| Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. And it is a normal and vital process in growth and development, such as:physiological angiogenesis plays an important role in embryogenesis, corpus luteum formation and wound healing, and in contrast, diabetes, proliferative retinopathy, atherosclerosis, psoriasis, rheumatoid arthritis, cancer and other diseases are associated with pathological angiogenesis. In particular, the increase of tumor volume and tumor cell migration depend on angiogenesis, so inhibition of tumor angiogenesis has become a hotspot of Anticancer Research, and the anti-angiogenic therapy has become a new strategy for cancer treatment.Studies showed that flavonoids may affect the activity of a variety of enzymes. It plays an important role in antibacteria, anti-inflammation, anti-mutation, decreasing blood pressure, detoxification and sedation. It is also exhibit prominent effect on antioxidation, anti-cancer and anti-angiogenesis etc. In this study, Kushecarpin D (KD), a new flavonoid isolated from the root of Sophora flavescens, was evaluated to its antiagiogenesis effect on the cultured human endothelial-like cell line (ECV304) as a model in vitro.There are two parts in this thesis:antiangiogenesis evalutation in vitro and the related mechanism. In the first part, SRB staining method and trypan blue exclusion assay were used to evaluate the effect of KD on cell proliferation and cytotoxicity, and cell adhesion, cell migration, tube-like structure formation were used to detect the antiangiogenesis effects. In the second part, flow cytometry was used to detect cell cycle, and DCFH-DA was used to detect the reactive oxygen species (ROS) generation in ECV304 cells treated with KD. Catalase activity and apoptosis were also checked with ammonium molybdate colorimetric assay and Hoechst 33258 staining method. The results are as follows:1. KD inhibited angiogenesis After treated with KD under concentrations of 10,20,30,40 and 50μg/ml for 12h, cell proliferation was apparently inhibited. However, it seemed that the inhibition rate has reached the maximum value when treated with 30μg/ml, and almost no increasing when treated with 40 and 50μg/ml. The results of cytotoxicity detected with Trypan blue exclusion assay indicated that cell proliferation inhibiting effect of KD was not through cytotoxicity mechanism, for no changing of cell viability was detected. The effect on cell migration was found after the treatment of 40μg/ml. cell adhesion was inhibited potently and under dose-dependent manner after treated with KD under the concentrations of 20,30,40 and 50μg/ml. Tube-like structure formation results also followed dose-depentdent manner and 10μg/ml has already exhibited the potent inhibition effect. In conclusion, our results indicated that KD exhibited the antiagiogenesis effect in vitro.2. The antiangiogenesis effect of KD may be related to cell cycle arrest at G2/M phase and its antioxidative functionCell cycle was arrested at G2/M phase (detected with flow cytometry) after treated with 40 and 50μg/ml of KD, but no apoptosis was found through Hochest 33258 staining method and flow cytometry. Intracellular H2O2 concentration decreased significantly after treated with KD, this was coincidence with the inhibition action of KD to the angiogenesis. So we postulated that the antiangiogenesis effect of KD may relate to its effect on cell cycle arrest and intracellular H2O2 inhibition. |
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