| Objective:To investigate the pro-angiogenic effects of paeoniflorin (PF) and the mechanisms of action.Methods:Transgenic zebrafish and human umbilical vein endothelial cells (HUVECs) were used to investigate the pro-angiogenic effects and the relative mechanisms of action of PF.1. The pro-angiogenic effects of PF in transgenic zebrafish.1) The pro-angiogenic effect of PF on Tg(fli-1:EGFP)ylzebrafish vascular insufficiency model. Zebrafish embryos of 24 hpf were pretreated with VRI for 3 h to establish the vascular insufficiency model and then post-treated with various concentrations of PF for 24 h. The formation of DLAV and ISVs were observed with a fluorescence microscope to evaluate the pro-angiogenic effect of PF.2) The pro-angiogenic effect of PF on Tg(fli-1:EGFP) y1 and Tg(fli-1:nEGFP)y7 transgenic zebrafish SIV. The 24 hpf Tg(fli-1:EGFP)yl, Tg(fli-1:nEGFP)y7 zebrafish embryos were treated with various concentrations of PF (25μM-100μM) for 48 h before observed with a fluorescence microscope. The number of SIV sproutings in Tg(fli-1:EGFP) yl zebrafish and endothelial cells in Tg(fli-1:nEGFP) y7 zebrafish were counted to evaluate the pro-angiogenic effect of PF.3) The pro-angiogenic effect of PF on the cerebral vessels in zebrafish. Tg(fli-1:EGFP; GATA-1:DsRED) zebrafish embryos were used to investigate the pro-angiogenic effect of PF on the cerebral vessels angiogenesis and blood flow. Zebrafish embryos of 24 hpf were pretreated with VRI (300 ng/ml,3 h) and then treated with PF (100μM) for 24 h before observed under a fluorescence microscope.4) The effect of PF on VEGF, FGF and EGF gene expression. In order to investigate PF’s mechanisms of action, zebrafish embryos of 24 hpf were treated with various concentrations of PF (6.25μM-100μM) for 6 h. Total mRNA was extracted and reverse transcribed into cDNA. Then real-time PCR was performed to evaluate the relative gene expression.5) The effect of PF on flt-1, kdr, kdrl and vWF gene expression. Zebrafish embryos of 24 hpf were pretreated with VRI for 2 h and then treated with various concentrations of PF (25μM- 100μM) for 6 h. Total mRNA was extracted and reverse transcribed into cDNA. Then real-time PCR was performed to evaluate the relative gene expression.2. In order to investigate the pro-angiogenic effect of PF on endothelial cells, we observed the pro-angiogenic effects of PF in HUVECs at which cell proliferation, migration and tube formation were assessed.1) The effect of PF on HUVECs proliferation. MTT assay:HUVECs were treated with various concentrations of PF. After 30 h, cells proliferation was determined by MTT assay with a microplate reader and the data were expressed as OD value. Hoechst staining: HUVECs were treated with various concentrations of PF (0.001,0.003,0.01,0.03μM). After 30 h, cells proliferation was determined by Hoechst 33258 staining assay(10μg/ml,15 min) which observed with a microscope and the data were expressed as cell count.2) The effect of PF on HUVECs migration. Wound healing assay:HUVECs were seeded in 48-well plate, and confluent HUVECs were scratched with a pipette tip. Then HUVECs were treated with PF (0.3,1 and 10μM) for 10 h. Migration was quantified as the difference value of the width of the area covered with cells and the area of the cell-free wound. Cell migration assay:xCELLigence RTCA DP Instrument was used to assess the promoting effect of PF on HUVECs migration. HUVECs were treated with different concentrations of PF (0.1,1 and 10 μM) and continuously monitored for 8 h. The data were collected every 15 min. Cell migration was expressed as cell index.3) The effect of PF on tube formation in HUVECs. Tube Formation Assay:In order to investigate the pro-angiogenic effect of PF on endothelial cells, tube formation assay was performed in HUVECs. HUVECs were treated with PF (0.3,1.0 and 10μM) for 4 h. The network-like structures were examined under an inverted microscope. The number of branching points in three random fields per well was quantified.3. In order to seek the possible therapeutic target of PF, the role of adenosine A1 receptor in the pro-angiogenic effect of PF was investigated.1) The role of adenosine A1 receptor in PF induced SIV angiogenesis. Zebrafish embryos of 24 hpf were co-treated with specific adenosine A1 receptor antagonist DPCPX (3μM) and PF (50μM). After 48 h, SIV sprouting counts were observed with a fluorescence microscope.2) The role of adenosine A1 receptor in PF induced ISVs angiogenesis. Zebrafish embryos of 24 hpf were pretreated with VRI and then co-treated with DPCPX (3μM) and