Screening Of Active Ingredient Of Anti-angiogenic Chinese Medicine And Studying Its Mechanism In Transgenic Zebrafish

Research background:With the social and economic development, improvement of living levels, as a result of changes of disease spectrum, the burden of infectious diseases relatively ease, and yet chronic non-infectious diseases and injuries including neoplasm are becoming a major portion of disease burden little by little between developed and developing countries. In the light of information provided by the Center of Tumor Registry in China, the result indicates that the morbidity and mortality of cancer in China is increasing year by year and malignancy was at the top of the list or a serious threat to human health.At present, surgery, chemotherapy and radiotherapy are clinically basic methods for remedy of cancer, however, because of its side-effects, efficacy and prognosis are always so poor. Consequently, research and development of drugs in relation to cure and precaution of cancer is alresdy facing serious challenge in the current medical profession.In recent years, in the wake of intensive study about tumor, it is found that the growth of primary tumor and metastasis of tumor cells are dependent on ingrowth of new blood vessels to provide adequate nutrition for its development. New blood vessels of tumor is formed by angiogenesis, if angiogenesis of tumor tissue could be inhibited and the supply of nutrition of cancer is cut off, it is bound to achieve the aim of inhibiting the growth and metastasis of tumor, thus,"anti-angiogenic therapy" and "hunger-cure" will be proposed soon as a new approach for the treatment of cancer. Along this line of thought, some studies in vitro show that suppressing the formation of new blood vessels of tumor can gain the goal of inhibition of proliferation of tumor cells.bevacizumab of anti-angiogenic medicine approved by FDA for application in2004was applied to colorectal cancer and can prolong the lifetime of patients. Therefore, these studies have confirmed that anti-angiogenic therapy is scientific and effective for the treatment of cancer.Angiogenesis is a complicated process controlled by a lot of growth factors, one of which called VEGF is deemed to the strongest action and highest specificity to this day. It can promote tumor vascular endothelial cell proliferation and migration as well as increase vascular permeability to achieve tumor angiogenesis. Taking VEGF and its receptor signaling pathway as a target or blocking VEGF-induced tumor vessel can inhibit tumor growth and metastasis.As a result, VEGF and its receptors is basic target for study of the mechanism of action of angiogenesis inhibitors.There has thousand years old for TCM and Chinese medicine applied for the treatment of diseases including cancer, accumulating rich clinical experience. With ample natural chinese herbal medicine resources in our motherland.Thus, searching for safe and effective tumor angiogenesis inhibitors from natural medicine in particular TCM is a major issue in the field of research and development of new drugs.In recent years,zebrafish has been gradually applied in screening anti angiogenic drugs,because of its biological characteristics of breeding and reproduction easily, embryonic development rapidly.Concretely,it has the following advantages:First, zebrafish assays will allow high throughput screening of compounds,operation is simple;second, short screening cycle, its rapid development leads to get the test results24-72hours;third, its embryos and larvae are transparent which means organs, cells and tissues are visualized in vivo and investigated in realtime;forth, Small amount of sample, and live animal experiments;fifth, Compounds are easily added to the water, absorbed and ingested once embryos are able to swallow or can be directly injected into the embryos;sixth, a high degree of conservation exists between the human and zebrafish genomes;seventh, low cost, its breeding, reproduction cost far less than other laboratory animals.In recent years, studies have showed that:ginsenoside Rg3, curcumin, norcantharidin, hydroxysafflor yellow-A, artesunate, indirubin have anti-angiogenic role. These active ingredients of chinese medicine have anti-angiogenic role in other models except zebrafish, it is considered that the occurrence of anti-angiogenic effect of these active ingredients is higher probability in zebrafish, Thus, six active ingredients of chinese medicine are selected for screening. In this study, transgenic zebrafish embryo is used as animal model, Stereo Fluorescence Microscope is utilized for observation of inhibitory effect of angiogenesis in zebrafish somite vessels treated with active ingredient of traditional chinese medicine, the anti-angiogenic mechanism is investigated from the viewpoint of molecular biology; meanwhile, it is provided for experimental evidence about the feasibility and applicability of screening of active ingredient of anti-angiogenic traditional chinese medicine in zebrafish.Experimental methods:1.Transgenic zebrafish (tgflil:eGFP) is used as experimental animal. The day before screening, homozygous transgenic zebrafish(tgflil:eGFP) and Wild type zebrafish(AB)are placed on the mating cartridge.Tomorrow morning,coupled zebrafish stimulated by the light will lead to spawning, embryos in half an hour are collected in a petri dish containing embryo culture medium,and then placed in a28.5℃incubator.To ten hours post fertilization1Ohpf),embrys are transferred into24-well plates containing embryo culture medium,each hole has a capacity of ten embrys. Prior to12hpf, Configured liquid drag is added to24-well plates successively. By means of liquid doubling dilution,every liquid drug will be diluted to three concentration gradient. Initially,effective drag which may inhibit angioge-nesis of somite vessels in zebrafish embryos is supposed to be chosen for father experiment,then paralleling five holes with the same concentration repeatedly.Meanwhile, positive control group(SU5416) and control group should be designed in the same culture plate. Active ingredient of Chinese medicine is dissolved with dimethyl sulfoxide (DMSO).Zebrafish embryos(48hpf) are observed by StereoFluorescence Microscope (OLYMPUS MVX10). Effective anti-angiogenic drug ought to be in accord with the following index:compared with zebrafish(AB) embryos contemporarily,morphological