疏肝健脾饮对乳腺癌癌前病变裸鼠淋巴管生成的影响研究

Effect of Shugan Jianpi Decoction on lymphangiogenesis in nude mice with precancerous lesions of breast cancer

目的研究疏肝健脾饮对乳腺癌癌前病变裸鼠淋巴管生成途径COX-2/VEGF通路中血管内皮生长因子-C(VEGF-C)、血管内皮生长因子受体3(VEGFR-3)、环氧合酶-2(COX-2)蛋白及mRNA表达的影响,探讨疏肝健脾饮对乳腺癌癌前病变淋巴管生成的干预作用机制,寻找中药复方抑制淋巴管生成的作用靶点。方法取120只BALB/c-nudemice裸鼠,采用乳腺原位移植MCF-10AT细胞株方法建立癌前病变模型,建模成功后随机分成6组,每组20只。模型对照组裸鼠予以0.9%氯化钠溶液灌胃,疏肝健脾饮组、疏肝理气组、健脾化痰组、活血化瘀组分别予以相应中药制剂灌胃,均1次/d;他莫昔芬组给予他莫昔芬腹腔注射,每周2次。各组均连续干预9周。至第9周周末处死各组裸鼠,分别采用Western-blot、RT-PCR技术检测肿瘤组织中VEGF-C、VEGFR-3、COX-2蛋白及mRNA表达情况。结果疏肝健脾饮组、疏肝理气组、活血化瘀组、他莫昔芬组VEGF-C蛋白及mRNA相对表达量均明显低于模型对照组(P均<0.05),且疏肝健脾饮组均明显低于活血化瘀组(P均<0.05)。疏肝健脾饮组、健脾化痰组、他莫昔芬组VEGFR-3蛋白相对表达量均明显低于模型对照组(P均<0.05),且疏肝健脾饮组及他莫昔芬组均明显低于疏肝理气组(P均<0.05);疏肝健脾饮组、疏肝理气组、他莫昔芬组COX-2蛋白相对表达量均明显低于模型对照组(P均<0.05),且疏肝健脾饮组及他莫昔芬组均明显低于健脾化痰组及活血化瘀组(P均<0.05)。疏肝健脾饮组、疏肝理气组、他莫昔芬组VEGFR-3mRNA相对表达量均明显低于模型对照组(P均<0.05),且疏肝健脾饮组和他莫昔芬组明显低于活血化瘀组(P均<0.05);疏肝健脾饮组、他莫昔芬组COX-2mRNA相对表达量均明显低于模型对照组和疏肝理气组(P均<0.05)。疏肝健脾饮组与他莫昔芬组VEGF-C、VEGFR-3、COX-2蛋白及mRNA相对表达量比较差异均无统计学意义(P均>0.05)。结论疏肝健脾饮可通过干预VEGF-C、VEGFR-3、COX-2的表达而影响淋巴管的生成,抑制乳腺癌癌前病变。

Objective It is to study the effects of Shugan Jianpi Decoction on the expression of vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR-3),cyclooxygenase-2 (COX-2) protein and mRNA in COX-2/VEGF pathway of lymphatic vessel formation in the mice with precancerous lesions of breast cancer,and to explore the intervention mechanism of Shugan Jianpiyin on lymphangiogenesis of breast cancer precancerous lesions,and seek for the target of the action of inhibition lymphangiogenesis with Chinese medicine compound.Methods A total of 120 BALB/c-nude mice were used to establish precancerous lesion model by orthotopic transplantation of MCF-10AT cells.After successful modeling,they were randomly divided into 6 groups,20 mice in each group.The model control group of nude mice were intragastrically administered with 0.9% sodium chloride solution,Shugan Jianpiyin group,Shugan Liqi group,Jianpi Huayu group,Huoxuehuayu group were given the corresponding Chinese medicine decoction,respectively,once a day,the tamoxifen group was given tamoxifen intraperitoneally,twice a week.Each group was continuously treated for 9 weeks.The nude mice of each group were sacrificed at the end of the 9th week.The expression of VEGF-C,VEGFR-3,COX-2 protein and mRNA in tumor tissues were detected by Western-blot and RT-PCR.Results The relative expressions of VEGF-C protein and mRNA in Shugan Jianpiyin group,Shugan Liqi group,Huoxuehuayu group and tamoxifen group were significantly lower than those in the model control group ( P <0.05),the expression in Jianpiyin group was significantly lower than the Huoxuehuayu group ( P <0.05).The relative expression levels of VEGFR-3 protein in Shugan Jianpiyin group,Jianpi Huatan group and tamoxifen group were significantly lower than those in the model control group ( P <0.05),and Shugan Jianpiyin group and tamoxifen group was significantly lower than Shugan Liqi group ( P <0.05).The relative expression of COX-2 protein in Shugan Jianpiyin group,Shugan Liqi group and tamoxifen group was significantly lower than that in the model control group ( P <0.05),and Shugan Jianpiyin group and tamoxifen group were significantly lower than Jianpi Huatan group and Huoxuehuayu group ( P <0.05).The relative expression levels of VEGFR-3 mRNA in Shugan Jianpiyin group,Shugan Liqi group and tamoxifen group were significantly lower than those in the model control group ( P <0.05),and Shugan Jianpiyin group and tamoxifen group was significantly lower than Huoxuehuayu group ( P <0.05);the relative expression of COX-2 mRNA in Shugan Jianpiyin group and tamoxifen group was significantly lower than that in the model control group and Shugan Liqi group ( P <0.05).There was no significant difference in the relative expressions of VEGF-C,VEGFR-3,COX-2 protein and mRNA between Shugan Jianpiyin group and tamoxifen group ( P >0.05).Conclusion Shugan Jianpi Decoction can affect the formation of lymphatic vessels and inhibit the precancerous lesions of breast cancer by intervention of the expression of VEGF-C,VEGFR-3 and COX-2.