PF (50μM). After 24 h, ISVs growth were observed with a fluorescence microscope.3) Determination of the effect of PF on ecto-5’nucleotidase activity. HUVECs were cultured in 6-well plate and treated with PF (0.001,0.01,0.1,1.0 and 10μM) for 24 h. Then washed with phosphate-free HEPES-buffered Ringer’s solution. Reaction buffer was added to each well and the plate was incubated at 37℃ for ten minutes. The reaction was stopped by 0.3 ml of 30%trichloroacetic acid. Ammonium molybdate (4.5 ml) was then added. After incubation at 45℃ for twenty minutes, the absorbance at 750 nm was measured. The inorganic phosphate production inhibited by 100μM α,β-methylene adenosine-5’-diphosphate, a specific ecto-5’nucleotidase inhibitor, represented the ecto-5’nucleotidase activity.4) The effect of PF on adenosine uptake. To study the adenosine uptake in HUVECs,300μl of [3H] adenosine and different concentrations of PF (0.001,0.01,0.1,1.0 and 10μM) were added to cells for 4 min at room temperature. To determine the passive uptake of adenosine, monolayers of cells were incubated in buffer containing [3H]adenosine in the presence of 0.5 mM nitrobenzylthioinosine (NBMPR). The uptake process was terminated by washing the plates rapidly with ice-cold phosphate-buffered saline buffer. Plates were air-dried and 500μl of 5% Triton-X added for lysing cells overnight. Cell lysates were mixed with 2 ml of scintillation liquid. Radioactivity was measured using a scintillation counter.Results:The effects and the underlying mechanisms of the pro-angiogenic effect of PF in zebrafish embryos in vivo and in HUVECs in vitro.1. PF promoted zebrafish angiogenesis. 1) The pro-angiogenic effect of PF on Tg(fli-1:EGFP) y1 zebrafish vascular insufficiency model. VRI treatment inhibited DLAV, ISVs growth and PF (6.25μM-100μM) treatment rescued the growth of DLAV, ISVs in a dose-dependent manner.2) The pro-angiogenic effect of PF on Tg(fli-1:EGFP) y1 and Tg(fli-1:nEGFP)y7 transgenic zebrafish SIV. PF (25μM-100μM,48 h) promoted Tg(fli-1:EGFP)yl zebrafish embryos SIV sprouting in a dose-dependent manner. PF (25μM-100 μM,48 h) elevated endothelial cell number of SIV in Tg(fli-1:nEGFP)y7 zebrafish embryos in a dose-dependent manner.3) The pro-angiogenic effect of PF on the cerebral vessels in zebrafish. PF (100μM) rescued VRI-induced PHS, PHBC, MCeV growth defect and cerebral blood flow decline. The effect of PF on angiogenesis related gene expression in zebrafish:4) The effect of PF on VEGF, FGF and EGF gene expression. PF (12.5μM-100μM) increased the mRNA expression of VEGFaE4E5 and bFGF, while has no effect on VEGFc, FGF1 and EGF mRNA expression.5) The effect of PF on flt-1, kdr, kdrl and vWF mRNA expression. VRI down-regulated the expression of flt-1, kdr, kdrl and vWF mRNA and PF rescued the down regulation effect of VRI.2. PF promoted angiogenesis in HUVECs.1) PF promoted HUVECs proliferation. MTT assay:PF (0.003μM-0.3μM) could promote HUVECs proliferation. Hoechst staining:PF (0.01 and 0.03μM) treated for 30 h elevated HUVECs cell count.2) PF promoted HUVECs migration. Wound healing assay:PF (1.0 and 10μM,10 h) enhanced the wound healing ability of HUVECs. Cell migration assay:PF (0.1,1.0 and 10 μM) promoted HUVECs migration, and the peak effect time point was at about 6.5 h.3) PF promoted HUVECs tube formation. PF (0.3μM) enhanced the ability of forming tube like structure in HUVECs.3. The role of adenosine A1 receptor in the pro-angiogenic effect of PF.1) The role of adenosine A1 receptor in PF induced SIV angiogenesis. PF (50μM) promoted SIV angiogenesis in zebrafish, and DPCPX (3μM) abolished the pro-angiogenic effect of PF on SIV sprouting. However, when used alone, DPCPX (3μM) didn’t inhibit SIV sprouting.2) The role of adenosine A1 receptor in PF induced ISVs angiogenesis. VRI (300 ng/ml) induced ISVs insufficiency in zebrafish, and PF (50μM) treatment rescued the down-regulating effect of VRI on ISVs growth. DPCPX (3μM) abolished the promoting effect of PF on ISVs growth, while it did not induced ISVs insufficiency when used alone.3) Determination of the effect of PF on ecto-5’nucleotidase activity. PF (0.001μM-10μM) exhibited no significant affection on ecto-5’nucleotidase activity in HUVECs.4) The effect of PF on adenosine uptake. PF (0.001μM-10μM) has no significant effect on adenosine uptake in HUVECs.Conclusion:Paeoniflorin could promote angiogenesis in zebrafish in vivo and in HUVECs in vitro, adenosine A1 receptor activation may be involved in the mechanisms of action of PF.
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