雄黄外用对三阴性乳腺癌上皮间质转化的调控作用研究

王春晖; 楚爱景; 朱振宇; 裴晓华

doi:10.14148/j.issn.1672-0482.2022.1027

雄黄外用对三阴性乳腺癌上皮间质转化的调控作用研究

doi: 10.14148/j.issn.1672-0482.2022.1027

王春晖^{1, 2,},
楚爱景³,
朱振宇¹,
裴晓华^2, ,

1.
北京中医药大学房山医院, 北京 102400
2.
北京中医药大学厦门医院, 福建厦门 361001
3.
北京中医药大学第三临床学院, 北京 100020

基金项目:

国家自然科学基金青年科学基金项目 82004367

详细信息

作者简介:
王春晖，男, 主治医师，E-mail：ghostrip@163.com

裴晓华，教授，博士生导师，北京中医药大学厦门医院院长，中华中医药学会外科分会主任委员，世教授，博士生导师，北京中医药大学厦门医院院长，中华中医药学会外科分会主任委员，世界中医药学会联合会疽证专业委员会会长，北京中医药学会副会长、外科专业委员会主任委员，国家中医药管理局十二五”中医药重点学科“中医乳腺病学”学科带头人，第七批全国老中医药专家学术经验继承工作指导老师，北京中医药大学国家双一流学科二级学科中医外科临床学系主任，北京市中医管理局“首届北京中医行业榜样”和“优秀名中医”，福建省临床重点专科(中医乳腺科)学术带头人，厦门市领军人才，厦门市中西医结合乳腺病重点专科学术带头人。作为第一完成人荣获中华中医药学会李时珍医药创新奖1项，中华中医药学会科技技术二等奖、三等奖各1项

通讯作者:
裴晓华，男, 主任医师, 主要从事中医药防治乳腺疾病和慢性难愈性创面研究，E-mail: pxh_127@163.com

中图分类号: R285.5
计量
- 文章访问数: 71
- HTML全文浏览量: 7
- PDF下载量: 27
- 被引次数: 0
出版历程
- 收稿日期: 2022-05-03
- 网络出版日期: 2022-11-19
- 发布日期: 2022-11-10

Study on the Regulatory Effect of Realgar for External Use on Epithelial Mesenchymal Transformation in Triple Negative Breast Cancer

1.
Fangshan Hospital, Beijing University of Chinese Medicine, Beijing 102400, China
2.
Xiamen Hospital, Beijing University of Chinese Medicine, Xiamen 361001, China
3.
The Third Clinical College, Beijing University of Chinese Medicine, Beijing 100020, China

摘要

摘要: 目的研究中药雄黄外用对三阴性乳腺癌上皮间质转化的作用及调控机制。方法采用人三阴性乳腺癌MDA-MB-231细胞构建裸鼠人乳腺癌移植瘤模型后, 将裸鼠随机分为模型组、雄黄外用组、卡培他滨片组, 干预6周后取材。通过HE染色、免疫组化、Western blot、qPCR等方法观察雄黄外用对乳腺癌肿瘤上皮间质转化的调控作用, 同时对动物血常规, 肝、肾功能指标进行检测。结果雄黄外用可使乳腺癌组织出现一定程度的坏死, 抑瘤率为30.2%;与模型组比较, 雄黄外用组裸鼠肝、肾功能及血常规检测等未见明显异常; 肿瘤组织免疫组化结果提示, 雄黄外用可增强上皮钙黏蛋白(E-cadherin)表达, 降低波形蛋白(Vimentin)表达; Western blot和qPCR结果提示: 与模型组比较, 雄黄外用可上调肿瘤组织中E-cadherin表达, 减弱Vimentin、Twist1、TGF-β1的表达, 差异具有统计学意义(P < 0.05)。结论雄黄外用可抑制人三阴性乳腺癌移植瘤在体生长, 其机制与增强E-cadherin表达, 降低Vimentin、Twist1、TGF-β1的表达, 实现对乳腺癌上皮间质转化的调控相关。
- 雄黄 /
- 外治法 /
- 乳腺癌 /
- 上皮间质转化
Abstract: OBJECTIVE To investigate the regulatory effect of realgar for external use on epithelial mesenchymal transformation in triple negative breast cancer. METHODS Human breast cancer nude mouse xenograft model was established by injecting MDA-MB-231 cells. The mice were randomly divided into model group, realgar group and capecitabine tablet group. Hematoxylin and eosin (HE), immunohistochemistry, Western blot and qPCR were used to observe the effect of realgar on epithelial-mesenchymal transition (EMT) of breast cancer. At the same time, the indexes of blood routine, liver and kidney function were detected. RESULTS External using realgar could induce necrosis of breast cancer tissue, the tumor inhibitory rate was 30.2%. Compared with the model group, the liver and kidney functions and blood routine tests of nude mice in realgar group were not significantly abnormal; The immunohistochemical results of tumor tissues suggested that realgar could enhance the expression of E-cadherin and reduce the expression of Vimentin; The results of Western blot and qPCR showed that realgar could enhance the expression of E-cadherin and reduce the expressions of Twist1 and TGF-β1 in tumor tissues (P < 0.05). CONCLUSION External using realgar can inhibit the growth of triple negative breast cancer and regulate EMT by increasing the expression of E-cadherin and decreasing the expressions of Vimentin, Twist1 and TGF-β1.
- realgar /
- external therapy /
- breast cancer /
- epithelial-mesenchymal transition

