| Objective To enrich the effective components of Huaier alcohol extract(TMTF),to explore the inhibitory effect of Huaier alcohol extract on metastasis of gastric cancer,and to focus on the mechanism of regulating EMT-related signal pathway of integrin β5 in combination with sirengil peptide.The drug toxicity and anti-tumor metastasis effect of Huaier alcohol extract were evaluated in vitro and in vivo.To provide a theoretical basis for the anti-tumor principle of traditional Chinese medicine combined with integrin inhibitors.Methods 1.Purification experiment of Huaier rude alcohol extract:total flavonoids were enriched by column chromatography and ultrasonic-ethanol extraction,and the content of total flavonoids was determined by NaNO2-Al(NO3)3 with rutin as the standard.2.Acute toxicity evaluation test:the wild type AB zebrafish was given 25μg/mL TMTF at one time.On the third day,the reaction of various organs of zebrafish was observed to determine the incidence of toxicity.3.Growth and metastasis inhibition experiment of gastric cancer:CM-Dil labeled human gastric cancer cells were microinjected into the yolk sac of zebrafish 2dpf normal wild type AB strain,and the drug was administered in the same acute toxicity evaluation test.The inhibitory effect of TMTF on the growth and metastasis of gastric cancer metastatic cells was evaluated by fluorescence intensity at 5th dpf.4.Cell clone formation,proliferation,migration and invasion experiment:with the help of CCK8 reagent,scratch plug-in and cell chamber,the size and number of cell clones,cell proliferation rate,migration distance and the number of cells penetrating the membrane of gastric cancer cells treated with sophora alcohol extract were observed,and the effect of the drug was evaluated.5.Western blotting experiment:after cleavage,the surviving cells under the action of Huaier alcohol extract and other drugs were collected,and the differences in the expression of integrin β5 and other proteins were confirmed by electrophoresis,membrane transfer and incubation of antibodies,and the drug action targets were determined.Results 1.The content of total flavonoids in the purified Huaier alcohol extract was more than 50%.2.TMTF showed no obvious toxicity of target organs,and the main toxic phenotype was developmental effects such as delayed absorption of yolk sac.3.TMTF mainly inhibited the metastasis of gastric cancer cells,but had no obvious inhibition on the growth of gastric cancer cells.4.High expression of integrin β5 promotes cell proliferation,migration and invasion.TMTF and Cilengitide can effectively inhibit its effect.5.TMTF inhibited the expression of integrin β5,ILK,FAK and EMT-related proteins,and the inhibitory effect was enhanced by the combination of TMTF and Cilengitide.Conclusion TMTF is mainly composed of total flavonoids and has advantages in anti-metastasis and low toxicity of gastric cancer cells.TMTF can inhibit the expression of integrin β5,ILK,FAK phosphorylation and EMT related proteins,and then inhibit the process of EMT.When TMTF combined with Cilengitide,the effect of blocking the metastasis of gastric cancer cells was more significant. |
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