乳岩宁对三阴乳腺癌荷瘤裸鼠Notch1信号通路的作用机制

Mechanism of Ruyanning Fomula(乳岩宁方)on Notch1 Signaling Pathway in Triple-Negative Breast Cancer Bearing Nude Mice

目的 观察乳岩宁方与卡培他滨联用对MDA-MB-231乳腺癌荷瘤裸鼠的瘤组织中Notch1信号通路的作用。方法 用对数生长期的MDA-MB-231细胞悬液,于裸鼠左侧胸壁接种,成功长出瘤组织后将其移植到未接种裸鼠的胸壁,建立三阴乳腺癌模型,共40只荷瘤裸鼠,分5组,每组8只,日1次灌胃,连续21 d灌药。观察裸鼠状态并计算肿瘤组织的抑制率;免疫印迹法测定Notch 1、Notch1受体的细胞内结构(Notch1 intracellular domain, NICD1)、与YRPW基序相关的发状分裂增强子1(Hairy and enhancer of split related with YRPW motif 1,Hey1)、发状分裂相关增强子1(Hairy and enhancer of split Homolog 1,Hes1)、髓细胞增生原癌基因(Cellular myelocytomatosis oncogene, C-myc)的蛋白表达量;实时荧光定量PCR测定Notch 1、Hey1、Hes1、C-myc的基因表达量。结果 与模型组对比,中药、西药和中药+西药组的肿瘤质量均有明显减轻,差异有统计学意义(P<0.01),3组的抑瘤率分别为33.11%、59.60%、65.93%。免疫印迹法结果表明,与模型组相比,中药组、西药组、中药+西药组的Notch 1、NICD1、Hey1、Hes1、C-myc蛋白水平明显降低,差异有统计学意义(P<0.01);而Notch 1和Hes1的蛋白表达水平在中药组和西药组中水平相当,差异无统计学意义(P>0.05);中药+西药组较西药组中Hes1蛋白表达水平明显减低,差异有统计学意义(P<0.05)。实时荧光定量结果表明,与模型组相比,中药组、西药组、中药+西药组Notch 1、Hey1、Hes1、C-myc的mRNA水平明显降低,差异有统计学意义(P<0.01);与西药组相比,中药组与西药组Notch 1、Hes1、C-myc的mRNA水平相当,差异无统计学意义(P>0.05);中药+西药组较西药组中Notch 1及Hey1的mRNA水平明显减低,差异有统计学意义(P<0.05)。结论 疏肝理气中药复方乳岩宁能够抑制三阴乳腺癌荷瘤裸鼠的肿瘤生长,其作用机制与三阴乳腺癌中的Notch1信号通路受到抑制相关。为疏肝理气中药治疗三阴乳腺癌,尤其是影响乳腺癌Notch1信号通路方面提供了实验依据。

Objective To observe the effect of Ruyanning Formula(乳岩宁方)combined with capecitabine on notch 1 signa-ling pathway in tumor tissue of MDA-MB-231 breast cancer cells bearing nude mice.Methods It used MDA-MB-231 cell suspension in logarithmic growth phase inoculated on the left chest wall of nude mice.After the tumor tissue successfully grew,it was transplanted to the chest wall of uninoculated nude mice to establish the triple-negative breast cancer model.A total of 40 tumor bearing nude mice were divided into 5 groups,with 8 in each group.The mice were administered once a day by gavage for 21 consecutive days.The state of nude mice was observed and the inhibition rate of tumor tissue was calculated.The protein ex-pressions of Notch 1,Notch1 intracellular domain(NICD1),Hairy and enhancer of split related with YRPW motif 1(Hey1),Hairy and enhancer of split Homolog 1(Hes1)and Cellular myelocytomatosis oncogene(C-myc)were measured by Western blot.The gene expressions of Notch 1,Hey1,Hes1 and C-myc were determined by RT-qPCR.Results The results of Western blot showed that compared with that of the model control group,the tumor weight of the Ruyanning Formula group,the capecitabine group and the Ruyanning Formula+capecitabine group were significantly reduced(P<0.01).The tumor inhibition rates of the three groups were 33.11%,59.60%and 65.93%,respectively.The results of Western blot showed that compared with those of the model control group,the levels of Notch 1,NICD1,Hey1,Hes1 and C-myc protein in the Ruyanning Formula group,the capecitabine group and the Ruyanning Formula+capecitabine group decreased significantly(P<0.01).The protein expression levels of Notch 1 and Hes1 were similar in the Ruyanning Formula group and the capecitabine group and there was no statistical significance(P>0.05).The expression level of Hes1 protein in the Ruyanning Formula+capecitabine group group was signifi-cantly lower than that in the capecitabine group(P<0.05).RT-qPCR results showed that compared with those of the model control group,the mRNA levels of Notch 1,Hey1,Hes1 and C-myc in the Ruyanning Formula group,the capecitabine group and the Ruyanning Formula+capecitabine group decreased significantly(P<0.01).Compared with those of the capecitabine group,the mRNA levels of Notch 1,Hes1 and C-myc in the Ruyanning Formula group and the capecitabine group were similar,with no statistical significance(P>0.05).The mRNA levels of Notch 1 and Hey1 in the Ruyanning Formula+capecitabine group were significantly lower than those in the capecitabine group(P<0.05).Conclusion Ruyanning Formula,a Chinese herbal compound for soothing liver and regulating Qi,can inhibit the tumor growth of nude mice bearing triple-negative breast cancer,and its mechanism is related to the inhibition of Notch 1 signaling pathway in triple-negative breast cancer.It provides an experimental basis for the treatment of the triple-negative breast cancer with Chinese herbs for soothing liver and regulating Qi.In particular,it has provided experimental evidence in terms of influencing notch 1 signaling pathway in breast cancer.