HTML全文

图 1 各组裸鼠体质量变化曲线

Figure 1. Body weight of nude mice at different time points

下载: 全尺寸图片幻灯片

图 2 各组裸鼠肿瘤体积变化曲线

注：Mod.模型组；Cap.卡培他滨片组；XHWY.雄黄外用组

Figure 2. Tumor volume at different time points

下载: 全尺寸图片幻灯片

图 3 取材后各组裸鼠肿瘤外观和质量比较

注：Mod.模型组；Cap.卡培他滨片组；XHWY.雄黄外用组；与模型组比较, ^*P < 0.05。

Figure 3. Tumor appearance and quality

下载: 全尺寸图片幻灯片

图 4 各组肿瘤组织HE染色比较

Figure 4. Morphological changes of tumor tissue

下载: 全尺寸图片幻灯片

图 5 肿瘤组织中E-cadherin、Vimentin免疫组化比较

注：Mod.模型组；Cap.卡培他滨片组；XHWY.雄黄外用组；与模型组比较, ^*P < 0.05；与卡培他滨片组比较, ^#P < 0.05。

Figure 5. Changes of E-cadherin and Vimentin in tumor tissues

下载: 全尺寸图片幻灯片

图 6 Western blot检测肿瘤组织中EMT相关因子蛋白表达情况比较

注：Mod.模型组；Cap.卡培他滨片组；XHWY.雄黄外用组；与模型组比较, ^*P < 0.05；与卡培他滨片组比较, ^#P < 0.05。

Figure 6. Effect of realgar on EMT relative protein expression in the breast tumor as determined by Western blot

下载: 全尺寸图片幻灯片

图 7 qPCR检测肿瘤组织中EMT相关因子mRNA表达情况比较

注：Mod.模型组；Cap.卡培他滨片组；XHWY.雄黄外用组；与模型组比较, ^*P < 0.05；与卡培他滨片组比较, ^#P < 0.05。

Figure 7. Effect of realgar on EMT relative gene expression in the breast tumor as determined by qPCR

下载: 全尺寸图片幻灯片

表 1 分组及干预措施

Table 1. Grouping and interventions

组别	n	干预措施
模型组	8	无菌去离子水0.2 mL·d^-1, 灌胃。
雄黄外用组	8	0.5 g·mL^-1醋调雄黄肿瘤处外敷, 约1 mm厚度，采用纱布包扎固定，8 h·d^-1。
卡培他滨片组	8	第2~3周, 第5~6周: 7.8 mg·mL^-1卡培他滨片混悬液0.2 mL·d^-1, 灌胃；第1周, 第4周: 无菌去离子水0.2 mL·d^-1, 灌胃。

