黄芪-莪术配伍对结肠癌原位移植瘤模型小鼠SDF-1/CXCR4/NF-κB信号通路的影响

Effect of Astragali Radix-Curcumae Rhizoma on SDF-1/CXCR4/NF-κB Signaling Pathway of Orthotopic Transplantation Model of Colon Cancer in Mice

目的:探讨黄芪-莪术抑制结肠癌原位移植瘤模型小鼠肿瘤生长的可能作用机制。方法:运用分子对接技术预测黄芪、莪术中主要活性成分与通路靶点蛋白基质细胞衍生因子-1(SDF-1),趋化因子受体4(CXCR4),核转录因子-κB p65(NF-κB p65)的分子间相互作用。建立CT26.WT结肠癌原位移植瘤模型进行体内实验验证。将60只BALB/c雄性小鼠随机分为假手术组,模型组,5-氟尿嘧啶(5-Fu)组、黄芪-莪术低、中、高剂量组,每组10只。假手术组和模型组给予生理盐水灌胃,5-Fu组(30 mg·kg^(-1))腹腔注射,黄芪-莪术低、中、高剂量组(0.32,0.64,1.28 g·kg^(-1))灌胃。干预15 d后,完整剥离原位瘤,取肿瘤相邻结肠组织5~6 cm。测量并计算肿瘤体积,苏木素-伊红(HE)染色观察肿瘤组织及结肠组织病理学变化,蛋白免疫印迹法(Western blot)检测结肠组织中趋化因子SDF-1及其受体CXCR4,磷酸化NF-κB p65(p-NF-κB p65)蛋白表达,Western blot和实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤组织趋化因子SDF-1及其受体CXCR4,NF-κB p65,细胞周期蛋白D1(Cyclin D1),癌基因c-Myc蛋白及mRNA表达水平。结果:与模型组比较,5-Fu与黄芪-莪术治疗均能降低原位瘤体积(P<0.05,P<0.01),5-Fu组抑瘤率最高(61.38±2.34)%,黄芪-莪术中剂量组抑瘤率(43.43±3.71)%。与模型组比较,各药物组肿瘤与结肠组织的病理学改变转轻。与模型组比较,黄芪-莪术显著下调了肿瘤小鼠结肠组织SDF-1,CXCR4,p-NF-κB p65蛋白表达水平(P<0.01),对肿瘤组织SDF-1,CXCR4,NF-κB p65,Cyclin D1,c-Myc的蛋白及mRNA表达明显下调(P<0.05,P<0.01)。结论:黄芪-莪术能够抑制结肠癌原位移植瘤的生长,其干预机制可能与调节SDF-1/CXCR4/NF-κB信号通路中相关蛋白及其mRNA表达有关。

Objective:To explore the possible mechanism of Astragali Radix-Curcumae Rhizoma(AC)in inhibiting tumor growth in the orthotopic transplantation model of colon cancer in mice.Method:The molecular docking technology was used to predict the intermolecular interaction between the main active components of AC and the pathway target proteins,such as stromal cell-derived factor-1(SDF-1),C-X-C motif chemokine receptor 4(CXCR4),and nuclear factor kappa-B p65(NF-κB p65).The orthotopic transplantation model of CT26.WT colon cancer was established in mice for in vivo experimental verification.Sixty BALB/c male mice were randomly divided into a sham operation group,a model group,a 5-fluorouracil(5-Fu,30 mg·kg^(-1))group,and low-(0.32 g·kg^(-1)),medium-(0.64 g·kg^(-1)),and high-dose(1.28 g·kg^(-1))AC groups,with 10 mice in each group.The sham operation group and the model group received normal saline by gavage.The corresponding drugs were administered by gavage in the 5-Fu group and by intraperitoneal injection in the AC groups.After intervention for 15 days,the tumor in situ was completely stripped,and the colon tissues 5-6 cm in length adjacent to the tumor were taken.The tumor volume was measured and calculated.The pathological changes of tumor tissues and colon tissues were observed by Hematoxylin-Eosin(HE)staining.Western blot was used to detect the protein expression of SDF-1,CXCR4,p-NF-κB p65 in colon tissues.Western blot and Real-time quantitative polymerase chain reaction(Real-time PCR)were used to detect SDF-1,CXCR4,NF-κB p65,Cyclin D1,oncogene c-Myc protein and mRNA expression in tumor tissues.Result:Compared with the model group,5-Fu and AC groups showed reduced tumor volumes in situ(P<0.05,P<0.01),with the tumor inhibition rate in the 5-Fu group as high as(61.38±2.34)%.The tumor-inhibiting effect was optimal in the medium-dose AC group,with the tumor inhibition rate of(43.43±3.71)%.Compared with the model group,5-Fu and AC groups showed relieved pathological changes of tumor and colon tissues.Specifically,AC down-regulated the protein expression levels of SDF-1,CXCR4,and p-NF-κB p65 in colon tissues(P<0.01),and down-regulated the protein and mRNA expression levels of SDF-1,CXCR4,NF-κB p65,Cyclin D1,and c-Myc in tumor tissues(P<0.05,P<0.01).Conclusion:AC can inhibit the growth of orthotopic transplantation tumor of colon cancer,and its intervention mechanism may be related to the regulation of related protein and mRNA expression in the SDF-1/CXCR4/NF-κB signaling pathway.