黄芪解毒汤干预Wnt/β-catenin通路抑制上皮间质转化抗乳腺癌肺转移机制研究

Study on Huangqi Jiedu Decoction for Wnt/β-catanin signaling pathway inhibiting EMT against lung metastasis of breast cancer

目的观察黄芪解毒汤对荷瘤裸鼠乳腺癌肺转移的抑制作用,探讨Wnt/β-连环蛋白(β-catenin)通路调控上皮间质转化(EMT)的作用机制。方法将30只裸鼠按随机数字表法分为模型组、阿霉素组及黄芪解毒汤低、中、高剂量组,每组6只。各组通过皮下注射小鼠乳腺癌4T1细胞制备荷瘤裸鼠模型。建模成功后,黄芪解毒汤低、中、高剂量组灌胃黄芪解毒汤17.82、35.64、71.28 g/kg,阿霉素组腹腔注射阿霉素0.05 g/kg,模型组灌胃等体积的生理盐水,均连续给药21 d。于造模后9、15、21 d测定瘤体积;给药结束后分离肿瘤组织,称瘤重、计算抑瘤率;分离肺组织,计数肺转移瘤结节数,计算肺转移抑制率;HE染色观察组织形态,评价模型的有效性;Western blot法检测肺组织β-catenin、E-钙黏蛋白(E-Cadherin)、波形纤维蛋白(Vimentin)表达;实时荧光定量PCR检测肺组织β-catenin、E-Cadherin、Vimentin mRNA水平。结果与模型组比较,黄芪解毒汤低、中、高剂量组瘤体积、重量降低(P<0.01);黄芪解毒汤高剂量组肺转移结节数减少(P<0.01);黄芪解毒汤低、中、高剂量组β-catenin、Vimentinm mRNA及蛋白表达降低(P<0.01),黄芪解毒汤高剂量组E-Cadherin蛋白和mRNA表达增加(P<0.01)。结论黄芪解毒汤可有效抑制乳腺癌荷瘤裸鼠移植瘤的生长及肺转移的进程,可能与下调Wnt/β-catenin信号通路关键分子表达,从而抑制EMT进程有关,以达到抑制乳腺癌肺转移的作用。

Objective To observe the inhibitory effects of Huangqi Jiedu Decoction on lung metastasis of breast cancer in nude mice;To explore the mechanism of intervening epithelial mesenchymal transformation(EMT)induced by Wnt/β-catenin signaling pathway.Methods Totally 30 nude mice were divided into model group,adriamycin group and Huangqi Jiedu Decoction low-,medium-,and high-dosage groups according to random number table method.Each group was injected subcutaneously with mouse breast cancer 4T1 cells to construct tumor-bearing nude mice model.Huangqi Jiedu Decoction low-,medium-and high-dosage groups were intragastrically administrated with Huangqi Jiedu Decoction 17.82,35.64 and 71.28 g/kg;adriamycin group was injected intraperitoneally adriamycin 0.05 g/kg;model group was intragastrically administrated with normal saline of the same volume for 21 d.Tumor volume was measured at 9,15,and 21 days after modeling.After the end of administration,the tumor tissue was separated,the tumor weight was measured,and the tumor inhibition rate was calculated.The lung tissue was Isolated,,the number of lung metastatic nodules and the inhibition rate of lung metastasis was counted.HE staining was used to observe the tissue morphology and evaluate the effectiveness of the model.The protein expressions ofβ-catenin,E-Cadherin and Vimentin in lung tissue were detected by Western Blot.The mRNA levels ofβ-catenin,E-Cadherin and Vimentin in lung tissue were detected by real-time fluorescent quantitative PCR.Results Compared with the model group,the tumor volume and mass of Huangqi Jiedu Decoction low-,medium-and high-dosage groups decreased(P<0.01);the number of pulmonary metastasis nodules in Huangqi Jiedu Decoction high-dosage group significantly decreased(P<0.01);the mRNA and protein expressions ofβ-catenin and Vimentinm decreased in the Huangqi Jiedu Decoction low-,medium-and high-dosage groups(P<0.01),and the protein and mRNA expressions of E-Cadherin increased in the Huangqi Jiedu Decoction high-dosage group(P<0.01).Conclusion Huangqi Jiedu Decoction can effectively inhibit the growth and lung metastasis of breast cancer transplanted tumor,and the mechanism may be to down-regulate the expression of key molecules in the Wnt/β-catanin signaling pathway,thereby inhibiting the EMT process,so as to inhibit the lung metastasis of breast cancer.