| Background and purposeSalvia Miltiorrhizae,a representative of traditional Chinese medicine for promoting blood circulation and removing blood stasis,is often clinically used to treat blood stasis syndromes,such as coronary heart disease,cerebral ischemic injury,and lower limb ischemia.The active ingredient group in salvia miltiorrhiza is mainly divided into fat-soluble ingredients(tanshinone ⅡA,cryptotanshinone,tanshinone I,etc.)and water-soluble ingredients(danshensu,salvianolic acid B,rosmarinic acid,etc.).The water-soluble components can inhibit platelet aggregation and thrombosis after entering into the blood circulation,and protect the cardiovascular system.Salvianolic acid B extracted from salvia miltiorrhiza and related preparations is commonly used as the quality control standard.This suggests that salvianolic acid B plays an important role in protecting vasculature.After investigating 1362 anti-cancer traditional Chinese medicine prescriptions and proven prescriptions,it was found that Danshen ranked fourth in the frequency of application,and ranked first in the traditional Chinese medicine for promoting blood circulation and removing stasis.Based on the research basis established by our research group on the intervention and treatment mechanism of tradional Chinese medicine for activating blood and removing blood stasis in all aspects of tumor occurrence and development,it has been verified that numerous active ingredients in Salvia miltiorrhiza can affect tumor proliferation,metastasis,and angiogenesis.To this end,can salvianolic acid B play a critical role in protecting vessels in tumors and improve tumor vascular structure and function?What is the underlying mechanism?This study was based on the regulation of salvianolic acid B on tumor blood vessels,and it explores the effects of salvianolic acid B on metastasis and analyses the underlying mechanisms.Research contents and resultsIn this study,4T1 mouse breast cancer cell line and MDA-MB-231 human cancer cell line were selected to establish orthotopic breast cancer models.Our previous study found that the main features of the models were excessive angiogenesis and the existence of a large number of immature blood vessels.We thus used these two models to investigate the effects of salvianolic acid B on tumor blood vessels.Through tumor volume measurement,immunohistochemistry,and H&E staining,it was found that salvianolic acid B failed to inhibit tumor growth and increase the area of necrosis in the tumor.Through immunofluorescence,immunohistochemistry,3D Doppler imaging,and scanning electron microscopy,it was shown that salvianolic acid B inhibited tumor angiogenesis,but it exhibited a more significant effect on normalizing tumor blood vessels and promoted the integrity of tumor vascular structure.The Mils assay was used to detect the permeability of tumor blood vessels,and the FITC-Lectin was used to access the perfusion of tumor blood vessels.It was found that salvianolic acid B could enhance vascular perfusion by improving the structure of tumor blood vessels.Using the in vivo hypoxia probe PIMO,immunohistochemistry for hypoxia marker HIF-lα,and in vivo photoacoustic imaging oxygen detection,we found that salvianolic acid B could enhance the oxygen-carrying capacity of tumor blood vessels and ameliorate the hypoxic state within the tumor.The 4T1 mouse breast cancer model was selected for the study of combination therapy of salvianolic acid B and chemotherapeutics.Through tumor volume measurement,immunohistochemistry,and immunofluorescence staining,it was revealed that the combination of salvianolic acid B with Cisplatin inhibited tumor growth more significantly Cisplatin alone.Through immunofluorescence,LC-MS,and flow cytometry,it was found that the combination effect was dependent on the enhancement of tumor vascular function.Salvianolic acid B could promote the accumulation of Cisplatin in tumors.Through virtual molecular docking,we further demonstrated the regulation mechanisms of salvianolic acid B on tumor blood vessels and found that salvianolic acid B could bind to PDGFR-β,thereby inhibiting its phosphorylation level.PDGFR-β is a marker of vascular pericytes,and the coverage of pericytes is a hallmark of vascular maturation.The level of PDGF-BB secreted by MDA-MB-231 cells was significantly higher than that of HUVECs based on PCR,ELISA,and western bot results.A large amount of PDGF-B can stimulate the migration of pericytes,and their shedding from as well as transformation into fibroblasts,thereby losing the protective effect on blood vessels.Salvianolic acid B could target PDGFR-β to block the stimulation effects of PDGF-B and promote the maturation of blood vessels.It was inferred that salvianolic acid B mediated the signaling crosstalk between tumor cells and pericytes to exert the effect onvascular regulation.Through micro-CT scanning and H&E staining of lung tissue,it was found that the lung metastasis of mice in the salvianolic acid B treated group was significantly inhibited.Transcriptome sequencing was used to further investigate the mechanism of salvianolic acid B to inhibit metastasis.Sequencing results revealed that transcription levels of Il31ra,Ezh2,116,Fgf7,and 1110 were significantly changed.PCR was used to confirmed the alterations in the these genes in the tumor tissues and tumor cells.Meanwhile,GO and KEGG and protein interaction analyses was performed on the results.It was shown that PDGF-B stimulated the secretion of PDGF-B in tumor cell and activates downstream Akt phosphorylation,thereby affecting Ezh2 transcription,and further promoting EMT to induce tumor metastasis.Salvianolic acid B could reverse this process by binding to PDGFR-β.WB was used to detect the protein levels of p-PDGFR-β,Akt,p-Akt,and the expression of EMT-related proteins to verify the prediction results.In addition to the activation of tumor cells,the conditioned medium of tumor cells was used to investigate their impact on the endothelial barrier.Based on the immunofluorescence staining and endothelial leakage experiments,it was found that salvianolic acid B could significantly prevent tumor cells from destroying the endothelial barrier.Conclusion and significanceBased on the above results,we found that salvianolic acid B improved the structure and function of tumor blood vessels.The role of salvianolic acid B in promoting the normalization of tumor blood vessels also provided a basis for the combination therapy of salvianolic acid B and chemotherapeutic drugs,which could enhance the delivery of chemotherapeutic drugs into the tumors and thus kill tumors.PDGF-B/PDGFR-β plays an important role in tumor vascular abnormalities and is the key to tumor cell-mediated signaling crosstalk between vascular endothelial cells and pericytes.Salvianolic acid B could block signal transduction through PDGFR-β,play a role in remodeling blood vessels,and inhibit tumor metastasis.It was first proposed to use the regine of promoting blood circulation and removing blood stasis to treat malignant tumors,which mainly focused on four aspects:inhibiting platelet aggregation,improving the state of microcirculation within the tumor,inhibiting tumor metastasis,and combining chemotherapy.In this study,we selected salvianolic acid B as the main pharmacologically active component of Salvia miltiorrhiza from traditional Chinese medicine for promoting blood circulation and removing blood stasis,and revealed that it targeted platelet-derived growth factor signaling,promoted tumor vascular normalization,and inhibited tumor metastasis.This study provided the basis for the clinical application of Salvia miltiorrhiza in the treatment of cancer. |
PDF Full Text Request