development is normal on the whole;the fluorescence intensity of flil gene expression is weakened,disappeared and fluorescence expressed in somite vessel is disorganized.2. Exploring the mechanism of anti-angiogenesis against effective drug. First of all, embryos(48hpf) are collected(method such as before) and divided into treatment group and control group. The expression of vascular endothelial growth factor receptor2(VEGFR2) was qualitatively detected by whole mount in situ hybridization(WISH).Related results are acquired by StereoFluorescence Microscope. Then embryos (48hpf) are collected(method such as before) and divided into treatment group and control group. The expression regularity of VEGFR2was quantitatively detected by RT-PCR assay. PCR products were detected by using a2%agarose gel electrophoresis, image was captured by gel image system, concrete data was obtained by using Band Scan5.0software.3.The results are expressed by mean±tandard deviation(X±S). SPSS13.0was applied into One-way ANOVA for mean comparison based on multiple samples.When the variance was not homogeneous,taking the Welch test and Brown-the Forsy test to adjust, then using Dunnet’ts T3and Dunnet’ts C.The value of P<0.01would mean significant difference.Major findings:1. Screening results of the active ingredients of traditional Chinese medicine (1) Ginsenoside Rg3and curcumin play no significant role in inhibiting angiogenesis.The concentration of Ginsenoside Rg3was between0.02～0.16μg/μl, all of embryos were death at24hpf. The density of curcumin from0.016μg/μl to0.064μg/μl would lead80%embryos to death at36hpf. However, both of them have no anti-angiogenesis function while less than death concentration.(2) Inhibitory role of norcantharidin in zebrafish was hardly any. Lethal concentration of norcantharidin must be more than0.025μg/μl.(3) The concentration from8μg/μl～10μg/μl, hydroxysafflor has not yet resulted in death, most of them give rise to spinal curvature. Repeat the experiment to be sure:only one fish vessel was clearly when observed,but inhibition of angiogenesis is unobvious.(4) Artesunate has slightly fuction in anti-angiogenesis, but no statistically significant. Beyond0.5μg/μl, all of embryos would be death at28hpf. Blow0.1μg/μl, influencing development of embryos.(5)When the density of indirubin is100μM, zebrafish embryos arise malformation or death.15μM,30μM,90μM, inhibintion of vessel by indirubin is in a dose-dependent manner, and its development is normal.2. Observation of anti-angiogenesis in zebrafish somite blood vessels treated with artesunate Compared with blank control group, DMSO group would mean no difference (P>0.05), Compared with blank control group, among the three groups dealt with artesunate exists difference but no significant (p<0.01) and does not show in a dose-dependent way.3.Observation of anti-angiogenesis in zebrafish somite blood vessels treated with indirubin Compared with blank control group, DMSO group would mean no difference (P>0.05), among the three groups dealt with indirubin is statistically significant (p<0.01)and in a dose-dependent way.4. Influence of VEGFR2gene expression in tgflil:eGFP zebrafish dealt with artesunate Compared with the blank control group,gene signal of VEGFR2reduced but not obviously; WISH and semi-quantitative RT-PCR showed that:Compared with the blank control group, low concentration group of artesunate is not statistically significant (p>0.05), middle^high concentration group of artesunate is statistically significant (p<0.01).5.1nfluence of VEGFR2gene expression in tgflil:eGFP zebrafish dealt with indirubin Compared with the blank control group,gene signal of VEGFR2reduced obviously. WISH and semi-quantitative RT-PCR showed that indirubin reduced the mRNA expression of VEGFR2in a dose-dependently way (p<0.01).Conclusion:1.The characteristics of small size,transparency,external fertilization and high fecundity during embryogenesis allow for easy manipulation and observation of developmental processes. Transgenic zebrafish tgflil:eGFP which has been used in our study that expresses green fluorescent protein (GFP) under the control of flil promoter in endothelial cell, blood vessels influenced by drug can be clearly observed under fluorescence microscope and quantitatively,fastly analyzed.Studies show that compared with human umbilical vein endothelial cell, chicken embryo allantoic membrane model. Zebrafish is well-suited for anti-angiogenic drug screening.2.The results about anti-angiogenesis affected by six drugs point out a fact that in addition to artesunate and indirubin.the remaining drugs have no obvious inhibition of angiogenesis and can lead to toxicity, malformation and developmental delay. Significant inhibition of angiogenesis can be observed in zebrafish embryos (48hpf) treated with indirubin,yet the concentration of which is more than100μM results teratogenicity and death equally.3. Compared with blank control group, artesunate and indirubin groups can inhibit angiogenesis in zebrafish embryo somite vessels,the latter will be in a dose-dependent manner.4. The result of WISH,RT-PCR show that gene signal of VEGFR2reduced. WISH and RT-PCR showed that artesunate reduced the mRNA expression of VEGFR2slightly, and did not show obviously in a dose-dependently manner. RT-PCR showed that indirubin reduced the mRNA expression of VEGFR2obviously in a dose-dependently manner, indirubin could inhibit angiogenesis of intersegmental vessels of the zebrafish embryos through inhibition of the phosphorylation of certain protein in the VEGF/VEGFR2pathway, which results in down-regulation of VEGFR2mRNA expressionIn conclusion,transgenic zebrafish has been taken as test animal in our study.Through observation of vascular fluorescence under a stereo fluorescence microscope and analysis of VEGFR2gene expression, it is found that in addition to indirubin and artesunate, the remaining drugs have no obvious inhibition of angiogenesis. The role of artesunate dose not show in a dose-dependent manner. The role of indirubin is present in a dose-dependent manner. With the increase of drug concentration, the expression of VEGFR2mRNA reduced, it is possible that indirubin could inhibit angiogenesis of intersegmental vessels of the zebrafish embryos through inhibition of the phosphorylation of certain protein in the VEGF/VEGFR2pathway, which results in down-regulation of VEGFR2mRNA expression.