基于PD-1/PD-L1及miR-200c/ZEB通路研究健脾消癌方治疗结肠癌肝转移的作用机制

Mechanism of action of Jianpi Xiaoai prescription in treatment of colorectal cancer liver metastasis based on the programmed death-1/programmed death-ligand 1 and microRNA-200c/zinc finger E-box-binding homeobox 1 pathways

目的:观察健脾消癌方对结肠癌肝转移荷瘤裸鼠肝组织中程序性死亡受体-1(PD-1)/程序性死亡配体-1(PD-L1)及微小RNA200c(miRNA-200c)/转录因子E盒结合锌指蛋白1(ZEB1)表达的影响。方法:在50只裸鼠中随机抽取10只作为假移植组,另取40只建立结肠癌肝转移模型。造模成功后再将其分为4组:模型组,健脾消癌方低、中、高剂量组(简称为低、中、高剂量组),每组各10只。假移植组和模型组灌胃等量0.9%氯化钠注射液,低、中、高剂量组灌胃健脾消癌方(5.4、10.8、21.6 g/kg),每天1次,连续3周。观察各组裸鼠肝转移瘤的情况,检测PD-1、PD-L1、miRNA-200c、ZEB1 mRNA的表达及PD-1、PD-L1、ZEB1蛋白的表达情况。结果:除假移植组外,其余各组均可在裸鼠的肝脏表面发现肝转移结节。各组肝转移结节数量、质量高剂量组与低、中剂量组比较,差异均有统计学意义(P<0.05)。肝脏组织病理学结果显示,与模型组比较,低、中、高剂量组癌细胞增长速度较慢。与假移植组比较,模型组miRNA-200c表达降低,PD-1、PD-L1、ZEB1 mRNA表达及PD-1、PD-L1、ZEB1蛋白表达升高(P<0.01)。与模型组比较,高剂量组miRNA-200c表达升高、PD-1蛋白表达降低(P<0.01),各剂量组PD-1、PD-L1、ZEB1mRNA表达及PD-L1蛋白表达降低(P<0.05或P<0.01),中、高剂量组ZEB1蛋白表达降低(P<0.01)。结论:健脾消癌方可有效抑制结肠癌肝转移,其扶正消癌的作用机制可能与升高miRNA-200c表达、降低PD-1、PD-L1、ZEB1蛋白表达相关。

Objective:To investigate the effect of Jianpi Xiaoai prescription on the expression of programmed death-1(PD-1)/programmed death-ligand 1(PD-L1)and microRNA-200c(miRNA-200c)/zinc finger E-box-binding homeobox 1(ZEB1)in liver tissue of nude mice bearing colon cancer liver metastasis.Methods:A to-tal of 50 nude mice were selected,among which 10 were randomly selected as sham transplantation group and the other 40 were used to establish a model of colon cancer liver metastasis,and after successful modeling,the 40 mice were divided into model group and low-,middle-,and high-dose Jianpi Xiaoai prescription groups(hereinafter re-ferred to as low-,middle-,and high-dose groups),with 10 mice in each group.The mice in the sham transplanta-tion group and the model group were given an equal volume of 0.9%sodium chloride injection by gavage,and those in the low-,middle-,and high-dose groups were given Jianpi Xiaoai prescription by gavage at a dose of 5.4,10.8,and 21.6 g/kg,respectively,once a day for three consecutive weeks.Liver metastasis was observed for each group,and the mRNA expression of PD-1,PD-L1,miRNA-200c,and ZEB1 and the protein expression of PD-1,PD-L1,and ZEB1 were measured.Results:Liver metastasis nodules were observed on the surface of the liver in all groups except the sham transplantation group.There were significant differences in the number and weight of liver metastasis nodules between the high-dose group and the low-/middle-dose groups(P<0.05).Liver histopatholog-ical examination showed that compared with the model group,the low-,middle-,and high-dose groups had a lower growth rate of cancer cells.Compared with the sham transplantation group,the model group had a significant reduc-tion in the expression of miRNA-200c and significant increases in the mRNA expression of PD-1,PD-L1,and ZEB1 and the protein expression of PD-1,PD-L1,and ZEB1(P<0.01).Compared with the model group,the high-dose group had a significant increase in the expression of miRNA-200c and a significant reduction in the pro-tein expression of PD-L1(P<0.01);all three dose groups had significant reductions in the mRNA expression of PD-1,PD-L1,and ZEB1 and the protein expression of PD-L1(P<0.05 or P<0.01);the middle-and high-dose groups had a significant reduction in the protein expression of ZEB1(P<0.01).Conclusion:Jianpi Xiaoai prescrip-tion can effectively inhibit the liver metastasis of colon cancer and exerts an effect of strengthening vital Qi and e-liminating cancer possibly by increasing the expression of miRNA-200c and reducing the protein expression of PD-1,PD-L1,and ZEB1.