健脾通络方通过Wnt/β-catenin信号通路调控结直肠癌细胞增殖、凋亡和迁移的机制研究

Mechanism research of Jianpitongluo Recipe regulating proliferation,apoptosis,and migration of colorectal cancer cells through Wnt/β-catenin signaling pathway

目的探究健脾通络方(JPTLR)通过Wnt/β-catenin信号通路调控结直肠癌(CRC)细胞增殖、凋亡和迁移的机制。方法分别给予人CRC细胞HCT116不同质量浓度的JPTLR(0、6.25、12.50、25.00、50.00、100.00、150.00、200.00 mg/mL)、5-氟尿嘧啶(5-FU)及两者联合进行干预,采用CCK-8法、AnnexinⅤ-FITC和碘化丙啶(PI)双染色法、细胞划痕实验检测各组细胞的增殖、凋亡和迁移能力。采用实时荧光定量PCR法及蛋白质印迹法检测JPTLR对Wnt/β-catenin信号通路及凋亡相关因子表达的影响。结果JPTLR具有抑制HCT116细胞增殖的作用,且这一作用具有时间及浓度依赖性。与空白对照组相比,JPTLR组的细胞凋亡率均显著升高,细胞划痕愈合率均显著降低,细胞中Wnt3a、β-catenin和Bcl-2的mRNA及蛋白表达水平均显著降低,而Bax、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)的mRNA及蛋白表达水平均显著升高,差异均有统计学意义(P均<0.01);JPTLR+5-FU组的细胞凋亡率最高,细胞划痕愈合率最低,细胞中Wnt3a、β-catenin和Bcl-2的mRNA及蛋白表达水平最低,而Bax、Caspase-3的mRNA及蛋白表达水平最高。结论JPTLR能够通过下调Wnt/β-catenin信号通路表达减弱CRC细胞的增殖、迁移和抗凋亡能力。本研究从促进肿瘤细胞凋亡的角度阐明了JPTLR的抗肿瘤作用机制,为JPTLR的临床应用转化提供了实验依据。

Objective This paper intends to investigate the mechanism of Jianpitongluo Recipe(JPTLR)in regulating proliferation,apoptosis and migration of colorectal cancer(CRC)cells through the Wnt/β-catenin signaling pathway.Methods Human CRC cell line HCT116 was treated with different concentrations of JPTLR(0,6.25,12.50,25.00,50.00,100.00,150.00,200.00 mg/mL),5-fluorouracil(5-FU)and the combination of the two.Cell proliferation,apoptosis and migration were detected by CCK-8,AnnexinⅤ-FITC and propidium iodide(PI)double staining and cell scratch test.The effects of JPTLR on Wnt/β-catenin signaling pathway and the expression of apoptosis-related factors were detected by real-time quantitative PCR and Western blotting.Results JPTLR can inhibit the proliferation of HCT116 cells in a time and concentration dependent manner.Compared with the blank control group,the apoptosis rate of the JPTLR group is increased,the scratch healing rate is decreased,and the mRNA and protein expression levels of Wnt3a,β-catenin,and Bcl-2 in the JPTLR group are decreased,the mRNA and protein expression levels of Bax and Caspase-3 are increased,with statistically significant differences(P<0.01).The JPTLR+5-FU group has the highest apoptosis rate and the lowest scratch healing rate.The mRNA and protein expression levels of Wnt3a,β-catenin,and Bcl-2 are the lowest in the JPTLR+5-FU group,while the mRNA and protein expression levels of Bax and Caspase-3 are the highest.Conclusions JPTLR can inhibit the proliferation,migration,and anti-apoptosis of colorectal cancer cells by down-regulating the expression of Wnt/β-catenin signaling pathway.This paper elucidates the anti-tumor mechanism of JPTLR from the perspective of promoting tumor cell apoptosis,and provides experimental evidence for the clinical application of JPTLR transformation.