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Objective:To explore the mechanism of Xiaojinwan in treating breast cancer bone metastases through cell experiments and bioinformatic analysis. Method:The inhibitory effect of Xiaojinwan on MCF-7 cell viability was detected by cell counting kit-8 （CCK-8） assay. The key components and targets responsible for Xiaojinwan in inhibiting breast cancer bone metastases were predicted by network pharmacology and molecular docking. The active components and targets of Xiaojinwan were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCSMP） and SwissTarget Prediction， and the breast cancer bone metastases-related targets from GeneCards and DisGeNET. The results were imported into STRING for constructing a protein-protein interaction （PPI） network， followed by Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis using DAVID. A network of the active components of Xiaojinwan-breast cancer bone metastases-related targets-pathways was constructed using Cytoscape 3.7.2. AutoDock 4 was employed for molecular docking. The protein expression levels of matrix metallopmteinase-9 （MMP-9）， hypoxia-inducible factor 1<italic>α </italic>（HIF1A）， and androgen receptor （AR） were assayed by Western blot. Result:Xiaojinwan inhibited the viability of MCF-7 cells and acted on breast cancer bone metastases through such processes as redox and protein autophosphorylation. KEGG enrichment analysis showed that HIF-1， vascular endothelial growth factor （VEGF） and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） pathways were involved. As verified by molecular docking， the active components such as eucalyptin stably bound to AR and MMP-9. Western blot indicated that Xiaojinwan dose-dependently inhibited the expression of MMP-9 and HIF1A proteins in MCF-7 cells. Conclusion:Xiaojinwan acts on AR and MMP-9 through HIF， VEGF and other related signaling pathways， thereby improving hypoxia in tumor microenvironment， inhibiting angiogenesis， and reducing cell invasion and viability.
RESUMO
<p><b>OBJECTIVE</b>To explore the effect of Yanghe Huayan Decoction (YHD, a representative recipe for warming yang mass dissipating) in inhibiting precancerosis of breast cancer (PBC) and on the protein and mRNA expression of ki67.</p><p><b>METHODS</b>The PBC rat model was established by dimethylbenzanthracene (DMBA), and 9 weeks later rats were randomly divided into the blank control group, the model group, the YHD group, the Sanjie Huatan Decoction group (SHD), the Pingxiao Tablet group (PT), and the tamoxifen group. Rats in the model group were administered with water by gastrogavage. Rats in the YHD group received YHD (deglued antler powder 12 g, prepared rhizome of rehmannia 9 g, cassia bark 6 g, white mustard seed 3 g, zedoary root 12 g, appendiculate cremastra pseudobulb 15 g, chekiang fritillary bulb 9 g, licorice root 6 g) at the daily dose of 7.2 g/kg by gastrogavage. Rats in the SHD group received SHD (oldenlandia diffusa 15 g, Scutellaria Barbata 15 g, Trichosanthes Kirilowii 15 g, pinellia 9 g) at the daily dose of 5.4 g/kg by gastrogavage. Rats in the PT group received PT at the daily dose of 144 mg/kg by gastrogavage. Those in the tamoxifen group received tamoxifen at the daily dose of 4 mg/kg by gastrogavage. Pathomorphological changes of the breast tissue were observed by HE staining. The positive rate and the gray value of ki67 expression were detected by immunohistochemical assay. And the expression of ki67 mRNA was detected by q-PCR.</p><p><b>RESULTS</b>Compared with the model group, the general hyperplasia and the occurrence rate of precancerous lesion were higher and the occurrence rate of invasive carcinoma was lower in each treatment group (P < 0.05). Except the SHD group, the intensity of ki67 grey value increased in each treatment group (P < 0.05, P < 0.01). Except the PT group, the positive rate of ki67 and mRNA expression of ki67 increased in the rest treatment groups (P < 0.05, P < 0.01). Compared with the YHD group, there was no statistical difference in the occurrence rate of infiltration or the occurrence rate of precancerous lesion (P > 0.05). The positive rate of ki67 expression and mRNA expression of ki67 increased in the PT group, showing statistical difference (P < 0.05).</p><p><b>CONCLUSIONS</b>YHD could partially inhibit and reverse canceration of breast cancer. It also could inhibit ki67 protein and mRNA expression. Its effect was similar to tamoxifen and superior to PT. So it was suitable for prevention and treatment of precancerous lesion of breast cancer.</p>