| Background and purposeThe morbidity and mortality of cancer are increasing year after year.Therefore,the search for new safe,effective and low-cost anti-cancer drugs and treatments is always a hot spot in the medical research field.Since tumor angiogenesis plays an important role in promoting tumor growth and metastasis,targeting tumor angiogenesis has become one of the main anti-tumor treatments.However,in actual clinical application,it has been found that all kinds of anti-angiogenesis drugs are easy to induce drug resistance,have limited efficacy,different degrees of toxic and side effects,and high economic cost.Increasing studies have indicated that endothelial cell metabolism is also the key to angiogenesis,and tumor angiogenesis may be effectively inhibited just by down-regulation of various metabolic pathways of endothelial cells.In Traditional Chinese medicine,blood stasis is considered to be one of the main pathogeneses inducing the occurrence and development of tumors.Many studies have shown that herbs of boosting blood circulation and defusing blood stasis have the effect of anti-tumor angiogenesis,and Carthamus tinctorius is a typical representative of them.However,there are only a few researches on the anti-tumor and anti-angiogenesis effects of Carthamus tinctorius decoction or water extract,without any necessary quality control for the experimental drugs used.As a new model organism,zebrafish has the advantages of large spawning capacity,rapid embryonic development,transparent embryo,and high homology with human genome,so it is especially suitable for the study of TCM anti-angiogenesis.The development of transcriptome technology provides a new research direction for the study of the mechanism of TCM drugs,as its comprehensive detection and analysis approaches are consistent with the characteristics of multi-target and multi-level effects of TCM.In this study,Carthamus tinctorius water extract(CTWE)that had been verified by ultra-high performance liquid chromatography fingerprint detection was used as the experimental drug,and zebrafish embryos were used as the experimental animal.The anti-angiogenesis effect of CTWE on zebrafish embryos was observed.Then,high flux m RNA sequencing and bioinformatics analysis were carried out to study the potential mechanism.At last,q PCR and PRM were used to verify the sequencing results and the potential mechanism at the transcriptome and proteomic levels,respectively.This study aims to provide the phenotype evidence of zebrafish embryo angiogenesis and some molecular biology research basis for the further study of the anti-angiogenesis effect of Carthamus tinctorius and its mechanisms.The results of this study may also provide some experimental research support for the clinical application of Carthamus tinctorius in the TCM treatment of cancer.Methods1.The CTWE was prepared by traditional decoction,concentration,and drying.Its fingerprint was detected by ultra-high performance liquid chromatography.2.CTWE was used to intervene in the critical period of blood vessel development of Tg(fl1a:EGFP)transgenic zebrafish embryos,and then the statement of blood vessel development of zebrafish embryos was observed by microscopes.3.High flux m RNA sequencing was performed on zebrafish embryos of experimental group with angiogenesis inhibitory phenotype after CTWE intervention and blank control group.The transcriptional changes of protein coding genes of zebrafish embryos were comprehensively detected,and bioinformatics analysis was carried out on the sequencing results.4.Differentially expressed genes cpt1 b,hadhaa,and acaa2 of zebrafish embryos encoding key catalytic enzymes in the fatty acid ?-oxidation pathway were selected as the subjects of further experiments.Their expression levels were determined by q PCR technology in order to verify the sequencing results and the mechanism at the transcriptome level.5.PRM technology was applied to detect the expression levels of proteins encoded by gene cpt1 b and hadhaa of zebrafish embryos in the fatty acid ?-oxidation pathway,so as to further verify the sequencing results and the mechanism at the proteomics level.Results1.The CTWE powder was 68.23 g,the paste yield rate was 34.12%,and the water content was less than 5%.The CTWE fingerprint showed characteristic peaks corresponding to all the standard reference substances,each peak with a similarity of no less than 0.95 comparing to its reference substance peak,indicating that the quality of CTWE was qualified and stable.2.The morphology of zebrafish embryos after CTWE intervention was normal,but the intersegmental vessels(ISVs)were poorly developed,and the dorsal longitudinal anastomotic vessel(DLAV)was not developed.At the same time,there was no teratogenic or lethal effect on zebrafish embryos of CTWE at 200?g/ml and 26 hours of intervention time.3.Compared with the blank control group,in the experimental group with angiogenesis inhibitory phenotype after CTWE intervention,888 differentially expressed genes of zebrafish embryos were significantly up-regulated and 965 ones were significantly down-regulated.Bioinformatics analysis indicated that the most likely mechanism of CTWE inhibiting angiogenesis of zebrafish embryos was the down-regulation of fatty acid ?-oxidation pathway of vascular endothelial cells.4.qPCR confirmed that CTWE down-regulated the expression of gene cpt1 b and hadhaa which encode key catalytic enzymes in the fatty acid ?-oxidation pathway of zebrafish embryos.5.PRM confirmed that CTWE could down-regulate the expression of related proteins encoded by gene cpt1 b and hadhaa in the fatty acid ?-oxidation pathway of zebrafish embryos.ConclusionCTWE significantly inhibited angiogenesis in zebrafish embryos.It downregulated the expression of gene cpt1 b and hadhaa of zebrafish embryos,and thus down-regulated the fatty acid ?-oxidation pathway.The mechanism of CTWE inhibiting angiogenesis of zebrafish embryos may be by down-regulating the fatty acid?-oxidation pathway of vascular endothelial cells. |
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