| Research background and purpose:The morbidity and mortality of malignant tumors are increasing yearly,causing a great medical burden.Tumor growth and metastasis depend on the formation of new blood vessels.Due to the rapid development of molecular medicine,targeted drugs have made great progress in anti-tumor angiogenesis.However,due to the multiple compensation mechanisms of vascular remodeling in tumors,traditional targeted anti-angiogenic drugs have limited therapeutic effects and are prone to drug resistance.Vascular endothelial cell metabolism is similar to that of tumor cells,for which glycolysis is the main mode of energy supply.Therefore,drugs targeting anti-tumor angiogenesis based on tumor vascular endothelial metabolism have become a new anti-tumor angiogenesis strategy.According to TCM,the basic pathogenesis of caner is a tangible mass formed by the accumulation of pathological products of deficiency of vital energy,phlegm,blood stasis and cancer toxin.After the tumor is formed,it is agglomerated into pieces and hard as a rock.Therefore,some doctors call it "rock" and put forward the treatment of softening and resolving hard mass.Traditional Chinese medicine(TCM)has played an important role in the treatment of malignant tumors.A large number of studies have confirmed that TCM has a significant anti-tumor angiogenesis effect,but the specific molecular mechanism needs to be further explored.High-throughput DNA gene sequencing technology has been widely used in the field of molecular biomedicine because of its high accuracy and high sensitivity.Zebrafish,as an emerging biological research model,has the characteristics of high-throughput and in vivo experiments.Moreover,during the early embryonic development,the embryo is transparent and can directly observe various tissues and organs,which has been widely used in the screening of anti-vascular drugs.In this study,RNA-seq sequencing technology was used to systematically and deeply study on turtle carapace,a traditional Chinese medicine with the effect of softening and resolving hard mass,so as to provide a certain research basis for the mechanism of turtle shell inhibiting tumor angiogenesis.Research methods:1.High performance liquid chromatography(HPLC)was used to analyze the chemical components in the TSWE and the fingerprint information was determined.2.Water extract of turtle shell was extracted by decoction,concentrating,sieving and drying.The TG strain flila-EGFP transgenic zebrafish was selected,and the water extract of turtle shell was added to fish water(working concentration 200 m g/ml),and the control group was added with PTK787(working concentration 5 m g/ml).The zebrafish embryos after fertilization 22(22 hpf)were intervened until 48 hpf,after the intervention,the vascular phenotypes of the embryos were observed and photographed under the fluorescence microscope.Then effects of each group on the formation of interstitial blood vessels(ISVs)in zebrafish were analyzed,and the effect of water extract of turtle shell on inhibiting angiogenesis phenotype was evaluated.3.Conduct genomics research on zebrafish with angiogenesis inhibition phenotype,extract RNA from the embryonic tissues of zebrafish with positive phenotype,construct a whole transcriptome library,conduct second-generation high-throughput sequencing,and analyze to determine all the RNA level regulation and expression genes.Comprehensively detect the dynamic changes of the zebrafish gene,explore the information from multiple angles,and conduct bioinformatics analysis on the results of genomics research.4.RNA was extracted from zebrafish embryo tissues after intervention of water extract of turtle shell,and the changes in differentially expressed genes related to ENO3,PFKM and glycolysis pathway were detected by real-time fluorescence quantitative PCR.Research results:1.The common peaks in the fingerprint of turtle shell had the same retention time as the corresponding reference chromatographic peaks,and the similarity was greater than 0.9.2.Compared with the blank control group,the fluorescence expression of ISVs in zebrafish embryos of the experimental group with the intervention of turtle shell water extract partially disappeared,and only a small amount of DLAV could be observed,suggesting that the aqueous extract of turtle shell could inhibit the neovascularization of zebrafish embryos.3.A high-throughput RNA-seq sequencing analysis revealed a total of 3821 differentially expressed genes in zebrafish embryo tissues with a neovascularization phenotype after intervention with aqueous extract of turtle shell.Among them,1781 differentially expressed genes were significantly down-regulated,and 2040 differentially expressed genes were significantly up-regulated,and differentially expressed genes in GO are mainly biological processes,mainly related to metabolism.Analysis of the KEGG pathway showed that up-regulated mRNA involved 10 pathways,down-regulated mRNA involved 32 pathways,and down-regulated glycolysis/gluconeogenesis signaling pathway involved 7 genes were significantly down-regulated.4.Rt-qPCR results showed that ENO3 and PFKM and other differentially expressed genes in the glycolytic pathway were significantly down-regulated,which was consistent with RNA-seq sequencing results.Conclusion:The water extract of turtle shell can inhibit the formation of new blood vessels in the transgenic zebrafish embryo of flila-egfp strain of TG,and its possible mechanism is related to the regulation of glycolysis pathway by down-regulating ENO3 and PFKM. |
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