下载: 导出CSV

表 2 引物序列

Table 2. Primer sequences

引物名称	正向序列(5'→3')	反向序列(5'→3')
TGF-β1	TACAGCAACAATTCCTGGCGATACC	CTCAACCACTGCCGCACAACTC
Vimentin	CCTTCGTGAATACCAAGACCTGCTC	AATCCTGCTCTCCTCGCCTTCC
CDH1	CTGATTCTGCTGCTCTTGCTGTTTC	CGTCCTCTTCTCCGCCTCCTC
GAPDH	TGACTTCAACAGCGACACCCA	CACCCTGTTGCTGTAGCCAAA
Twist1	CCTCGGACAAGCTGAGCAAGATTC	GTCGCTCTGGAGGACCTGGTAG
Slug	CTGTGACAAGGAATATGTGAGC	CTAATGTGTCCTTGAAGCAACC

下载: 导出CSV

参考文献(24)

[1]	MAIRIN O, HARRY C, TONY F, et al. Interventions for raising breast cancer awareness in women[J]. Cochrane Database Syst Rev, 2017, 2: CD011396. doi: 10.1111/ijn.12582
[2]	LIU X, LI JJ, CADILHA BL, et al. Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis[J]. Sci Adv, 2019, 5(6): eaav4275.
[3]	KHALED N, BIDET Y. New insights into the implication of epigenetic alterations in the EMT of triple negative breast cancer[J]. Cancers, 2019, 11(4): 559. doi: 10.3390/cancers11040559
[4]	FOLKMAN J. The role of angiogenesis in tumor growth[J]. Semin Cancer Biol, 1992, 3(2): 65-71. https://www.sciencedirect.com/science/article/pii/S0093775402700651
[5]	GOODMAN CR, SEAGLE BLL, FRIEDL TWP, et al. Association of circulating tumor cell status with benefit of radiotherapy and survival in early-stage breast cancer[J]. JAMA Oncol, 2018, 4(8): e180163. https://www.scholars.northwestern.edu/en/publications/association-of-circulating-tumor-cell-status-with-benefit-of-radi
[6]	LIU F, GU LN, SHAN BE, et al. Biomarkers for EMT and MET in breast cancer: An update[J]. Oncol Lett, 2016, 12(6): 4869-4876. doi: 10.3892/ol.2016.5369
[7]	吴晓龙, 徐瑞荣, 沈明月, 等. 徐瑞荣治疗骨髓增殖性肿瘤经验[J]. 辽宁中医杂志, 2019, 46(5): 944-946. https://www.cnki.com.cn/Article/CJFDTOTAL-LNZY201905015.htm WU XL, XU RR, SHEN MY, et al. Professor XU Ruirong's experience in treatment of myeloproliferative neoplasms[J]. Liaoning J Tradit Chin Med, 2019, 46(5): 944-946. https://www.cnki.com.cn/Article/CJFDTOTAL-LNZY201905015.htm
[8]	裴可, 齐元富, 李运伦, 等. 中西医结合治疗恶性肿瘤体表破溃创面进展[J]. 中华中医药杂志, 2019, 34(12): 5790-5792. https://www.cnki.com.cn/Article/CJFDTOTAL-BXYY201912073.htm PEI K, QI YF, LI YL, et al. Research progress of combination of Chinese and western medicine for the treatment of malignant tumor rupture wound[J]. China J Tradit Chin Med Pharm, 2019, 34(12): 5790-5792. https://www.cnki.com.cn/Article/CJFDTOTAL-BXYY201912073.htm
[9]	王春晖. 二硫化二砷和粉防己碱对三阴性乳腺癌联合作用的研究[D]. 北京: 北京中医药大学, 2018. WANG CH. Study on the combined effect of arsenic disulfide and tetrandrine on triple negative breast cancer[D]. Beijing: Beijing University of Chinese Medicine, 2018.
[10]	王洪图. 黄帝内经灵枢白话解[M]. 北京: 人民卫生出版社, 2004: 491. WANG HT. The Vernacular Explanation for The Yellow Emperor's Inner Classic: The Spiritual Pivot[M]. Beijing: People's medical publishing house, 2004: 491.
[11]	杨群柳, 全建峰. 中医外治法治疗恶性肿瘤及其相关病症的研究进展[J]. 江苏中医药, 2022, 54(2): 77-81. https://www.cnki.com.cn/Article/CJFDTOTAL-JSZY202202024.htm YANG QL, QUAN JF. Review of treatment of malignant tumors and related diseases with external therapies in Chinese medicine[J]. Jiangsu J Tradit Chin Med, 2022, 54(2): 77-81. https://www.cnki.com.cn/Article/CJFDTOTAL-JSZY202202024.htm
