基于PI3K/AKT通路介导EMT程序启动探讨补肾活血汤对乳腺癌的骨转移影响

Effect of Bushen Huoxue Decoction(补肾活血汤)on Bone Metastasis of Breast Cancer Based on PI3K/AKT Pathway Mediated Epithelial-mesenchymal Transition

目的探讨补肾活血汤对裸鼠乳腺癌的骨转移影响及机制研究。方法建立裸鼠乳腺癌模型,分空白组、模型组、补肾活血汤组、LY294002(PI3K抑制剂)组、联合组(补肾活血汤+LY294002组)。给药期间记录裸鼠体质量变化,药物干预后处死动物,取裸鼠乳腺肿瘤和骨组织,X线、CT影像学观察裸鼠骨质影响;苏木素-伊红(HE)观察裸鼠乳腺癌和肿瘤骨转移情况;免疫组化、蛋白免疫印迹法(western blot)检测骨组织破骨细胞活化因子T转录因子(nuclear factor-active T cell NFATC1)、E-钙黏蛋白(E-cadherin)、N-钙蛋白(N-cadherin)、锌指E盒结合蛋白1(zinc finger E-box binding Protein 1)、磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)的蛋白表达。结果与空白组相比,模型组活动少,精神状态差,体质量降低(P<0.05),骨质破坏明显,骨转移率87%,E-cadherin蛋白表达下调,NFATC1、N-cadherin、ZEB1、PI3K/AKT等蛋白表达提高;与模型组相比,补肾活血汤组、联合组裸鼠精神可、体质量增加(P<0.05),LY294002组体质量与模型组相比未见明显增重(P>0.05);各实验组骨破坏均减少,其中补肾活血汤组抑制NFATC1活化,与模型组相比,能减少24%骨转移;western blot结果提示LY294002和补肾活血汤提高E-cadherin蛋白表达,降低N-cadherin、ZEB1、PI3K/AKT等蛋白表达(P<0.05),其作用在联合组表现更为显著(P<0.01)。结论补肾活血汤对乳腺癌裸鼠有骨保护且预防骨转移作用;补肾活血汤预防副转移作用可能减弱PI3K/AKT的活化蛋白表达相关。

Objective To investigate the effect of Bushen Huoxue Decoction(补肾活血汤)on bone metastasis of breast cancer in nude mice and its mechanism.Methods The nude mouse model of breast cancer was established and divided into blank group,model group,Bushen Huoxue Decoction group,LY294002(PI3K inhibitor)group,and combined group(Bushen Huoxue Decoction+LY294002 group).During the administration period,the weight change of nude mice was recorded.After the drug intervention,the animals were killed,and the breast tumor and bone tissue of nude mice were taken.The bone effect of nude mice was observed by X-ray and CT imaging,and the breast cancer and tumor bone metastasis of nude mice were observed by hematoxylin eosin(HE).Immunohistochemistry and Western blot were used to detect nuclear factor active T cell NFATC1,E-cadherin,N-cadherin,zinc finger E-box binding protein 1phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)protein expression.Results Compared with the blank group,the model group showed less activity,poor mental state,weight loss(P<0.05),significant bone destruction,bone metastasis rate of 87%,down-regulation of E-cadherin protein expression,and increased protein expression of NFATC1,N-cadherin,ZEB1,PI3K/AKT;Compared with the model group,the nude mice in the Bushen Huoxue Decoction group and the combination group had good spirit and weight gain(P<0.05),and the weight of LY294002 group had no significant weight gain compared with the model group(P>0.05);However,the bone destruction in each experimental group was reduced,and the activation of NFATC1 in the Bushen Huoxue Decoction group was the lowest,which could reduce bone metastasis by 24%compared with the model group;Western blot results showed that LY294002 and Bushen Huoxue Decoction increased the expression of E-cadherin protein and decreased the expression of N-cadherin,ZEB1,PI3K/AKT protein(P<0.05),which was more significant in the combination group(P<0.01).Conclusion Bushen Huoxue Decoction can protect bone and prevent bone metastasis in nude mice with breast cancer;The effect of Bushen HuoxueDecoction on preventing metastasis may weaken the expression of PI3K/AKT activated protein.