Preliminary Establishment And Application Of Angiogenesis Intervention Drug Screening And Activity Detection Technology Platform (1)

Objective:Cardiovascular system is the first functional organ system in the embryo development of vertebrates.Over 800 projects on angiogenesis have been approved by NSFC(National Natural Science Foundation of China),with the funding amount of more than 300 million,and many evaluation methods have been established.However,the existing evaluation mode relying on in vitro screening limits the repeatability and reliability while traditional Chinese medicine(TCM)is selected as the research object.Thus,it is urgent to establish efficient and objective methods.This paper used quail embryo model(qCAM),microvascular 3D imaging analysis system,DESI-MSI mass spectrometry,classic vascular endothelial cell experiments,metabonomics and other multidisciplinary methods for mutual corroboration to establish one platform for screening and activity evaluation for angiogenic drugs suitable for TCM,aiming to provide technical support and methodology reference for relevant research.Methods:I Platform establishment.This platform was initially established,and the methodology investigation and research process examples were completed.This platform includes three consecutive evaluation parts:(1)Drug screening preliminarily based on qCAM model(qCAM screening);(2)Activity verification based on HUVEC assays(HUVEC validation);(3)Mechanism research based on omics analysis(omics analysis).1.Methodology investigation.The similarity,repeatability,reliability,applicability and controllability,feasibility,and economy of qCAM model were investigated.The main indexes of qCAM model includes embryo mortality,angiogenesis morphology observation,vascular pathology and ultrastructure,expression of qCAM VEGFR2 mRNA and NO content,and so on,from five levels of whole,tissue,cell,subcellular and molecular to comprehensively characterize the intervention effects of subjects on angiogenesis.(1)Optimization of drug delivery carrier.The effects of gelatin sponge,rice grain,molecular sieve,silica gel,macroporous resin and methylcellulose as drug delivery carriers on the observation indexes of qCAM model were compared.(2)The choice of time for administration.The development of qCAM and the changes of vascular distribution were observed for 7 consecutive days after window opening operation,and the time point of sampling was investigated.(3)Model stability.The results of qCAM microvascular count(third+fourth)were compared under the same experimental conditions in different batches(January,March,May,July,September,and November in 2018).(4)Model repeatability.The effects of DG-15(5,20?g,for 36 h)on the number of microvessels in qCAM was evaluated in a confirmatory way.2.Research process examples.Taking Sorafenib tosylate(ST)as the research object,the establishment and research process of the platform were expounded from experimental design,selection principle,methodology connotation and data analysis.(1)qCAM screening.The effects of ST(40,80 ?g)on the number of micro vessels,EN-face vascular distribution,vascular ultrastructure,VEGFR2 expression and NO content were evaluated by using 40 ?g Dovitinib(Dov)as the positive control.(2)HUVEC validation.The effect of 40 ?M ST treatment for 24 h on the inhibition rate of HUVEC cell activity and the percentage of scratch healing were evaluated by using 2.5 ?M Dov as the positive control.(3)Omics analysis.The main differential metabolites and pathways of 40 ?g ST for 36 h were analyzed by UPLC-QTOF-MS-based non-target metabolism.? Application examples.This platform is used to solve the specific scientific problems of representative research cases,in order to clarify the characteristics and advantages of this platform.1.Application of this platform in the study of TCM.Based on this platform and qCAM model,the property rules of the cold and hot drugs were studied based on angiogenesis,taking 30 kinds of Chinese herbs,crude Curcumae Rhizoma(Curcuma phaeocaulis Val.,Curcuma kwangsiensis S.G.Lee et C.F.Liang,Curcuma wenyujin Y.H.Chen et C.Ling)and vinegar Curcumae Rhizoma(VCR)as research objects,in order to preliminarily reveal the biological connotation of cold and hot property of TCM based on angiogenesis.(1)Literature mining.The CNKI national academy treasure database was used for retrieval of single library ancient books;the retrieval formula was set as "FT=(‘blood vessel'+‘generation')*'cold and hot'";the retrieval type was "full text";the retrieval time was unlimited.The CNKI national academy treasure database was used for retrieval of Cross-database literature;the retrieval formula was set as "SU=(‘blood vessel'+‘generation')*‘cold and hot'";the retrieval type was“subject”;the retrieval time was unlimited.(2)Evaluation using qCAM model.The effects of 30 cold and hot Chinese herbs on the relative vascular area,microvascular number and total vessel number were evaluated by T-test.(3)Platform verification.qCAM screening:The microvessel number,VEGFR2 mRNA and NO content of qCAM were detected after 36 h of administration of 83.33 mg/mL crude Curcumae Rhizoma and VCR decoction;HBMEC validation:The cell activity and ROS levels of HBMEC damaged by 40 ?M t-BHP were detected after 24 h of treatment with 83.33 ?g/?L crude Curcumae Rhizoma and VCR decoction;omics analysis:The main differential metabolites and pathways of 83.33 ?g/?L crude Curcumae Rhizoma and VCR decoction for 36 h were analyzed by GC-MS-based non-targeted metabolomic metabolism.The main differential components were identified by GC-MS-based metabolomics as the quality marker of VCR for promoting angiogenesis.2.Application of this platform in the study of active components of TCM.The effects and mechanisms of tetrahydropalmatine(THP)on angiogenesis were revealed using this platform.