[12]	李琦玮, 于明薇, 王笑民. 癌毒理论研究现状[J]. 中医杂志, 2015, 56(4): 347-350, 354. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ201504030.htm LI QW, YU MW, WANG XM. Research status of cancer toxin theory[J]. J Tradit Chin Med, 2015, 56(4): 347-350, 354. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ201504030.htm
[13]	李慧杰, 齐元富, 李秀荣. 基于癌毒理论探讨攻毒治法抗肿瘤转移[J]. 中医药信息, 2018, 35(4): 81-83. LI HJ, QI YF, LI XR. Exploration of anti-tumor metastasis by therapeutics of eliminating toxin based on theory of cancer toxin[J]. Inf Tradit Chin Med, 2018, 35(4): 81-83.
[14]	郑文利, 李慧杰, 裴可, 等. 基于癌毒理论探讨攻毒类中药在恶性肿瘤治疗中的应用[J]. 中医药信息, 2019, 36(4): 98-102. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXN201904028.htm ZHENG WL, LI HJ, PEI K, et al. Chinese medicinal of counteracting toxic substances in the treatment of malignant tumor based on cancer toxin theory[J]. Inf Tradit Chin Med, 2019, 36(4): 98-102. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXN201904028.htm
[15]	李经纬, 余瀛鳌, 蔡景峰, 等. 中医大辞典[M]. 北京: 人民卫生出版社, 1995: 397. LI JW, YU YA, CAI JF, et al. Dictionary of Traditional Chinese Medicine[M]. Beijing: People's medical publishing house, 1995: 397.
[16]	HYAFIL F, BABINET C, JACOB F. Cell-cell interactions in early embryogenesis: A molecular approach to the role of calcium[J]. Cell, 1981, 26(3): 447-454. https://www.sciencedirect.com/science/article/pii/0092867481902142
[17]	YANG L, WANG XW, ZHU LP, et al. Significance and prognosis of epithelial-cadherin expression in invasive breast carcinoma[J]. Oncol Lett, 2018, 16(2): 1659-1665. https://www.spandidos-publications.com/ol/16/2/1659
[18]	ESLAMI AMIRABADI H, TUERLINGS M, HOLLESTELLE A, et al. Characterizing the invasion of different breast cancer cell lines with distinct E-cadherin status in 3D using a microfluidic system[J]. Biomed Microdevices, 2019, 21(4): 101.
[19]	HUGO HJ, GUNASINGHE NPAD, HOLLIER BG, et al. Epithelial requirement for in vitro proliferation and xenograft growth and metastasis of MDA-MB-468 human breast cancer cells: Oncogenic rather than tumor-suppressive role of E-cadherin[J]. Breast Cancer Res, 2017, 19(1): 86. doi: 10.1186/s13058-017-0880-z
[20]	PATEL NA, PATEL PS, VORA HH. Role of PRL-3, Snail, Cytokeratin and Vimentin expression in epithelial mesenchymal transition in breast carcinoma[J]. Breast Dis, 2015, 35(2): 113-127. https://content.iospress.com/articles/breast-disease/bd395
[21]	NIETO MA. The snail superfamily of zinc-finger transcription factors[J]. Nat Rev Mol Cell Biol, 2002, 3(3): 155-166. https://www.nature.com/articles/nrm757/
[22]	YANG J, MANI SA, DONAHER JL, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis[J]. Cell, 2004, 117(7): 927-939. https://www.sciencedirect.com/science/article/pii/S0092867404005768
[23]	THIERY JP, SLEEMAN JP. Complex networks orchestrate epithelial-mesenchymal transitions[J]. Nat Rev Mol Cell Biol, 2006, 7(2): 131-142. https://www.nature.com/articles/nrm1835
[24]	ZHENG H, KANG Y. Multilayer control of the EMT master regulators[J]. Oncogene, 2014, 33(14): 1755-1763. https://www.nature.com/articles/onc2013128