胞活力,引起细胞形态改变,呈浓度依赖性;与空白组比较,参白解毒方(1、2、4 g·L^(-1))组克隆形成率均明显下降(P<0.05,P<0.01),参白解毒方(2、4 g·L^(-1))组诱导HCT116细胞发生G0/G1期阻滞(P<0.05,P<0.01)。与空白组比较,参白解毒方(1、2、4 g·L^(-1))组周期蛋白D(1 CyclinD1)表达明显下降(P<0.05,P<0.01),参白解毒方(2、4 g·L^(-1))组细胞周期蛋白A(2 CyclinA2),周期蛋白依赖性激酶4(CDK4)蛋白表达明显下降(P<0.05,P<0.01),参白解毒方(4 g·L^(-1))组周期蛋白依赖性激酶1(CDK1)表达显著下降(P<0.01)。与空白组比较,参白解毒方(2、4 g·L^(-1))组课诱导HCT116细胞凋亡,并下调抗凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关xl蛋白(Bcl-xl)表达(P<0.05,P<0.01),降低HCT116细胞线粒体膜电位;与空白组比较,参白解毒方(2、4 g·L^(-1))组可下调NF-κB信号通路相关蛋白IκB激酶α(IKKα)、NF-κB抑制蛋白α(IκBα)、磷酸化p65蛋白(p-p65)的表达(P<0.05,P<0.01)。结论:参白解毒方有效抑制HCT116细胞增殖,其机制可能通过抑制HCT116细胞NF-κB信号通路的激活,引起细胞周期阻滞,诱导细胞凋亡。

Objective:To investigate the mechanism by which Shenbai Jiedu prescription(SBJDF)inhibits the proliferation of colorectal cancer(CRC)HCT116 cells.Method:After 48 h treatment of HCT116 cells with SBJDF(0,0.25,0.5,1,2,4 g·L^(-1)),the viability of HCT116 cells were determined by methyl thiazolyl tetrazolium(MTT)colorimetry.Following the classification of cells into blank control group and SBJDF(1,2,4 g·L^(-1))groups,the effect of SBJDF on HCT116 cell morphology was observed under an inverted microscope.The effects of SBJDF on the proliferation of HCT116 cells and mitochondrial membrane potential(Δψm)were detected by colony formation assay and JC-1 probe,respectively.The flow cytometry was then performed for determining cell cycle distribution and apoptosis.The effects of SBJDF on cell cycle-,apoptosis-,and nuclear factor kappa-B(NF-κB)signaling pathway-related proteins were determined by Western blot.Result:SBJDF effectively inhibited the vitality of HCT116 cells and changed their morphology in a concentration-dependent manner.Compared with the blank control group,SBJDF at 1,2,4 g·L^(-1)significantly reduced cell colony formation(P<0.05,P<0.01),and SBJDF at 2 and 4 g·L^(-1)arrested the HCT116 cell cycle at G0/G1 phase(P<0.05,P<0.01).Compared with the blank control group,SBJDF at 1,2,4 g·L^(-1)remarkably down-regulated the protein expression of CyclinD1(P<0.05,P<0.01).SBJDF at 2 and 4 g·L^(-1)lowered the CyclinA2 and cyclin-dependent kinase 4(CDK4)(P<0.05,P<0.01).SBJDF at 4 g·L^(-1)reduced the cyclindependent kinase 1(CDK1)(P<0.01).Compared with the blank control group,SBJDF at 2 and 4 g·L^(-1)induced HCT116 cell apoptosis,down-regulated the protein expression of anti-apoptosis-related proteins Bcl-2 and Bcl-xl as well as the NF-κB signaling pathway-related proteins IκB kinaseα(IKKα),inhibitorαof NF-κB(IκBα),and phospho-NF-κB p65(p-p65)(P<0.05,P<0.01),and diminished the mitochondrial membrane potential of HCT116 cells.Conclusion:SBJDF inhibits the proliferation of HCT116 cells,which may be related to its inhibition of the activation of NF-κB signaling pathway and the induction of cell cycle arrest and apoptosis.