参白解毒方通过调控PTEN/PI3K/Akt信号通路抑制结直肠癌细胞增殖  

Shenbai Jiedu Prescription Inhibits Proliferation of Colorectal Cancer Cells by Regulating PTEN/PI3K/Akt Signaling Pathway

作　　者：刘见荣 黄敏[1] 范旻旻 程海波[1,3] 沈卫星 肖君[4] 徐长亮 谭佳妮 赖岳阳[1,3] 余成涛 孙东东 李柳 LIU Jianrong;HUANG Min;FAN Minmin;CHENG Haibo;SHEN Weixing;XIAO Jun;XU Changliang;TAN Jiani;LAI Yueyang;YU Chengtao;SUN Dongdong;LI Liu(The First School of Clinical Medical,Nanjing University of Chinese Medicine,Nanjing 210023,China;Nanjing Hospital of Traditional Chinese Medicine(TCM),Nanjing 210012,China;Jiangsu Collaborative Innovation Center of TCM Prevention and Treatment of Tumor,Nanjing 210023,China;Affiliated Hospital of Nanjing University of Chinese Medicine,Jiangsu Province Hospital of China Medicine,Nanjing 210029,China)

机构地区：[1]南京中医药大学第一临床医学院,南京210023 [2]南京市中医院,南京210012 [3]江苏省中医药防治肿瘤协同创新中心,南京210023 [4]南京中医药大学附属医院江苏省中医院,南京210029

出　　处：《中国实验方剂学杂志》2022年第14期36-43,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金重点项目(81930117);江苏高校优势学科建设工程项目(PAPD)。

摘　　要：目的:研究参白解毒方基于磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)信号通路抑制结直肠癌细胞HCT116增殖的作用机制。方法:采用水提醇沉法提取参白解毒方有效部位进行冻干粉制备;体外培养HCT116细胞,用参白解毒方(2、4、8、16 g·L^(-1))对HCT116细胞进行处理,采用噻唑蓝(MTT)比色法检测参白解毒方对HCT116细胞增殖的抑制作用;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞PTEN、PI3K、Akt、糖原合成酶激酶-3β(GSK-3β)、c-核蛋白类基因(c-Myc)、生存素(Survivin)和细胞周期蛋白D1(CyclinD1)mRNA表达水平;蛋白免疫印迹法(Western blot)检测PTEN、磷酸化磷酸酶及张力蛋白同源物(p-PTEN)、PI3K、Akt、磷酸化蛋白激酶B(p-Akt)、GSK-3β、磷酸化糖原合成酶激酶-3(p-GSK-3β)、c-Myc、Survivin、CyclinD1的蛋白表达水平;免疫荧光检测β-连环蛋白(β-catenin)入核情况。结果:与空白组比较,参白解毒方各质量浓度均可以显著抑制HCT116细胞的增殖(P<0.01),且具有浓度依赖性。与空白组比较,参白解毒方处理细胞后PTEN、GSK-3βmRNA表达水平均上调,PI3K、Akt、c-Myc、Survivin、CyclinD1 mRNA表达水平均下调(P<0.01);细胞PTEN、p-PTEN和GSK-3β蛋白表达水平上调,PI3K、Akt、p-Akt、GSK-3β、p-GSK-3β、c-Myc、Survivin、CyclinD1蛋白表达水平均下调(P<0.05,P<0.01)。免疫荧光结果显示参白解毒方可以抑制结直肠癌细胞HCT116中β-catenin的入核。结论:参白解毒方可以抑制结直肠癌细胞HCT116的增殖,其作用机制可能是通过调节PTEN/PI3K/Akt信号通路。Objective:To study the mechanism of Shenbai Jiedu prescription inhibiting the proliferation of HCT116 colorectal cancer(CRC) cells by regulating the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.Method: Shenbai Jiedu prescription was extracted by water extraction and alcohol precipitation to prepare freeze-dried powder. HCT116 cells were cultured in vitro,and treated with different concentrations of Shenbai Jiedu prescription(2,4,8,16 g·L^(-1)). The inhibitory effect of Shenbai Jiedu prescription on the proliferation of HCT116 cells was tested by methyl thiazolyl tetrazolium(MTT). Real-time quantitative PCR was used to detect the mRNA expression levels of PTEN,PI3K,Akt,glycogen synthase kinase-3β(GSK-3β),c-Myc,survivin and Cyclin D1. Western blot was employed to measure the protein expression levels of PTEN,phosphorylated PTEN(p-PTEN),PI3K,Akt,phosphorylated Akt(p-Akt),GSK-3β,phosphorylated GSK-3β(p-GSK-3β),c-Myc,survivin and Cyclin D1,β-catenin nuclear import was explored by immunofluorescence assay. Result:Compared with the control group,Shenbai Jiedu prescription inhibited the proliferation of HCT116 cells in a dose-dependent manner(P<0.01). Compared with the control group,the mRNA expression levels of PTEN and GSK-3β were up-regulated whereas those of PI3K,Akt,c-Myc,survivin and CyclinD1were down-regulated after treatment with Shenbai Jiedu prescription(P<0.01). The protein expression levels of PTEN,p-PTEN and GSK-3β were up-regulated whereas those of PI3K,Akt,p-Akt,GSK-3β,p-GSK-3β,c-Myc,survivin and CyclinD1were down-regulated(P<0.05, P<0.01). Immunofluorescence assay showed that Shenbai Jiedu prescription suppressed β-catenin nuclear import in HCT116 cells. Conclusion: Shenbai Jiedu prescription inhibited the proliferation of HCT116 cells via the mechanism of regulating the PTEN/PI3K/Akt signaling pathway.

关 键 词：参白解毒方 结直肠癌 细胞增殖 磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(PKB/Akt)信号通路 β-连环蛋白(β-catenin)入核 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5