Studying The Regulating Effect Of WCWE And USRWE On Zebrafish's COX2-MMPs And ANGPT-TIE Signaling Pathway

Background and objective:It is valuable to research the traditional Chinese medicine, especially in how to inhibite the angiogenesis of tumor tissue. Finding the medicine inhibiting the angiogenesis of tumor tissue and elucidating the mechanisms become a new direction of new drug research. As the representative medicine of clearring away the heat and toxins, Wild Chrysanthemum and Uniflower Swisscentaury Root are Widely used in the clinical anti-tumor therapy, and many clinical and experimental experience about these two medicine have been got in the anti-tumor therapy. However, there are a few researchs about the mechanisms of inhibiting the angiogenesis of tumor tissue with these two medicine. In most experiments, the cancer cells in vitro and the rats are used as biological research model, which cannot reflect the angiogenesis inhibiting effect of these two medicine directly.The blood vessels of transgenic zebrafish with fluorescent tags can image more clearly, which is advantageous to count ISVs and visually analyse the effect of inhibiting the angiogenesis. Choosing the TG transgenic flila-EGFP zebrafish as biological research model, Water Extracts of Wild Chrysanthemum and Uniflower Swisscentaury Root are evaluated the angiogenesis inhibiting effect directly. Using realtime-PCR to analyse the differences of COX2, MMPs and ANGPT-TIE signaling genes transcript levels in zebrafish embryos treated with these two kinds of chinese traditional medicine compared to blank control and positive control, we can evaluate the relevant mechanisms of inhibiting the angiogenesis.Methods:1.Using High performance liquid chromatography (HPLC) to determine the fingerprints of Wild Chrysanthemum and Uniflower Swisscentaury Root, in order to provide an effective reference and repeatability for the experiments afterwards.2. Choosing the transgenic flila-EGFP zebrafish as biological research model.180 Zebrafish embryos were randomly divided into four groups:blank control group (0.1% DMSO), positive control group (5 ?g/ml PTK787), WCWE(Wild Chrysanthemum Water Extracts)group and USRWE(Uniflower Swisscentaury Root Water Extracts) group, and each group had 30 embryos. The two TCM water extracts which were diluted in 0.1% dimethyl sulfoxide (DMSO), were added to each well at a concentration of 200 ?g/ml.22 hour post-fertilization (hpf) embryos were distributed into 12-well plates for a treatment period of 26 hours. At 48hpf embryos were tricaine anesthetized and imaged by Fluorescence microscope to analyse the differences of ISVs in each group.3.72 Zebrafish embryos were randomly divided into four groups:blank control group (0.1% DMSO), positive control group (5?g/ml PTK787), WCWE(Wild Chrysanthemum Water Extracts)group and USRWE(Uniflower Swisscentaury Root Water Extracts) group, and each group had 18 embryos. The two TCM water extracts which were diluted in 0.1% dimethyl sulfoxide (DMSO), were added to each well at a concentration of 200 u g/m1.22 hour post-fertilization (hpf)embryos were distributed into 12-well plates for a treatment period of 26 hours. At 48hpf embryos were made into Homogenate. Using realtime-PCR to analyse the differences of COX2?MMP9?MMP2 genes transcript levels in zebrafish embryos treated with these four kinds of chinese traditional medicine compared to blank control and positive control.4.96 Zebrafish embryos were randomly divided into four groups:blank control group (0.1% DMSO), positive control group (5?g/ml PTK787) WCWE(Wild Chrysanthemum Water Extracts)group and USRWE(Uniflower Swisscentaury Root Water Extracts) group, and each group had 24 embryos. The two TCM water extracts which were diluted in 0.1% dimethyl sulfoxide (DMSO), were added to each well at a concentration of 200 u g/ml.22 hour post-fertilization (hpf)embryos were distributed into 12-well plates for a treatment period of 26 hours. At 48hpf embryos were made into Homogenate. Using realtime-PCR to analyse the differences of ANGPT1?ANGPT2b?TIE1?TIE2 genes transcript levels in zebrafish embryos treated with these four kinds of chinese traditional medicine compared to blank control and positive control.Results:1. There were 7 common peaks among Wild Chrysanthemum, and the peak3 was similar to Chlorogenic acid with the similarity of greater than 0.90, the peak4 was similar to Luteol in-7-O-glucoside with the similarity of greater than 0.90. There were 7 common peaks among Uniflower Swisscentaury Root, and the peak6 was similar to ecdysterone with the similarity of greater than 0.90.2. The ISVs of Zebrafish embryos in blank control group displayed intactly and clearly. Compared to the blank control group, The ISVs and DLAV of Zebrafish embryos in positive control group disappeared intactly. Embryos treated with these two kinds of TCM present a lower number of complete ISVs and DLAV.3. Compared to the blank control group, COX2 gene was significantly reduced in these two kinds of TCM groups(P<0.01). However, MMP2 and MMP9 genes was reduced just in WCWE groups (P<0.05).4. Compared to the blank control group, ANGPT1 gene was not alterant in these two kinds of TCM groups (P>0.05). However, TIE1 gene was just reduced in USRWE group(P<0.05). The data of ANGPT2b and TIE2 was given fall into disuse because of the few genes transcript levels.Conclusion:1.WCWE and USRWE have anti-angiogenic effects in the transgenic flila-EGFP zebrafish embryos.2. WCWE downregulated the expression of COX2, MMP9 and MMP2 gene, which could be the mechanisms of inhibiting the angiogenesis of transgenic zebrafish embryos.3. USRWE upregulated the expression of TIE1 gene, which could be the mechanisms of inhibiting the angiogenesis of transgenic zebrafish embryos.