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ObjectiveTo analyze and determine the differential components of freeze-dried and sun-dried Panacis Quinquefolii Radix（PQR）， and to compare the differences in their pro-angiogenic activities. MethodFingerprints of freeze-dried and sun-dried PQR were established based on ultra performance liquid chromatography（UPLC）， and chemometrics methods such as principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were combined to determine the differential saponin composition of the two decoction pieces， and six representative saponins were selected and their contents in freeze-dried and sun-dried PQR were determined by UPLC. Transgenic zebrafish line Tg（fli1a∶EGFP） embryos fertilized for 24 h were selected， and different doses of 70% methanol extracts of freeze-dried and sun-dried PQR（10， 30 mg·L-1） were used to intervene in normal zebrafish and in a zebrafish model of intersegmental vascular（ISV） injury induced by vascular endothelial growth factor（VEGF） receptor tyrosine kinase inhibitor Ⅱ（PTK787）， then the development of subintestinal vein（SIV） and ISV of zebrafish was observed， SIV diameter， mean number of crossings and mean number of germinations were determined， and the ISV vascular index was calculated， in order to compare the pro-angiogenic activities of the two decoction pieces. ResultThe similarity of the fingerprints of freeze-dried and sun-dried PQR decoction pieces was>0.950， and 17 common peaks were identified， of which 6 common peaks were designated as peak 6（ginsenoside Rg1）， peak 7（ginsenoside Re）， peak 8（ginsenoside Rb1）， peak 11（ginsenoside Rc）， peak 13（ginsenoside Rb2）， and peak 16（ginsenoside Rd）， respectively. A total of 11 differential saponin components were screened by PCA and OPLS-DA， indicating that there were some differences in the contents of the components in the two decoction pieces. The results of determination showed that the contents of ginsenosides Rg1， Re， Rb1 and Rb2 in freeze-dried PQR were higher than those in sun-dried PQR， while the contents of ginsenosides Rc and Rd were lower than those in sun-dried PQR（P<0.05， P<0.01）. In the study of the pro-angiogenic effect on normal zebrafish embryos， compared with the blank group， and the SIV vessel diameter， mean germination rate and mean crossover rate were significantly higher in the high-dose groups of freeze-dried and sun-dried PQR（P<0.01）， and the vessel diameter， mean numbers of crossings and germinations in the freeze-dried PQR group were higher than those of the sun-dried PQR group（P<0.05）. In the study of the pro-angiogenic effect on zebrafish embryos with ISV injury， the development of ISV in the model group was significantly inhibited when compared with the blank group， compared with the model group， different dose groups of freeze-dried and sun-dried PQR could promote the growth and sprouting of ISV， and the number of normal blood vessels in the freeze-dried PQR group was significantly higher than that in the sun-dried PQR group at the same dosage（P<0.05）. ConclusionFreeze-drying can effectively avoid the loss and secondary transformation of ginsenosides in PQR， and its angiogenic activity is better than that of sun-dried PQR， which can provide a reference for the production and development of high-quality PQR decoction pieces.
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BACKGROUND:Combining seed cells with 3D bioprinting technology enables the specific construction of various tissues and organs to meet the demands of tissue repair.However,further research is needed on the promotion of angiogenesis in damaged tissues. OBJECTIVE:By cultivating a 3D scaffold structure of methacrylated gelatin loaded with fibroblasts,obtaining the supernatant,and mixing it in different proportions with a complete culture medium to simulate the cellular microenvironment during tissue repair,this study aimed to explore the role of various cellular microenvironments in promoting angiogenesis in endothelial cells. METHODS:A methacrylated gelatin scaffold structure loaded with fibroblasts was prepared using an extrusion-based 3D bioprinting process.Hydrogel scaffold extract was prepared and mixed with a complete culture medium in ratios of 1:1,1:2,and 1:4 to obtain conditioned medium.Mouse embryonic fibroblasts BALB3T3 and human umbilical vein endothelial cells were co-cultured with complete medium(control group)and hydrogel scaffold extract,respectively.Cell proliferation was assessed using the CCK-8 assay and cell viability was analyzed using live/dead staining.Three kinds of conditioned medium and complete medium(control group)were used to co-culture with human umbilical vein endothelial cells for tube formulation assay,vascular genetic testing,and immunofluorescence staining of CD31. RESULTS AND CONCLUSION:(1)Scanning electron microscopy revealed that the methacrylated gelatin scaffold exhibited a porous structure,and rheological results demonstrated excellent mechanical properties of the hydrogel.CCK-8 assay and live/dead cell staining showed that the hydrogel scaffold extract had no obvious cytotoxicity.(2)Tube formulation assay indicated that the hydrogel showed the total length of cell tubules in 1:1 conditioned medium group was smaller than that in the control group(P<0.05).There were no statistical differences among the four groups in the number of vascular branches formed by endothelial cells(P>0.05).(3)qRT-PCR results showed that for vascular endothelial growth factor mRNA expression,the 1:2 conditioned medium group was lower than the 1:1 conditioned medium group on day 1(P<0.01).On day 3,the expression level of vascular endothelial growth factor in the 1:2 conditioned medium group was higher than that in the control group(P<0.01).On day 5,the cytokine expression level in the 1:2 conditioned medium group was significantly higher than that in the other three groups(P<0.01 or P<0.000 1).The expression in the 1:1 conditioned medium group was significantly lower than that in the other three groups(P<0.05 or P<0.01).On day 1,the expression level of basic fibroblast growth factor in the 1:1 conditioned medium group was significantly higher than that in the control group and 1:4 conditioned medium group(P<0.01,P<0.05).The expression was higher in the 1:2 conditioned medium group than that in the control group(P<0.05).On day 3,the expression levels of cytokines in the 1:4 conditioned medium group was higher than that in the control group(P<0.05).(4)On day 3,the expression of CD31 in the 1:2 conditioned medium group was higher than that in the control group and the 1:4 conditioned medium group(P<0.05).(5)The results indicate that the resulting conditioned media can simulate the microenvironment of vascular regeneration after tissue damage,promoting the vascularization process of endothelial cells.The best promotion of vascularization in endothelial cells was observed when the ratio of supernatant to complete culture medium was 1:2.