散结消瘤方调控TGF-β_(1)/Smads介导的上皮间质转化对乳腺癌皮下移植瘤的影响

Inhibitory effect of decoction for dissipating binds and dispersing tumor on subcutaneous graft tumor of breast cancer via TGF-β_(1)/Smads-mediated epithelial mesenchymal transition

目的探讨散结消瘤方通过调节转化生长因子-β_(1)(TGF-β_(1))/Smads信号通路及其介导的上皮间质转化(EMT)对小鼠乳腺癌皮下移植瘤的影响。方法雌性BALB/cA裸小鼠40只,按体重随机分为模型组、环磷酰胺组、散结消瘤方低剂量组、散结消瘤方中剂量组、散结消瘤方高剂量组,每组8只。各组均采用人乳腺癌细胞株(MCF-7)接种法制备乳腺癌皮下移植瘤模型。造模第6天,环磷酰胺组给予环磷酰胺溶液30 mg/kg腹腔注射,散结消瘤方低、中、高剂量组分别给予7.8 g/kg、15.6 g/kg、31.2 g/kg散结消瘤方灌胃,模型组给予生理盐水灌胃,均持续干预4周。比较各组小鼠瘤重和抑瘤率,免疫组化染色观察瘤组织中基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)阳性表达情况,Western blot法检测肿瘤组织中E-cadherin、Vimentin、twist、Snail、TGF-β_(1)、Smad2、p-Smad2、Smad3、p-Smad3蛋白表达情况,RT-PCR法检测肿瘤组织中TGF-β_(1)、Smad2、Smad3 mRNA表达情况。结果环磷酰胺组及散结消瘤方各组瘤重均明显低于模型组(P均<0.05);散结消瘤方高剂量组和环磷酰胺组瘤重均明显低于散结消瘤方低、中剂量组(P均<0.05),抑瘤率均明显高于散结消瘤方低、中剂量组(P均<0.05)。环磷酰胺组及散结消瘤方各组瘤组织中MMP-2、MMP-9阳性表达平均光密度值,Vimentin、twist、Snail、TGF-β_(1)蛋白相对表达量及p-Smad2/Smad2、p-Smad3/Smad3比值,TGF-β_(1)、Smad2、Smad3 mRNA相对表达量均明显低于模型组(P均<0.05),瘤组织中E-cadherin蛋白相对表达量均明显高于模型组(P均<0.05);散结消瘤方高剂量组和环磷酰胺组上述各指标改善情况均明显优于散结消瘤方低、中剂量组(P均<0.05),散结消瘤方高剂量组与环磷酰胺组各指标比较差异均无统计学意义(P均>0.05)。结论散结消瘤方对乳腺癌皮下移植瘤具有明显的抑瘤作用,其作用机制可能与调控TGF-β_(1)/Smads信号通路,逆转其所介导的EMT相关。

Objective It is to explore the effect of decoction for dissipating binds and dispersing tumor on subcutaneous graft tumor of breast cancer via regulating tumortransforming growth factor-β_(1)(TGF-β_(1))/Smads signaling pathway and its mediated epithelial mesenchymal transition(EMT)in rats.Methods Forty female BALB/cA nude mice were randomly divided into model group,cyclophosphamide group and low,medium and high dose groups of decoction for dissipating binds and dispersing tumor according to their body weight,with 8 rats in each group.The models of subcutaneous transplanted tumor of breast cancer were prepared by inoculating human breast cancer cell line(MCF-7)in each group.On the 6th day of modeling,the cyclophosphamide group was given intraperitoneal injection of cyclophosphamide solution 30 mg/kg,the low,medium and high dose groups of decoction for dissipating binds and dispersing tumor were given decoction for dissipating binds and dispersing tumor 7.8 g/kg,15.6 g/kg and 31.2 g/kg by gavage,respectively,and the model group was given normal saline by gavage,all of which were continuously treated for 4 weeks.The tumor weight and tumor inhibition rate of mice in each group were compared,the positive expressions of matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9)in tumor tissues of each group were observed by immunohistochemical staining,the protein expressions of E-cadherin,Vimentin,twist,Snail,TGF-β_(1),Smad2,p-Smad2,Smad3 and p-Smad3 in tumor tissues were detected by Western-blot,and the mRNA expression of TGF-β_(1),Smad2 and Smad3 in tumor tissues were detected by RT-PCR.Results The tumor weights of the cyclophosphamide group and each group of decoction for dissipating binds and dispersing tumor were significantly lower than that of the model group(all P<0.05),the tumor weights of the high-dose group of decoction for dissipating binds and dispersing tumor and the cyclophosphamide group were significantly lower than that of the low and medium-dose groups of decoction for dissipating binds and dispersing tumor(all P<0.05),and the tumor inhibition rates were significantly higher than those of the low and medium-dose groups(all P<0.05).The mean optical density values of MMP-2 and MMP-9 positive expressions,relative expressions of Vimentin,twist,Snail,TGF-β_(1)protein and p-Smad2/Smad2 and p-Smad3/Smad3 ratios,relative expressions of TGF-β_(1),Smad2 and Smad3 mRNA in tumor tissues of the cyclophosphamide group and each group of decoction for dissipating binds and dispersing tumor were significantly lower than those of the model group(all P<0.05),and the relative expression of E-cadherin protein in tumor tissues were significantly higher than that in the model group(all P<0.05).The improvement of the above indexes was significantly better in the high-dose group of decoction for dissipating binds and dispersing tumor and the cyclophosphamide group than those in the low and medium-dose groups(P<0.05),and the differences between the high-dose group and the cyclophosphamide group were not statistically significant(all P>0.05).Conclusion Decoction for dissipating binds and dispersing tumor has obvious antitumor effect on subcutaneous transplanted tumor of breast cancer,and its mechanism may be related to regulating TGF-β_(1)/smads signaling pathway and reversing its mediated EMT.