Study On The Anti-angiogenic Activity Of Tripterygium Wilfordii Based On The Zebrafish Mode

Objectives:Rhizoma Sparganii,a traditional Chinese medicine can break the blood Qi and relieve swelling or pain,is the dried rhizome of Sparganium stoloniferum Buch.-Ham.(SsBH).It is used on treatment of blood stasis,amenorrhea,functional dysperpsia and cancers.Based on background researches,SsBH has anti-angiogenic activity,but its active material basis has not yet been clarified,and mostly gathered in the physiological anti-angiogenic activity.There are four aims in this study.First,this extracts with anti-angiogenic activity was picked out among different polar extracts of SsBH using a fluorescent transgenic zebrafish model.Second,the entried components in zebrafish larvae of SsBH were analyzed using UPLC/Q-TOF-MSE techniques and speculated its anti-angiogenic active components.Third,the zebrafish patientderived tumor xenograft(PDTX)was used to investigate the effects of SsBH on tumor angiogenesis,and to determine the anti-tumor angiogenesis of SsBH in vivo.Last,the proteomics technique was used to analyze the change of protein expression before and after SsBH administration,and its anti-tumor angiogenesis mechanism was initially determined.Methods:1)SsBH were sequential extracted with petroleum ether,dichloromethane,ethyl acetate,methanol,and water.The anti-angiogenic activity of separate extracts was assessed by the number of intersegmental blood vessels in Tg(flkl:mCherry)zebrafish model.We then selected the most active extract for further study.2)UPLC/Q-TOF-MSE was adopted to make a full component characterization on SsBH active extract.Transgenic zebrafishes used for the physiological angiogenesis model were taken as the object of study to be treated in the blank group and the SsBH group for 72h,respectively.After the zebrafish larvae were processed with a high-pressure homogenizer,UPLC/Q-TOF-MSE was used to analyze the entered components of SsBH,and thereby to rapidly screen out the anti-angiogenic components from SsBH.3)The zebrafish patient-derived tumor xenograft(PDTX)was used as a pathological angiogenesis model.The 2dpf zebrafish juveniles were selected and the Bel7402 human hepatoma cells were injected into the center of yolk sac.The IOD values of green fluorescence-labeled tumor and red fluorescence-labeled angiogenesis in the zebrafish before and after administration were calculated using Image Pro Plus software.Then,we used the gross of tumor and vessels near the tumor in the yolk sac to evaluate the effect of SsBH on tumor angiogenesis.4)Taking advantage of TMT quantitative proteomics technique,we compare the protein expression profiles of the PDTX and SsBH administration groups.Then we screened the differentially expressed proteins(DEPs)among the treatments and performed a Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the DEPs.Results:1)At the same crude drug dosage,the ethyl acetate extract,methanol extract and water extract all had significant anti-angiogenic effects.The anti-angiogenic activity of methanol extract and water extract was significantly higher than ethyl acetate extract.Compared with MNLC(maximum non-lethal concentration),the anti-angiogenic activity of the methanol extract and water extract was significantly higher than the petroleum ether extract,dichloromethane extract and ethyl acetate extract,and the methanol extract and water extract had no difference in activity.Then,we mixed the methanol extract and water extract at the same crude drug dosage,and compared the difference between the anti-angiogenic activity of the mixture and the separate administration.The results showed that the anti-angiogenic activity of the mixture of methanol and water extract was significantly better than two separate extracts.Therefore,the following study used the mixture of methanol and water extract mixture(SSMW)as the study subject.2)By analyzing the mass spectrometry results of SSMW,42 chemical components were inferred in SsBH.Through the comparison of mass spectrum between the blank group and the SSMW group,a total of 6 entered components were founded.3)In low,medium and high concentration solution of SSMW,the gross of yolk sac tumors and blood vessels were significantly decrease.It was shown that SSMW can inhibit the growth of tumor and tumor blood vessels in PDTX with a concentration-dependent manner.4)The analysis of DEPs showed that there were 554 differential proteins between the model and SSMW treatment,and 37 up-regulated proteins and 17 down-regulated proteins in SSMW treatment were detected.According to GO and KEGG analysis,these genes were enriched in Adrenergic signaling in cardiomyocytes and MAPK signaling pathway.Conclusions:In this study,we use in vivo zebrafish model to screen the anti-angiogenic activity of different polar solvent extracts.The result showed that compare with the SSME and SSW extract,SSMW had the most inhibitory effect on physiological angiogenesis.By analyzing the mass spectrometry results of SSMW,42 chemical componentswere inferred in SsBH.Subsequently,we discovered 5 metabolites of Acyl compounds of sucrose and 1 prototype constitute(1-O-feruloyl-3-O-p-coumarylglycerol)in zebrafish’ s biology samples.The tumor xenograft model was used as a pathological angiogenesis model to study the antitumor angiogenesis and anti-angiogenesis of SSMW.The results showed that SSMW had significant inhibition on the growth of tumor and vessels near the tumor in yolk sac in zebrafish.It indicated that SSMW indeed has anti-tumor and anti-tumor angiogenic activity in vivo.The Proteomics experiment demonstrates that SsBH can exert anti-tumor angiogenesis and anti-tumor activity by modulating Gs-AC-cAMP-PKA signaling pathway and calcium channel in Adrenergic signaling in cardiomyocytes and MAPK/ERK in MAPK signaling pathway.