| [Background and Purpose]A key and difficult problem in traditional Chinese medicine research is to understand the interaction between complex chemical system in traditional Chinese medicine prescription and biological system of diseases development, and to clarify the molecular mechanism of traditional Chinese medicine prescription.In recent years, there has made great achievements in the study of multi component and multi targeted effect of the Chinese herbal medicine in molecular and cellular levels and lay a solid foundation for the study of traditional Chinese medicine prescriptions.promoting blood circulation for removing blood stasis is the law of TCM for the treatment of cancer.Danshen,as a representative medicine,is mainly not only used for the treatment of cardiovascular diseases but also cancer in clinic.All the traditional Chinese medicine compound used for treatment of liver cancer almost contain Salvia miltiorrhiza.Clinical application is the aqueous extract or injection of Salvia miltiorrhiza,so what is the matter foundation in water soluble components of Salvia miltiorrhiza based on "single component used in a variety of disease" theory? Whether it plays a role through the common factors of two kinds of diseases?The previous study exploring related target of effective components in Salvia miltiorrhiza applied to treatment of tumor through data mining tools found that angiotensin Ⅱ type1receptor AT1R (Angiotensin II type1receptor) has an important role in both cardiovascular disease and tumor growth, metastasis especially tumor angiogenesis. Therefore, we expect to explore whether the water soluble components of Salvia miltiorrhiza can interference tumor angiogenesis by affecting AT1R of tumor cells and to further elucidate molecular mechanism of Danshen applied to treatment of multiple disease.[Research contents]Liver cancer,a high degree of malignancy tumor,was used to analysis whether water soluble components of Salvia miltiorrhiza can affect tumor blood vessels by regulaing expression of ATIR protein in SMMC-7721cells in this paper.The first chapter we examined the correlation among AT1R protein and VEGFA and MVD (microvessel density) in clinical HCC samples.The second chapter we screened the effective components from water-soluble of Salvia miltiorrhiza which could influence AngⅡ-AT1R axis.Using a mouse sponge implant model to screen four kinds of water-soluble components of Salvia miltiorrhiza and we preliminarily confirmed the effective component through the detection of hemoglobin in the sponge,VEGFA concentration in mouse blood serum and vessel count by HE staining and immunohistochemistry.Then we further confirmed the effect and mechanism of this component through in-vivo and in-vitro experiments.The third chapter we examined the role of the effective component in liver cancer angiogenesis.We screened liver cancer cell line which ATIR highly express using Western bloting and immunofluorescence technique.MTS method was used to investigate the effect of effective components on the proliferation of hepatocellular carcinoma cell induced by Angll.WB and ELISA method were used to detect the effect of effective components on the concentration of VEGFA and ATIR protein expression of hepatocellular carcinoma cells induced by Angll.We examined the effects of active components on the volume and angiogenesis of SMMC7721transplant tumor in nude mice.[Research results]The first chapter we examined40cases of HCC samples by immunohistochemistry and found that expression of AT1R were positively correlated with expression of VEGFA and MVD in hepatocellular carcinoma tissues,indicating that ATIR is one of the most important factors in liver cancer angiogenesis.The second chapter,we found that just only salvianolic acid B2mg/Kg among4water-soluble components can effectively inhibit angiogenesis induced by Ang Ⅱ.Immunohistochemistry sresults howed that the salvianolic acid B can downregulate expression of VEGFA and ATIR in vascular endothelial.To further confirm the role of salvianolic acid B and found that salvianolic acid B had no significant effect on the proliferation of the primary human umbilical vein endothelial cells (HUVEC) induced by AngII,but it can effectively inhibit migration and tube formation of HUVEC induced by AngⅡ.WB experimental results showed that salvianolic acid B could express ATIR protein dose dependently downregulated the Ang II induced HUVEC.ELISA assy showed that salvianolic acid B can dose-dependendly reduce VEGFA concentration in cell supernatant. We further confirmed the effect of salvianolic acid B using the zebrafish angiogenesis model, experiments showed that salvianolic acid B can effectively inhibit the zebrafish angiogenesis induced by Ang II. The third chapter we screened the hepatocellular carcinoma cell lines which ATIR was high expression and the highest expression of two:SMMC7721and HepG2.We found that salvianolic acid B had no significant effect on the proliferation of the two hepatoma cells induced by AngⅡ,but it can dose-dependently downregulated expression ATIR and VEGFA in cell supernatant.Finally nude mice experiments showed that salvianolic acid B can effectively inhibit the growth of SMMC7721transplanted tumor,but in-vitro experiments confirmed that it had no significant effect on the proliferation of hepatoma cells,then whether it can affect angiogenesis of licer cancer?By means of immunohistochemical experiments we found that salvianolic acid B could dose-dependently downregulated the expression of AT1R protein and VEGFA in transplanted tumor, while reducing the expression of CD34in hepatocellular carcinoma.[Conclusion and significance]The experimental results show that the water-soluble component of Danshen salvianolic acid B reduced VEGF autocrine of vascular endothelial cells by downregulating the expression of ATIR protein in vascular endothelial cells,thereby inhibiting the migration and tube formation induced by AngⅡ in endothelial cell.In HCC, salvianolic acid B reduced the VEGF secretion of liver cancer cell by downregulating the expression of AT1R in hepatoma cells,thus inhibition of angiogenesis in hepatocellular carcinoma.The significance of this study:①To provide a basis for understanding the molecular mechanism of Danshen multi-component multi target for the treatment of diseases;②To reveal the important factors of hepatocellular carcinoma angiogenesis and provide the reference for the clinical treatment of liver cancer. |
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