Screening And Efficacy Study Of Angiogenesis Drug Monomers In Three Traditional Tibetan Medicine Prescriptions

Marrow mesenchymal stem cells(MSCs)are originated from mesodermal pluripotent stem cells and often isolated from bone marrow.They have the characteristics of attachment,self-renewal and differentiation of all mesoderm cell-derived and partial ectodermal cells(Such as the differentiation of muscle cells,neurons and epithelial cells)in vitro and vivo.They can release some growth factors or cytokines to promote endogenous angiogenesis through paracrine effects.The angiogenesis process generally includes the degradation of the vascular endothelial matrix,the proliferation and migration of endothelial cells,the formation of vascular loops and new basement membranes by the endothelialization of the endothelial cells.The endothelial and smooth muscle cells constitute the major components of the blood vessel wall.?-SMA and VEGF are specific marker genes of vascular smooth muscle cells and vascular endothelial cells,respectively.CD31 is an adhesion molecule and is often present in tight junctions between endothelial cells.The expression of three specific marker genes ?-SMA,VEGF and CD31 reflects the entire process of angiogenesis.This study established the positive control of simvastatin in vitro cell screening model of the VEGF promoter that regulates the reporter gene luciferase.Three Tibetan drugs(Wilwishfui-treaurepill;Ershiwuwei shanhu pill;20-Herb Chenxiang)with high content and high frequency were used for safety evaluation in vitro.The maximum concentration of 10?M was selected as the drug for further screening and 10 drug monomers with different concentrations were determined.The effect of compounds on luciferase transcriptional activity regulated by the VEGF promoter revealed that the four monomeric compounds of Alantolactone,Crocin II,Costunolide and Mangiferin can upregulate transcriptional expression of VEGF promoter-regulated reporter gene significantly.It demonstrated the expression of intracellular specificity genes can be upregulated simultaneously and the drug has the potential to induce rMSCs to differentiate into the cell types represented by this specific gene.In the case of compounds that were initially screened positive,the expression of vascular marker molecules induced by rat bone marrow mesenchymal stem cells(rMSCs)was investigated in vitro.RT-PCR and qPCR showed that the four compounds couldpromote to varying degrees.rMSCs transcriptionally synthesizes mRNA of ?-SMA,VEGF and CD31 genes,in which VEGF and CD31 have higher relative expression of mRNA at 2days while ?-SMA at 4 days.For higher expression levels,the timing of the expression of the three genes is in line with the general process of angiogenesis.rMSCs expressing ?-SMA,VEGF,and CD31 protein were detected by immunofluorescence in situ.The results showed that four compounds could significantly promote the expression in situ of VEGF protein(**P?0.01)in rMSCs,as well as increase the expression of a-SMA and CD31 protein in rMSCs to varying degrees,verifying the potential of rMSCs to differentiate into the cell types represented by this specific gene,and also demonstrate the four drugs monomeric compounds could partly promote angiogenesis.In this study,four monomer compounds including alantolactone,crocin ?woody lactone,and mangiferin were screened and verified from promoter-regulated reporter gene transcription expression activities,relative mRNA expression levels,and protein expression levels.Not only it provides more methods for rapid screening and quantitative assessment of drug efficacy,but also validates the potential of rMSCs to differentiate into blood vessels,providing a theoretical basis for rMSCs to vascular phenotype conversion and drug promotion of angiogenesis.