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胡玉蝶,王晓丽,焦方敏,杨争,袁博,胡金辉.基于Src/VEGF轴探究补肾活血汤治疗乳腺癌骨转移的作用机制[J].湖南中医药大学学报,2023,43(1):59-68[点击复制] |
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| 基于Src/VEGF轴探究补肾活血汤治疗乳腺癌骨转移的作用机制 |
| 胡玉蝶,王晓丽,焦方敏,杨争,袁博,胡金辉 |
| (湖南中医药大学, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 探讨Src/VEGF信号通路对补肾活血汤治疗乳腺癌骨转移的影响及作用机制。方法 通过TCMSP、GeneCards数据库，挖掘出补肾活血汤治疗乳腺癌骨转移的潜在靶点，并将靶点进行拓扑分析和GO、KEGG富集分析，对拓扑分析排第一的关键核心靶点进行实验验证。15只裸鼠随机分为对照组、模型组、补肾活血汤组，5只/组，模型组、补肾活血汤组通过胫骨注射人乳腺癌4T1-luc细胞构建裸鼠乳腺癌骨转移模型，补肾活血汤组按照36.67g/（kg·d）予以补肾活血汤灌胃，对照组、模型组按照0.01mL/（g·d）予以生理盐水灌胃，连续给药14d。监测并记录裸鼠体重变化；HE染色法检测裸鼠骨组织病理学变化；X线、微CT检测裸鼠胫骨情况；胫骨组织Trap染色观察破骨细胞；Western blot检测裸鼠胫骨组织细胞原癌基因（Src）、血管内皮生长因子抗体（vascular endothlial growth factor,VEGF）、金属基质蛋白1（matrix metalloproteinase 1,MMP1）蛋白表达水平；免疫组化法检测裸鼠胫骨组织SRC、VEGF、MMP1、活化T细胞核因子抗体（nuclear factor of activated T-Cells 1,NFATc1）基因表达水平。结果 补肾活血汤治疗乳腺癌骨转移潜在靶点共949个，其中核心靶点15个，关键核心靶点为Src；KEGG富集分析显示，补肾活血汤可能调控197个信号通路；GO富集分析得到生物过程（biological process,BP）605个条目、细胞组成（cellular component,CC）52个条目、分子功能（molecular function,MF)117个条目。与对照组相比，模型组裸鼠体重明显减轻（P<0.01），HE染色可见大量肿瘤细胞和炎性细胞浸润，X线、微CT下观测胫骨骨破坏面积百分比显著增加（P<0.01），Trap染色可见成熟破骨细胞数量明显增加（P<0.01），胫骨组织Src、VEGF、MMP1、NFATc1表达水平显著升高（P<0.01）；与模型组相比，补肾活血汤组裸鼠体重明显增加（P<0.01），肿瘤细胞和炎性细胞浸润情况好转，X线、微CT下观测胫骨骨破坏面积百分比显著降低（P<0.05），成熟破骨细胞数量明显减少（P<0.05），胫骨组织SRC、VEGF、MMP1、NFATc1表达水平显著降低（P<0.05或P<0.01）。结论 补肾活血汤治疗乳腺癌骨转移是多成分、多靶点、多通路共同作用的结果，其可能通过下调Src/VEGF轴及其下游MMP1、NFATc1基因的表达，抑制破骨细胞活化、改善骨破坏，发挥治疗乳腺癌骨转移的作用。 |
| 关键词: 乳腺癌 骨转移 骨破坏 补肾活血汤 Src/VEGF轴 实验验证 作用机制 |
| DOI：10.3969/j.issn.1674-070X.2023.01.010 |
| 投稿时间：2022-05-21 |
| 基金项目:湖南省教育厅重点项目（20A373）；湖南省科技厅科普专项（2021ZK4108）；湖南省中医药管理局重点项目（C2022020）；长沙市科技局一般项目（kq2202461）；校级研究生创新课题（2021CX68）；校级课题重点课题（2019XJJJ039）。 |
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| Bushen Huoxue Decoction's mechanisms of action in treating bone metastasis of breast cancer based on Src/VEGF axis |
| HU Yudie,WANG Xiaoli,JIAO Fangmin,YANG Zheng,YUAN Bo,HU Jinhui |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To explore the mechanisms of Bushen Huoxue Decoction in treating bone metastasis of breast cancer based on Src/VEGF signaling pathway. Methods Potential targets for treating bone metastasis of breast cancer by Bushen Huoxue Decoction were identified from TCMSP database and GeneCards database. Topological analysis and GO and KEGG enrichment analysis were performed on the targets, and the hub core targets that were ranked the first were further verified in topological analysis. Then 15 nude mice were randomly divided into control group, model group and Bushen Huoxue Decoction group, with 5 mice per group. The model group and Bushen Huoxue Decoction group were injected human breast cancer 4T1-LUC cells into the tibia to construct the nude mouse breast cancer bone metastasis model, and bushen Huoxue Decoction group was given Bushen Huoxue Decoction by 36.67 g/ (kg·d). Control group and model group were given intragastric normal saline according to 0.1 mL/(g·d) for 14 consecutive days, and the changes of body weight were monitored and recorded. The histopathological changes of bone in nude mice was measured by HE staining. The tibial bone of nude mice was detected by X-ray and mic-CT. The number and size of osteoclasts were observed by Trap staining. The expression levels of Src, vascular endothlial growth factor (VEGF) and matrix metalloproteinase 1 (MMP1) in tibial cells of nude mice were measured by Western blot. The expression levels of Src, VEGF, MMP1 and nuclear factor of activated T-cells 1 (NFATc1) in tibial tissues of nude mice were detected by immunohistochemical method. Results A total of 949 potential targets of Bushen Huoxue Decoction in treating bone metastasis of breast cancer were identified, among which 15 were core targets and Src was the hub target. KEGG enrichment analysis showed that Bushen Huoxue Decoction may regulate 197 signaling pathways. GO enrichment analysis revealed 605 items of biological process (BP), 52 items of cellular component (CC), and 117 items of molecular function (MF). Compared with control group, the model group showed significant lower body weight (P<0.01). Moreover, for model group, HE staining showed a large number of tumor cells and inflammatory cells; X-ray and microCT revealed the significant higher percentage of tibial bone damage area (P<0.01); Trap staining showed the significantly larger number of mature osteoclasts (P<0.01) and higher expression levels of Src, VEGF, MMP1 and NFATc1 in tibia tissues (P<0.01). Compared with model group, Bushen Huoxue Decoction group had the significantly higher body weight of nude mice (P<0.01) and the infiltration of tumor cells and inflammatory cells was improved. X-ray and microCT observation showed the lower percentage of damaged area of tibia bone (P<0.05), less number of mature osteoclasts (P<0.05) and decreased expression levels of Src, VEGF, MMP1 and NFATc1 in tibia tissue (P<0.05 or P<0.01) for Buyshen Huoxue Decoction group in comparision to model group. Conclusion Bushen Huoxue Decoction treats the bone metastasis of breast cancer by the interaction of multiple components, multiple targets and multiple pathways. It may inhibit osteoclast activation, improve bone destruction by down-regulating Src/VEGF axis and the expression of downstream MMP1 and NFATc1 genes, thus taking effects on bone metastasis of breast cancer. |
| Key words: breast cancer bone metastases bone destruction Bushen Huoxue Decoction Src/VEGF axis experimental verification mechanisms of action |
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