基于肿瘤微环境中TLR4/NF-κB通路的变化探讨乳癌术后方对乳腺癌小鼠肺转移的影响

Investigation on Ruai Shuhou Prescription on Breast Cancer Mice Lung Metastasis based on Tumor Microenvironment TLR4/NF-κB Pathway Changes

目的:观察乳癌术后方对乳腺癌肺转移模型小鼠移植瘤生长的影响,探讨其可能的作用机制。方法:萤火虫荧光素酶标记的小鼠乳腺癌肺高转移细胞4T1(4T1-luc)接种于小鼠第三对乳房脂肪垫建立乳腺癌肺转移模型;于接种后7 d随机分组:NS组、扶正组、解毒组、全方组及环磷酰胺组,每组6只,连续给药21 d。计算肿瘤抑制率,以生物发光成像系统监测肿瘤细胞在体内的生长过程,通过肺转移灶定量分析观察小鼠肺转移情况,并采用蛋白印迹法(Western blotting)检测原位瘤中TLR4、NF-κB及IκB等蛋白的表达。结果:经过21 d给药,除了扶正组小鼠体重变化不明显,其余各组体重均有不同程度的降低。与NS组相比,各用药组瘤体积、肺转移抑制率均有所降低;全方组瘤体积、肺转移抑制率均较扶正组低(P<0.05),与解毒组相比仅瘤体积差异有统计学意义(P<0.05)。各组小鼠原位瘤组织中NF-κB蛋白表达水平无明显差异,但是与NS组相比,给药组的磷酸化的NF-κB蛋白(p-NF-κB)及磷酸化的IκB蛋白(p-IκB)均有明显降低,且全方组较扶正组、解毒组表达更低。另外,与NS组比较,乳癌术后方全方组及其拆方组IκB蛋白表达均有所上升,而TLR4蛋白表达水平均有所下降。结论:乳癌术后方对乳腺癌肺转移模型小鼠均有抑瘤和抗肿瘤转移作用,其机制可能与调节肿瘤微环境中TLR4/NF-κB通路相关蛋白表达有关。

Objective：To investigate the effect of Ruai Shuhou Prescription on growth of xenotransplants in pulmonary metastasis of mice with breast cancer and explore the possible mechanism.Methods：The pulmonary metastasis model of breast cancer was set up through subcutaneous injection of 4T1-luc cells in the third breast fat pad.7 days after inoculation,forty mice bearing breast cancer were randomly divided into five groups（in each group there were 6 nude mice）：control group,whole formula group,Fuzheng group,Jiedu group,cyclophosphane group.Each group except the model group was treated with Ruai Shuhou Prescription and its disassembled prescriptions for 21 days.The tumor inhibitory rate was calculated.The growth of tumor cells in the body was monitored by bioluminescent imaging system.The pulmonary metastasis was evaluated by lung metastases quantitative analysis.The expression levels of TLR4,NF-ΚB and IκB proteins in xenografts were detected by Western blotting.Results：After 21 days of administration,in addition changes in body weight of mice were obvious,other groups had different levels of reduced weights.Each treatment group＇s tumor volume and lung metastasis inhibition rate decreased compared with those of NS group.The whole formula group＇s tumor volume and lung metastasis inhibition rate were lower than those of Fuzheng group（P〈0.05） and the whole formula group＇s tumor volume was significant compared with that of the Jiedu group（P〈0.05）.Each group＇s mice tumor tissues in situ NF-κB protein expression had no significant difference,but compared with the NS group,phosphorylated NF-κB protein（p-NF-κB） and phosphorylated IκB protein（p-IκB） were significantly reduced,and the whole formula group＇s was lower than those in the Fuzheng group and Jiedu group.In addition,compared with NS group,after operation the whole formula group as well as the disassembled prescriptions groups＇ IκB protein expression was increased and TLR4 protein levels were decreased.Conclusion： Ruai Shuhou Prescription has effect of tumor suppression and resisting tumor metastasis and its mechanism may be related to microenvironment of TLR4,NF-κB and IκB.