(1)qCAM screening.The effects of THP(16,32,64 ?g)on the number of microvessels,EN-face vascular distribution,and VEGFR2 expression were evaluated by using 16 ?g gastrodin as the positive control.(2)HUVEC validation.Five HUVECs cell injury models were used.The model conditions were 7.2 ?M H2O2 for 12 h,3.6 ?M H2O2 for 12 h,11 ?M tert-butylhydroperoxide(t-BHP)for 4 h,5.5 ?M t-BHP for 4 hours and 320 ?M oxidized low density lipoprotein(oxLDL)for 12 h,which resulted in different degree of cell damage.The effects of 20,40 and 80 ?M THP on the cell activity and the percentage of scratch healing of HUVEC were detected by using 80 ?M gastrodin as the positive control.(3)Omics analysis.The differential metabolites and pathways regulated by 64 ?g/80 ?M THP were analyzed by DESI-MSI/GC-MS-based non-target metabolomics.3.Application of this platform in the study of chemical drugs.The effects and mechanisms of BA-12 on angiogenesis were revealed by this platform.(1)qCAM screening.The effects of BA-12(20,40,80 ?g)on the number of microvessels,and VEGFR2 expression were evaluated by using 40 ?g Dov as the positive control.(2)HUVEC validation.The effects of 1.25 ?M,2.5?M and 5 ?M BA-12 treatment for 24 h on HUVEC cell activity,percentage of scratch healing and protein expression of VEGFR2 were evaluated by using 2.5 ?M Dov as the positive control.The effects of 2.5 ?M,5 ?M and 10 ?M BA-12 treatment for 24 h on T24 cell activity,percentage of scratch healing,cell cycle and apoptosis rate were evaluated by using 2.5 ?M Dov as the positive control by MTT,scratch assay and flow cytometry.(3)Omics analysis.The differential metabolites and pathways regulated by BA-12 were analyzed by UPLC-QTOF-MS/GC-MS-based non-target metabolomics.Results:I Platform establishment1.Methodology investigation.(1)Optimization of drug delivery carrier.Gelatin sponge did not affect the drug and could be accurately quantified.(2)The choice of time for administration.The time for administration of decreased false positive rate was 36 h after opening the window.(3)The stability of the model.There was no significant difference in microvascular count between the groups of qCAM in different batches.(4)Model repeatability.Compared with the control group,the high dose(20?g)DG-15 administration significantly(P<0.05)increased the number of qCAM microvessels,which was consistent with the previous results.2.Research process examples.(1)qCAM screening.Compared with the control group,80?g ST administration significantly(P<0.05)inhibited the microvascular number,VEGFR2 mRNA expression and NO content,with the increase of the intercellular space of vascular endothelial cells and swelling of mitochondria.(2)HUVEC validation.Compared with the solvent group,40 ?M ST treatment could significantly(P<0.05)inhibit cell activity and scratch healing percentage after 24 h.(3)Omics analysis.ST inhibited angiogenesis by regulating glycerin metabolism(P<0.05).II Application examples1.Application of this platform in the study of TCM.(1)Literature mining.Most hot drugs with sweet flavor promote angiogenesis,and most cold drugs with bitter flavor inhibit angiogenesis.eNOS pathway related to cold and hot property involves in angiogenesis.(2)Evaluation using qCAM model.The difference of the effects of cold and hot Chinese herbs on angiogenesis was significant(P<0.05).(3)Platform verification.Compared with the control group,the decoction of VCR(83.33 mg/ml)could significantly(P<0.05)promote the microvessels,expression of VEGFR2 mRNA and NO content in qCAM;compared with the model group,VCR treatment for 24 h could protect the activity of injured cells and ROS overexpression;VCR promoted angiogenesis by regulating metabolism pathways such as tricarboxylic acid cycle(P<0.05).2.Application of this platform in the study of active components of TCM.(1)qCAM screening.Compared with the control group,64 ?g THP significantly(P<0.05)increased the number of qCAM microvessels and the expression level of VEGFR2 mRNA.(2)HUVEC validation.Compared with the model group,80 ?M THP treatment could significantly(P<0.05)protect the cell activity and the percentage of scratch healing after 24 hours.(3)Omics analysis.THP promoted angiogenesis by regulating the transformation of citrulline to arginine and affecting arginine biosynthesis(P<0.05).3.Application of this platform in the study of chemical drugs.(1)qCAM screening.Compared with the control group,BA-12 of 80 ?g significantly(P<0.05)inhibited the expression of microvascular number and VEGFR2 mRNA in qCAM.(2)HUVEC validation.Compared with the solvent group,5 ?M BA-12 significantly(P<0.05)inhibited the cell activity and the percentage of scratch healing,and decreased the expression of VEGFR2.(3)Omics analysis.BA-12 inhibited angiogenesis by regulating GSH and glycerophospholipids metabolism(P<0.05).Conclusion:This manuscript applied multidisciplinary methods for mutual corroboration,established one platform of screening and activity detection of angiogenic intervention drugs,and proposed the basic principles,operation process and guiding principles of this platform based on three continuous parts(qCAM screening,HUVEC validation and omics analysis).This platform has the following advantages:to realize the qualitative/quantitative detection of complex research objects such as TCM as one comprehensive method;to realize real-time detection in vivo and in vitro by obtaining multi-dimensional,real-time and fast biological effect information generated by the intervention of the subjects on angiogenesis;to identify more accurately the object to promote/suppress efficacy and molecular mechanism on angiogenesis in vivo and in vitro using multiomics analysis.This subject will provide technical support and methodology reference for the R&D of related new drugs.