Proangiogenesis effects of compound danshen dripping pills in zebrafish

Fig. 1. Screening of potential target genes andpathways of CDDP. A We obtained 7,100 coronary heart disease-related genes from GeneCards, 108 target genes of Panax notoginseng and 162 target genes of Radix Salviae from the TCMSP database. The 65 intersecting genes were considered important targets and were further studied. B Among the top 28 potential target genes predicted to be regulated by CDDP, AKT1 and JUN ranked highest. C Protein–protein interactions (PPIs) of the 65 targets (interaction score ≥ 0.950). The separated proteins were predicted to be less related and then deleted. D, E GO enrichment analysis showing the top 20 molecular biological functions of the target genes. This result demonstrated that DNA-binding transcription factor binding was the most common gene function. The abscissa represents the ratio of related genes, and the ordinate lists the functions corresponding to proteins. The bubble size represents the number of genes, whereas the color represents the P value. F, G KEGG pathway enrichment analysis showing the 20 most likely pathways involved; the PI3K-AKT signaling pathway ranked first

Fig. 2. The proangiogenic effect of CDDP on subintestinal vessels (SIVs). A Twenty-four hpf embryos were treated with various concentrations of CDDP (0.3, 0.6, 0.9 and 1.2 mg/mL) for 48 h. The survival rate was recorded. B The toxicity screening of CDDP did not significantly affect the early-stage appearance of embryos. Scale bar, 50 μm. C Body length was not affected by CDDP treatment, indicating that embryo development was normal. D The yolk sac area was also unchanged, which meant that the nutritional status was maintained at a similar level. E Heart rates of the treatment groups were not notably different from those of the control group. F Schematic representation of the experimental design for G-, I. Twenty-four hpf transgenic zebrafish Tg(fli1: EGFP) embryos were cotreated with CDDP (0.3, 0.6, 0.9 and 1.2 mg/mL) for 24 h. After washing with culture water, the embryos were treated with the same concentration of CDDP for another 24 h. The box in 72 hpf larvae indicates the position photographed and displayed in G. G Representative pictures of each group; arrows indicate branch points and sprouting vessels of SIVs induced by CDDP in zebrafish. Scale bar, 50 μm. H, I The branch point…

Fig. 3. Endothelial layer thickening and ectopic growth of optical vessels in the CDDP treatment group. A-E Different treatments with CDDP led to thickening of the endothelial layer in the optical arteries of Tg(fli1:EGFP) larvae. Scale bar, 50 μm. In the 1.2 mg/mL CDDP treatment group, ectopic sprouts F and tumor-like growth G could be observed. H Endothelial layer thickness (T) was calculated by subtracting inner diameter (Di) from outer diameter (Do) using the formula T = (Do—Di)/2. I Changes in the thickness of the endothelial layer were quantified and are presented as the mean ± SEM. CDDP promoted thickening of the endothelial layer, although only the 1.2 mg/mL group showed statistical significance. N = 15 for each group, and the experiment was replicated 4 times. J Twenty-four hpf Tg(fli1: EGFP) embryos treated with or without CDDP (1.2 mg/mL) for 48 h. The mRNA extracted from 20 embryos from the same group was pooled, and the expressions of VEGF receptors (Flt1, Kdrl and Flt4) and VEGF (Vegfaa) were analyzed by RT–qPCR. Both VEGF receptor 2 (Vegfaa) and Kdrl were significantly upregulated in the CDDP treatment group. The relative expression level of each gene was normalized to the…

Fig. 4. Analysis of FGF, MAPK/ERK, SREBP 1 and 2 signaling pathways in CDDP-treated embryos. Expression levels were quantified using RT–qPCR and normalized against β-actin. Data are represented by relative fold changes ± SEM with respect to control embryos (arbitrarily set to 1). A Relative mRNA expression levels of Fgf1a, Fgf2, Fgf4, Fgf10a and Fgf11b, as well as the receptors Fgfr1a and Fgfr2. None were statistically changed. B The relative mRNA expression of bRaf, cplα2, mknk2b, JUN, Egf and Btc was also detected. Only JUN expression was significantly upregulated. C Relative mRNA expression levels of the SREBP 1 and 2 signaling pathways showed that Srebp2 might be activated and that as a downstream factor, Fasn expression was elevated. N = 6, and the data are presented as the mean ± SEM

Fig. 5. Pro-angiogenesis effect of CDDP in VRI-induced ISV deficiency in zebrafish. A Schematic representation of the experimental design. Twenty-four hpf Tg(fli1:EGFP) zebrafish embryos were pretreated with VRI (100 nM) for 24 h and then treated with 1.2 mg/mL CDDP or fresh VRI for another 24 h. Ten consecutive ISVs in the framed area were photographed, observed in each embryo, and then displayed in B. Fifteen embryos were included in each group, and the experiment was replicated three times. B Embryos treated with CDDP alone showed obvious enlarged posterior blood islands (PBIs, indicated by the dotted circle), whereas VRI treatment inhibited the development of ISVs, which could be restored by CDDP treatment. Arrows indicate deficient ISVs. Scale bar, 100 μm. C The numbers of intact and deficient vessels were calculated. D The ISV index was used to quantify the growth of ISVs, which was calculated using the formula ISV index = (intact vessel number × 1) + (defective vessel number × 0.5). The results showed that there was no obvious defect in the development of ISVs in the CDDP and DMSO treatment groups, but the ISVs of the VRI-treated embryos were significantly shorter, and the VRI +…

Fig. 6. The effect of the VEGF/PI3K/AKT signaling pathway on the proangiogenic effect of CDDP. A Schematic representation of the experimental design. Twenty-four hpf Tg(fli1:EGFP) embryos were pretreated with various concentrations of the PI3K inhibitor LY294002 (LY, 0.3–3 µM) and the AKT inhibitor VIII (AKTi, 0.03–0.3 µM) for 30 min and were then cotreated with VRI (100 nM) and CDDP (1.2 mg/mL) for 24 h. Ten consecutive ISVs in the framed area were observed in each embryo. Fifteen embryos were analyzed in each group, and the experiment was replicated four times. B CDDP relieved the abnormal development of ISVs, which could be inhibited by 3 µM LY, 0.1 or 0.3 µM AKTi. C Twenty-four hpf embryos were treated with various concentrations of LY (0.3–3 µM) and AKTi (0.03–0.3 µM) for 24 h. Both inhibitors could promote angiogenesis of ISVs at specific concentrations. D The angiogenic efficacy of CDDP could be weakened by LY or AKTi at specific concentrations. The ISV index of each zebrafish embryo was calculated as (the number of intact vessels × 1) + (the number of defective vessels × 0.5). Data are presented as the mean ± SEM